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DOI:10.1111/bph.13397 www.brjpharmacol.org
British Journal of Pharmacology
RESEARCH PAPER Genipin alleviates sepsisinduced liver injury by restoring autophagy
Correspondence Sun-Mee Lee, School of Pharmacy, Sungkyunkwan University, Suwon 440–746, Korea. E-mail: [email protected] ---------------------------------------------------------
Received 10 October 2015
Revised 18 November 2015
Accepted
Hong-Ik Cho, So-Jin Kim, Joo-Wan Choi and Sun-Mee Lee
30 November 2015
School of Pharmacy, Sungkyunkwan University, Suwon 440–746, South Korea
BACKGROUND AND PURPOSE Autophagy is an essential cytoprotective system that is rapidly activated in response to various stimuli including inflammation and microbial infection. Genipin, an aglycon of geniposide found in gardenia fruit, is well known to have anti-inflammatory, antibacterial and antioxidative properties. This study examined the protective mechanisms of genipin against sepsis, with particular focus on the autophagic signalling pathway.
EXPERIMENTAL APPROACH Mice were subjected to sepsis by caecal ligation and puncture (CLP). Genipin (1, 2.5 and 5 mg·kg 1) or vehicle (saline) was injected i.v. immediately (0 h) after CLP, and chloroquine (60 mg·kg 1), an autophagy inhibitor, was injected i.p. 1 h before CLP. Blood and liver tissues were isolated 6 h after CLP.
KEY RESULTS Genipin improved survival rate and decreased serum levels of aminotransferases and pro-inflammatory cytokines after CLP; effects abolished by chloroquine. The liver expression of autophagy-related protein (Atg)12-Atg5 conjugate increased after CLP, and this increase was enhanced by genipin. CLP decreased Atg3 protein liver expression, and genipin attenuated this decrease. CLP impaired autophagic flux, as indicated by increased liver expression of microtubule-associated protein-1 light chain 3-II and sequestosome-1/p62 protein; this impaired autophagic flux was restored by genipin, and chloroquine abolished this effect. Genipin also attenuated the decreased expression of lysosome-associated membrane protein-2 and Rab7 protein and increased expression of calpain 1 protein induced by CLP in the liver.
CONCLUSIONS AND IMPLICATIONS Our findings suggest that genipin protects against septic injury by restoring impaired autophagic flux. Therefore, genipin might be a potential therapeutic agent for the treatment of sepsis.
Abbreviations ALT, serum alanine aminotransferase; AST, serum aspartate aminotransferase; Atg, autophagy-related protein; CLP, caecal ligation and puncture; LAMP, lysosome-associated membrane protein; LC3, microtubule-associated protein 1 light chain 3; mTOR, mammalian target of rapamycin; PE, phosphatidylethanolamine; phospho, phosphorylated; p62, sequestosome1/p62; p70S6K, p70S6 kinase; TBS/T, 0.1% Tween 20 in 1× Tris-buffered saline; TEM, transmission electron microscopy; 4E-BP1, eukaryotic translation initiation factor 4E binding protein 1
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Genipin alleviates liver injury by restoring autophagy
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Tables of Links TARGETS Calpain 1 Calpain 2
LIGANDS Cathepsin B
Chloroquine
mTOR
IL-6
TNF-α
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology. org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).
Introduction Sepsis is a massive systemic inflammatory syndrome that is triggered by severe infection, trauma or toxin and leads to multiple organ failure and death. During the development of sepsis, bacterial components lead to excessive secretion of pro-inflammatory cytokines, which induce endothelial and epithelial injury, vasodilatation and oedema, subsequently causing the development of multiple organ dysfunction syndromes (Taylor et al. 1995). Despite advances in therapeutic care and an increased understanding of the pathophysiology of sepsis, it remains a leading cause of mortality in intensive care units. There is a substantial need for an effective therapy that will decrease the morbidity and mortality associated with sepsis. The liver is a critical organ for host survival in response to sepsis through its role in scavenging bacteria and producing inflammatory mediators; however, it might also become damaged in the pathogenesis of sepsis as the result of a dysregulated inflammatory response (Recknagel et al. 2012; Gonnert et al. 2013). Clinical evidence has shown that hepatic dysfunction is an early event in sepsis and is a specific risk factor for poor outcome (Kramer et al. 2007). Autophagy is an evolutionarily conserved self-eating–recycling process in which cytoplasmic proteins or organelles are sequestered by a double-membraned autophagosome and delivered to lysosomes for proteolytic degradation. Autophagy has recently been recognized as a crucial modulator of immune responses as it participates in pathogen killing or antigen processing and presentation by immune cells, thus highlighting its potential as a therapeutic target in infectious diseases, including sepsis (Valdor and Macian 2012). Several studies have demonstrated the critical association between autophagy and sepsis. Autophagy was activated in patients with sepsis and in caecal ligation and puncture (CLP) animal models of sepsis (Hsieh et al. 2011; Lo et al. 2013). Moreover, polymorphisms in the autophagy-related protein (Atg) 16 L1 or autophagy-related human immunity-related GTPase gene affect the clinical outcome of patients with severe sepsis (Kimura et al. 2014; Savva et al. 2014). Accumulating evidence from human and animal studies indicates that increased autophagic vacuolization in the liver, particularly in the hepatocytes, is possibly associated with mitochondrial injury (Watanabe et al. 2009). In the liver of the CLP animal model, the number of autophagosomes was increased, as demonstrated by microtubule-associated protein-1 light chain 3 (LC3)-II accumulation and electron microscopic examination, and its inhibition resulted in elevated serum transaminase levels (Takahashi et al. 2013). However, there is
limited information on the core molecular machinery of autophagic flux and its regulatory signalling in sepsis. Genipin, an aglycone derived from an iridoid glycoside called geniposide, is a major component of the fruit of Gardenia jasminoides, which has been widely used for the treatment of inflammatory diseases and hepatic disorders in traditional medicine (Koo et al. 2004). A plethora of studies also showed that genipin has various pharmacological effects including antioxidative, anticancer and antifungal activities (Lelono et al. 2009; Kim et al. 2012a; Kim et al. 2013). Genipin reduced mortality induced by D-galactosamine/LPS-induced acute liver injury by preventing apoptotic cell death and the inflammatory response (Takeuchi et al. 2005). Recently, our group reported that genipin attenuates mortality and multiple organ injury in sepsis through inhibition of toll-like receptor-dependent inflammatory signalling pathways (Kim et al. 2012b). Therefore, we investigated the hepatoprotective mechanisms of genipin in septic injury, in particular focusing on the autophagy machinery. Our present study may provide a novel strategy to counteract sepsis, which is currently the most challenging problem in intensive care.
Methods Group sizes Ten mice were used per each group for the survival test and eight mice per each group for biochemical assay.
Randomization Randomization was conducted by an individual other than the operator. The animals with similar degrees of body weight were selected randomly from the pool of all cages eligible for inclusion in the study and divided into groups.
Statistical comparison Results are presented as mean ± SEM. Survival data were analysed by Kaplan–Meier curves and the log-rank test. The overall significance of results was analysed by one-way ANOVA. Differences between compared groups were considered statistically significant at P < 0.05 with the appropriate Bonferroni correction for multiple comparisons. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). British Journal of Pharmacology (2016) 173 980–991
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Validity of animal species or model selection CLP in rodents has become the most widely used and clinically relevant model for experimental sepsis and is currently considered as the gold standard in sepsis research. The CLP model has been considered to be a realistic model for the induction of polymicrobial sepsis in experimental settings to study the underlying mechanisms of sepsis.
Ethical statement All experiments were approved by the Animal Care Committee of the Sungkyunkwan University School of Pharmacy (SUSP14-27) and performed in accordance with the guidelines of the National Institutes of Health (NIH publication No. 86–23, revised 1985) with adherence to the 3Rs (Replacement, Refinement and Reduction). Animal studies follow the ARRIVE guidelines (Kilkenny et al., 2010; McGrath & Lilley, 2015). Animals. Male imprinting control region mice weighing 27–29 g were obtained from Orient Bio Inc. (Seongnam, Korea).
Experimental procedures Polymicrobial sepsis was induced by CLP as described previously by Chaudry et al. (1979. Mice were anaesthetized by i.m. injection of ketamine (55 mg·kg 1, Yuhan Corporation, Seoul, Korea) and xylazine (7 mg·kg 1, Bayer, Germany), a midline abdominal incision was then made and the caecum was carefully exposed avoiding damage to the blood vessels. The caecum was ligated just distal to the ileocecal valve without intestinal obstruction, punctured twice using a 20-gauge needle and squeezed to expel a small amount of faecal material to ensure patency of the puncture sites. The caecum was returned to its normal intra-abdominal position, and the abdominal incision was closed with two layers of running sutures. Shamoperated animals were subjected to laparotomy and intestinal manipulation without ligation and puncture. All animals received 1 mL of normal saline s.c. immediately after the operation for fluid resuscitation. For the survival experiment, mice received an i.v. injection of genipin (Wako Pure Chemical Industries, Ltd., Osaka, Japan; 1, 2.5, and 5 mg·kg 1) or saline (vehicle) via the tail vein immediately (0 h) after CLP. The dose and timing of genipin treatment was selected based on our previous study (Kim et al. 2012b). Mortality was monitored up to 10 days after the operation, and the mice were followed for 3 weeks to ensure that no late mortalities occurred. On the basis of a survival test, 2.5 mg·kg 1 genipin was selected as the optimally effective dose for further biochemical studies. Animals were randomly divided into five groups as follows: (1) vehicle-treated sham-operated (sham), (2) 2.5 mg·kg 1 genipin-treated sham-operated (genipin), (3) vehicle-treated CLP (CLP), (4) 2.5 mg·kg 1 genipin-treated CLP (CLP + genipin), (5) 60 mg·kg 1 chloroquine (CQ) and 2.5 mg·kg 1 genipintreated CLP (CQ + CLP + genipin). Chloroquine was dissolved in saline and administered i.p. (60 mg·kg 1) 1 h before the CLP for confirming the involvement of autophagy in the protective effects of genipin on sepsis. Under ketamine (55 mg·kg 1) and xylazine (7 mg·kg 1) anaesthesia, mice were killed at 6 h after CLP. Blood was collected from the inferior vena cava 6 h after CLP, centrifuged (10 000 rpm, 10 min, 4°C) to obtain a serum sample and stored at 75°C until assayed. Liver tissue was 982
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simultaneously isolated at 6 h after CLP and stored at until assayed.
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Housing and husbandry Mice (n = 5 animals per cage) were kept in a temperaturecontrolled and humidity-controlled room (25 ± 1°C and 55 ± 5%, respectively) under a 12 h light–dark cycle, with water and food provided ad libitum. Mice were given 7 days to acclimatize to the housing conditions and reverse light cycle and habituated to handling before starting the experiments. Mice were monitored for confirming abnormal symptoms or wound that would be reasons for removal of the animal from the experiment prematurely.
Interpretation This study thoroughly considered the 3Rs. Assessment of hepatic damage. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, the representative biomarkers of liver damage, were assayed at 37°C by monitoring decrease in absorbance at 340 nm for 1 min because of the disappearance of NADH with ChemiLab ALT and AST assay kit (IVDLab Co., Uiwang, Korea), respectively, using a Hitachi 7600 automatic analyzer (Hitachi, Tokyo, Japan). Serum cytokine levels. Serum TNF-α and IL-6 levels were quantified using commercial mouse ELISA kits (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Western blot immunoassay. Fresh liver tissue was isolated and homogenized in PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology In., Seongnam, Korea) for whole protein samples according to the manufacturer’s instructions. After being kept in an ice-cold bath for a period of 30 min for cell lysis, the whole homogenate was centrifuged at 13 000 x g for 5 min. Protein concentration was determined using a BCA Protein Assay kit (Pierce Biotechnology Inc., Rockford, IL, USA). Protein samples (20 μg) were separated by 10–15% SDS-PAGE and transferred to PVDF membranes using the Semi-Dry Trans-Blot Cell (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were washed with 0.1% Tween 20 in 1× Tris-buffered saline (TBS/T) and blocked for 1 h at room temperature with 5% (w/v 1) skimmed milk powder or 5% BSA in TBS/T. Blocked membranes were incubated overnight at 4°C with primary antibodies, washed five times for 7 min each in TBS/T and incubated with the appropriate secondary antibodies for 1 h at room temperature, followed by detection using an ECL detection system (iNtRON Biotechnology Inc.) according to the manufacturer’s instructions. The intensity of the immunoreactive bands was determined using TOTALLAB TL 120 software (Nonlinear Dynamics Ltd., Newcastle, UK). The following primary antibodies were used: Atg3, Atg12–5 complex, Atg7, phosphorylated (phospho)-mammalian target of rapamycin (mTOR), mTOR, phospho-eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), 4E-BP1, phospho-p70S6 kinase (p70S6K), and 70S6K (all from Cell Signaling Technology, Beverly, MA, USA); sequestosome-1/p62 (p62) (Abcam, Cambridge, MA, USA); beclin-1, cleaved Atg5, calpain 1, calpain 2, Rab7, and
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cathepsin B (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); LC3 and lysosome-associated membrane protein (LAMP)2 (Novus Biologicals, Littleton, CO, USA); and β-actin (Sigma-Aldrich, St. Louis, MO, USA). Protein densities were standardised to those of β-actin, and phosphorylated protein densities were standardised to those of total protein for total lysates. Transmission electron microscopy (TEM). Liver tissues were fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in 100 mM sodium phosphate (pH 7.2). Samples were washed with 100 mM Na cacodylate (pH 7.4), postfixed in 2% osmium tetroxide and then washed again. The samples were dehydrated in a graded series of ethanol and propylene oxide and embedded in epoxy resin (TAAB 812 Resin; Marivac Industries, Montreal, QC, Canada). Ultrathin (60–70 nm) sections were counterstained with uranyl acetate and lead citrate and viewed using a Hitachi 7600 TEM (Hitachi High-Technologies America, Inc., Schaumburg, IL, USA) equipped with a Macrofire monochrome progressive scan CCD camera (Optronics, Inc., Muskogee, OK, USA) and AMTV image capture software (Advanced Microscopy Techniques, Corp., Danvers, MA, USA). Immunohistochemistry. Formalin-fixed, paraffin-embedded liver tissues were prepared for immunohistochemistry assay. Each sample was fixed by immersion in 10% neutralbuffered formalin. The liver sections were deparaffinised
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with xylene, hydrated in serial dilutions of alcohol and microwaved in citrate buffer for antigen retrieval. The sections were immersed in 3% hydrogen peroxide solution to inhibit endogenous peroxidase activity and then incubated with monoclonal antibodies against LC3 (Novus Biologicals; 1:200) or LAMP-2 (Santa Cruz Biotechnology; 1:1000) followed by Polink-2 Plus HRP anti-mouse or rabbit DAB detection kit (Golden Bridge International, Inc., Bothell, WA, USA) according to the manufacturer’s instructions. The sections were stained with diaminobenzidine (Dako, Glostrup, Denmark) and counterstained with Mayer haematoxylin (ScyTek Laboratories, Inc., West Logan, UT, USA), washed again, dehydrated in alcohol, cleared in xylene, mounted with MM-24 (Leica Microsystems, Wetzlar, Germany) and coverslipped. Histological changes were evaluated in randomly chosen histological fields at 400× magnification.
Results Effect of genipin on CLP-induced lethality and hepatocellular damage In the CLP group, the survival rate was 70% on the first day after CLP and stabilized at 20% on the fifth day of observation. Treatment with genipin at a dose of 2.5 or 5 mg·kg 1 immediately
Figure 1 Effect of genipin on sepsis-induced lethality (A) and serum ALT and AST (B) levels. (A) Mice were i.v. administered vehicle or various doses of 1 genipin (1, 2.5 or 5 mg) immediately after CLP (n = 10). (B) Mice were administered 2.5 mg·kg genipin i.v. immediately after CLP (n = 8). Serum + was obtained and assayed for ALT and AST levels at 6 h after CLP. *P < 0.05 versus sham group; P < 0.05 versus CLP group. British Journal of Pharmacology (2016) 173 980–991
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after CLP significantly improved the survival rate compared with the CLP only group (P = 0.0131 and 0.0492, respectively; Figure 1A). The levels of serum ALT and AST in the sham group were 29.8 ± 2.7 and 84.9 ± 5.1 U·L 1, respectively. At 6 h after CLP, the levels of serum ALT and AST significantly increased and these effects were attenuated by treatment with genipin at 2.5 mg·kg 1 (Figure 1B).
Effect of genipin on CLP-induced release of serum cytokines The levels of serum TNF-α and IL-6 in the sham group were 31.6 ± 3.3 and 27.6 ± 5.5 pg.·mL 1, respectively. At 6 h after CLP, the levels of serum TNF-α and IL-6 dramatically increased and these effects were were attenuated by treatment with genipin at 2.5 mg·kg 1 (Figure 2).
Effect of genipin on autophagic flux The protein expression levels of LC3-II, a reliable marker of the autophagosome, and p62, a selective substrate for autophagy, significantly increased 2.2- and 2.6-fold, respectively, compared with those of the sham group at 6 h after CLP. Genipin enhanced the increase in LC3-II and attenuated the increase in p62 (Figure 3A). Moreover, we utilized autophagic flux inhibitor chloroquine to confirm the autophagy activation by genipin and its possible contribution to attenuation of liver damage triggered by sepsis. Treatment with chloroquine enhanced the increased level of LC3-II and reversed the attenuated level of p62 by genipin (Figure3B). Furthermore, chloroquine reversed the protective effects of genipin against sepsis-induced mortality and hepatocellular damage as indicated by decreased survival rate and increased ALT and AST activities compared with genipintreated CLP animals (Figure 3C and D). To confirm our Western blot results, we observed autophagic vacuoles, including autophagosomes and autolysosomes, by TEM. Compared with the basal level of autophagic vacuoles in the sham group, the number of autophagic vacuoles was markedly increased in the CLP group and was further increased by genipin (Figure 4).
Effect of genipin on autophagosome formation Beclin-1 complex formation leads to nucleation of a phagophore, which is initially elongated by the Atg12–516 L1 complex and completed by LC3-II to yield the mature
autophagosome. Protein expression of the Atg12–5 complex increased to 1.6-fold that of the sham group at 6 h after CLP, and this increase was enhanced by genipin treatment. However, the level of beclin-1 protein expression was not affected by sepsis or treatment with genipin (Figure 5A). Next, we assessed two hallmarks of LC3 lipidation, Atg3 and Atg7. The level of Atg3 protein expression decreased to approximately 53% that of the sham group, and this decrease was attenuated by genipin treatment. The level of Atg7 protein expression was not affected by sepsis, but was increased by genipin treatment (Figure 5B).
Effect of genipin on autophagosome–lysosome fusion and degradation Fusion of the autophagosome with a lysosome to form the autolysosome is a prerequisite for complete degradation of the cargo contents. We investigated the expression of the lysosomal membrane proteins LAMP-2 and Rab7 and the lysosomal protease cathepsin B. As shown in Figure 6A, in the CLP group, the expression levels of LAMP-2 and Rab7 protein significantly decreased to approximately 74% and 67% that of the sham group, respectively, and these decreases were attenuated by genipin. There were no significant changes in cathepsin B protein expression in any of the experimental groups. To further confirm that genipin restores CLP-induced impairment of autophagosome–lysosome fusion, we analysed the distribution of respective LC3 and LAMP-2 by immunohistochemistry assay. CLP group exhibited the increase in the distribution of LC3 and the decrease that of LAMP-2 compared with the sham group. Genipin treatment enhanced the increase in LC3 distribution and attenuated the decrease in LAMP-2 distribution (Figure 6B).
Effect of genipin on CLP-induced mTOR and calpain activation To elucidate the mechanisms by which genipin affects autophagy activation in sepsis, we investigated the involvement of the mTOR-dependent pathway and calpain system, which is independent of mTOR. The phosphorylation levels of mTOR and its downstream substrates, 4E-BP1 (Ser65) and p70S6K (Thr389), significantly increased 2.1-, 1.4-, and 1.6fold, respectively, compared with those of the sham group after CLP. Genipin did not affect these phosphorylations (Figure 7A). Calpain 1 protein expression significantly increased 2.6-fold compared with that of the sham group after
Figure 2 1
Effect of genipin on serum TNF-α and IL-6 levels at 6 h after CLP. Mice were administered 2.5 mg·kg genipin i.v. immediately after CLP (n = 8). + Serum was obtained and assayed for TNF-α and IL-6 levels at 6 h after CLP. * P < 0.05 versus sham group; P < 0.05 versus CLP group. 984
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Figure 3 Effect of genipin on LC3-II and p62 protein expressions in the liver (A), effect of chloroquine (CQ) in the presence of genipin on LC3-II and p62 protein expressions in the liver (B), sepsis-induced lethality (C) and serum ALT and AST levels (D) at 6 h after CLP. Mice were administered 1 2.5 mg·kg genipin i.v. immediately after CLP (n = 8). (B–D) Mice were pretreated with chloroquine i.p. 1 h before CLP. * P < 0.05 versus + # sham group; P < 0.05 versus CLP group; P < 0.05 versus CLP + genipin group.
Figure 4 Effect of genipin on autophagic vacuoles in the liver at 6 h after CLP. Autophagic vacuoles (arrows indicated) were observed from TEM images. * + P < 0.05 versus sham group; P < 0.05 versus CLP group. British Journal of Pharmacology (2016) 173 980–991
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Figure 5 Effect of genipin on beclin-1 and Atg12–5 (A), Atg3 and Atg7 (B) protein expressions in the liver at 6 h after CLP. Mice were administered 1 2.5 mg·kg genipin i.v. immediately after CLP (n = 8). The protein expressions were measured by Western blot analysis at 6 h after CLP. + * P < 0.05 versus sham group; P < 0.05 versus CLP group.
CLP. Genipin attenuated the increase in calpain 1; however, the level of calpain 2 protein expression was not affected by sepsis or genipin (Figure 7B). The protein expression level of cleaved Atg5, which is cleaved by the calpain system, significantly increased to 1.6-fold that of the sham group at 6 h after CLP, and this increase was attenuated by genipin treatment (Figure 7B).
Discussion The participation of autophagy in the dysfunction of multiple organs during sepsis makes autophagy an attractive target for therapeutic manipulation after sepsis. Previous studies reported that complete activation of autophagy by rapamycin exerts a cardioprotective effect by restoring depressed cardiac performance and decreasing inflammatory responses (Hsieh et al. 2011). More recently, it has been shown that activation of autophagy inhibits hepatocyte death and ultimately prevents liver injury (Takahashi et al. 2013; Tang et al. 2013). Genipin is the hydrolytic product of geniposide that has potent antioxidant, anti-inflammatory and anti-apoptotic properties. We previously showed that genipin prevents oxidative stress and subsequent apoptotic cell death in a mouse model of fulminant hepatic failure (Kim et al. 2010). Genipin exhibits anti-inflammatory and antibacterial effects, which protect against LPS-induced acute systemic inflammation and CLP-induced sepsis (Kim et al. 2012b). Furthermore, 986
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Dando et al. (2013 demonstrated that genipin has anticancer activity through induction of autophagic cell death in a pancreatic adenocarcinoma cell line. However, there is limited information on the effect of genipin on autophagy in in vivo animal studies. In the present study, we found that genipin enhanced autophagic flux, which may have contributed to increased survival in murine sepsis. Earlier reports indicated that autophagy is transiently increased in response to septic insult, based on findings of increased LC3-II accumulation and the autophagic vacuoles observed in hepatocytes by TEM (Watanabe et al. 2009; Takahashi et al. 2013). However, it is not clear whether increased levels of LC3-II can be translated as acceleration of autophagy in this situation or as partial or complete impairment of autophagic flux by blockage of lysosomal fusion and degradation. LC3II is known to only exist on mature autophagosomes. An increased level of LC3-II is interpreted as evidence for autophagy activation, although it could also indicate autophagy suppression caused by decreased autolysosomal degradation of LC3. For example, in a rat model of pancreatitis, LC3-II levels significantly increased after LPS administration; however, this was not the result of induction of autophagy but rather a blockade of fusion between autophagosomes and lysosomes (Fortunato et al. 2009). The autophagic substrate p62 is an ubiquitinbinding protein that recognizes ubiquitinated substrates and recruits phagophores through direct interaction with LC3-II (Pankiv et al. 2007). Because LC3-II and p62 are both degraded with the autophagic cargo in the autolysosome, the accumulation of LC3-II and p62 aggregates is regarded as a robust marker
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Figure 6 Effect of genipin on LAMP-2, Rab7 and cathepsin B protein expressions (A) and immunohistochemistry with LC3 and LAMP-2 (B) in the liver at 6 h 1 after CLP. Mice were administered 2.5 mg·kg genipin i.v. immediately after CLP (n = 8). (A) The protein expressions were measured by Western blot analysis at 6 h after CLP. (B) Liver sections were fixed and immunostained with anti-LC3 (1:200; brown) and anti-LAMP-2 (1:1000; brown) + antibodies. Original magnification 400×. * P < 0.05 versus sham group; P < 0.05 versus CLP group.
of impaired autophagic flux (Lee et al. 2012). In this study, we initially observed crosstalk between autophagy and liver injury during sepsis. The hepatic levels of LC3-II and p62 protein expression significantly increased 6 h after CLP. Treatment with the autophagy inhibitor chloroquine further increased the levels of LC3-II and p62 protein expression. In addition, chloroquine exacerbated the liver injury, as evidenced by a decreased
survival rate and increased levels of serum ALT and AST (Supporting Information Figure SS1). These results indicate that impaired autophagic flux is responsible for sepsis-induced liver injury. Genipin further increased LC3-II protein expression and reduced p62 accumulation, attenuated the liver injury by decreasing serum ALT, AST and inflammatory cytokine levels and improved the survival rate. TEM images revealed that British Journal of Pharmacology (2016) 173 980–991
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Figure 7 Effect of genipin on phospho-mTOR, phospho-4E-BP1 and phospho-p70S6K (A), calpain 1, calpain 2 and cleaved Atg5 (B) protein expressions in 1 the liver at 6 h after CLP. Mice were administered 2.5 mg·kg genipin i.v. immediately after CLP (n = 8). The protein expressions were measured + by Western blot analysis at 6 h after CLP. *P < 0.05 versus sham group; P < 0.05 versus CLP group.
genipin further increased the number of autophagic vacuoles in the livers of mice during sepsis, which correlated with our Western blot results. In order to further examine the autophagy activation by genipin and its possible contribution to attenuation of liver damage triggered by sepsis, we utilized autophagy inhibitor chloroquine. Treatment with chloroquine blocked the autophagic flux restoration by genipin and reversed the hepatoprotective effects of genipin during sepsis. Collectively, our data suggest that genipin restores the autophagic flux that is impaired by sepsis and septic liver injury. Correct interactions between the autophagy machinery and autophagy receptors are important for the execution of the complete autophagy process. The process of autophagy involves some complex key steps: membrane isolation, phagophore elongation, autophagosome formation, autophagosome–lysosome fusion and degradation (Kang et al. 2011). After the beclin-1/Vps34 complex initiates membrane isolation from varied intracellular origins and gathers autophagic proteins essential for pre-autophagosomal structure, two conjugation systems play essential roles in the formation of the autophagosome: an Atg12-Atg5 conjugate and membrane-associated LC3-II. The Atg12-Atg5 conjugate binds to the outer membrane of the phagophore and dissociates after mature autophagosome formation, and LC3-II subsequently binds to the Atg12–5-associated phagophore and forms the mature autophagosome. The cytosolic pre-LC3 form is cleaved by Atg4 to LC3-I, which binds covalently to PE by the action of Atg7 and Atg3 enzymes and finally becomes LC3-II. Several reports have shown that gene silencing of Atg5, Atg3 or Atg7 leads to impairment of autophagosome formation and causes cellular damage in several in vitro and in vivo tests (Cutting et al. 2014; Diakopoulos et al. 2014; Lamoureux et al. 2013; Lin et al. 2014). 988
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In the present study, sepsis significantly increased the level of the Atg12–5 complex and decreased the level of Atg3; however, it did not affect the level of Atg7. Genipin enhanced the Atg12–5 complex level, attenuated the decreased Atg3 level and increased the Atg7 levels, indicating that genipin enhances the phagophore elongation and increases mature autophagosome formation during sepsis. The level of beclin-1 protein expression was not affected by sepsis. Considering that beclin-1 modulates autophagy through assembly and disassembly with a variety of activator and/or inhibitor proteins, such as Vps34, Bcl-2 Vps15, HMGB1, Ambra1 and rubicon, the precise effect of sepsis on the formation of beclin-1 complex should be studied further. Fusion of the lysosome with a mature autophagosome in the late stage of the autophagy process is a critical step for degradation of its contents (Yu et al. 2010). Mature autophagosomes initially fuse with endosomal vesicles and acquire LAMP-1 and LAMP-2, thus gaining the ability to combine with lysosomes. These structures then fuse with lysosomes (autolysosomes) and acquire the lysosomal proteases cathespins and acid phosphatases required for substrate degradation (Kirkegaard et al. 2004). Gene silencing of LAMP-2 is characterized by a disturbance in lysosomal positioning, which leads to dysfunctional fusion and accumulation of autophagosomes (Noda and Klionsky 2008). Furthermore, recent studies demonstrated that Rab7, a member of the small GTPase family, is also required for the fusion of autophagosomes and lysosomes (Ganley et al. 2011; Wang et al. 2014). In sepsis-induced kidney injury, down-regulation of the expression of Rab7 leads to incomplete activation of the autophagic process (Yen et al. 2013). In this study, sepsis significantly decreased the levels of LAMP-2 and Rab7 protein expression, whereas it did not
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affect the level of cathepsin B protein. Genipin treatment attenuated the decrease in LAMP-2 and Rab7 protein levels. Furthermore, genipin enhanced the distribution of LC3 protein and attenuated the decreased distribution of LAMP-2 after CLP in liver tissue. These results suggested that genipin restores autophagosome–lysosome fusion in the liver during sepsis. Numerous components, such as mTOR, intracellular Ca2+ and oxidative stress, appear to be involved in the regulation of autophagy in mammalian cells (Hoyer-Hansen et al. 2007). Among them, mTOR, a serine/threonine protein kinase that regulates diverse cellular functions, is the most studied negative modulator of autophagy (Ravikumar et al. 2004). In the normal state, mTOR directly interacts with the Atg1-Atg13 complex and mediates phosphorylation-dependent inhibition of its kinase activities. Laufenberg et al. (2014 showed that sepsis decreased basal protein synthesis, in association with a reduction in mTOR activation, in skeletal muscle. Furthermore, the mTOR inhibitor temsirolimus induced autophagy and protected against endotoxaemia-induced acute kidney injury (Howell et al. 2013). Calpain is a Ca2+-dependent cysteine protease that plays a crucial role in a variety of cellular processes, including different signal transduction pathways, the regulation of apoptosis and autophagic degradation. The two main isoforms of calpain—micromolar Ca2+-requiring calpain (μ-calpain, calpain 1) and millimolar Ca2+-requiring calpain (m-calpain, calpain 2)—differ primarily in their calcium requirements, and deregulation of calpain activity following loss of Ca2+ homeostasis leads to tissue damage in various pathophysiological events including myocardial infarcts and stroke (Goll et al. 2003). Recent studies have suggested that the calpain system could inhibit the autophagy process by directly cleaving several Atg proteins involved in all steps of autophagy from autophagosome formation to autolysosome degradation (Kim et al. 2008; Russo et al. 2011; Villalpando Rodriguez and Torriglia 2013). Among the Atg proteins, it has been recognized that the Atg5 is the specific target: full-length Atg5 is cleaved into the truncated Atg5 by activated calpains, which play important roles in the crosstalk between autophagy and apoptosis. Actually, several studies have shown the cleaved Atg5 levels with consistent calpain activation as the evidence for calpainmediated autophagy impairment (Zhu et al. 2015; Cha et al. 2014). Moreover, calpain activity increased in endotoxaemiainduced or CLP-induced sepsis animal models, suggesting that this increased protease activity could be a negative autophagy regulatory mechanism during sepsis (Zafrani et al. 2012; Li et al. 2014). In our study, the levels of mTOR, calpain 1 and cleaved Atg5 protein expression significantly increased in sepsis, and genipin attenuated the levels of calpain 1 and cleaved Atg5, but not mTOR. Collectively, our data suggest that genipin activates autophagic flux through the downregulation of calpain.
Conclusion In conclusion, these findings suggest that genipin protects against septic injury by restoring the impaired autophagic flux via inhibition of the calpain system. Thus, we propose that genipin may provide new a pharmacological intervention strategy for septic injury.
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Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2013R1A1A3008145). H-I. C. (NRF-2012H1A2A1016419) and So-Jin Kim (NRF-2013H1A2A1034472) received ‘Global Ph.D. Fellowship Program’ support from the NRF funded by the Ministry of Education, Science, and Technology (MEST) in Korea.
Author contributions H-I. C., S. J. K. and J. W. C. performed the research. H- I. C., S J. K. and S. M. L. designed the research. H-I. C. and S. J. K. analysed the data. H-I. C. and S. M. L. wrote the paper.
Conflicts of interest None to declare.
References Alexander SPH, Fabbro D, Kelly E, Marrion N, Peters JA, Benson HE, et al. (2016). The Concise Guide to PHARMACOLOGY 2015/16: Enzymes. Br J Pharmacol 172: 6024–6109. Cha HH, Hwang JR, Kim HY, Choi SJ, Oh SY, Roh CR (2014). Autophagy induced by tumor necrosis factor α mediates intrinsic apoptosis in trophoblastic cells. Reprod Sci 21: 612–622. Chaudry IH, Wichterman KA, Baue AE (1979). Effect of sepsis on tissue adenine nucleotide levels. Surgery 85: 205–211. Curtis MJ, Bond RA, Spina D, Ahluwalia A, Alexander SP, Giembycz MA, et al. (2015). Experimental design and analysis and their reporting: new guidance for publication in BJP. Br J Pharmacol 172: 3461–3471. Cutting AS, Del Rosario Y, Mu R, Rodriguez A, Till A, Subramani S, et al. (2014). The role of autophagy during group B streptococcus infection of blood–brain barrier endothelium. J Biol Chem 289: 35711–35723. Dando I, Fiorini C, Pozza ED, Padroni C, Costanzo C, Palmieri M, et al. (2013). UCP2 inhibition triggers ROS-dependent nuclear translocation of GAPDH and autophagic cell death in pancreatic adenocarcinoma cells. Biochim Biophys Acta 1833: 672–679. Diakopoulos KN, Lesina M, Wormann S, Song L, Aichler M, Schild L, et al. (2014). Impaired autophagy induces chronic atrophic pancreatitis in mice via sex- and nutrition-dependent processes. Gastroenterology 148: 626–638. Fortunato F, Burgers H, Bergmann F, Rieger P, Buchler MW, Kroemer G, et al. (2009). Impaired autolysosome formation correlates with Lamp2 depletion: role of apoptosis, autophagy, and necrosis in pancreatitis. Gastroenterology 137: 350–360. Ganley IG, Wong PM, Gammoh N, Jiang X (2011). Distinct autophagosomal–lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest. Mol Cell 42: 731–743. Goll DE, Thompson VF, Li H, Wei W, Cong J (2003). The calpain system. Physiol Rev 83: 731–801.
British Journal of Pharmacology (2016) 173 980–991
989
BJP
H-I Cho et al.
Gonnert FA, Recknagel P, Hilger I, Claus RA, Bauer M, Kortgen A (2013). Hepatic excretory function in sepsis: implications from biophotonic analysis of transcellular xenobiotic transport in a rodent model. Crit Care 17: R67. Howell GM, Gomez H, Collage RD, Loughran P, Zhang X, Escobar DA, et al. (2013). Augmenting autophagy to treat acute kidney injury during endotoxemia in mice. PLoS One 8: e69520. Hoyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, et al. (2007). Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell 25: 193–205. Hsieh CH, Pai PY, Hsueh HW, Yuan SS, Hsieh YC (2011). Complete induction of autophagy is essential for cardioprotection in sepsis. Ann Surg 253: 1190–1200.
Lelono RA, Tachibana S, Itoh K (2009). Isolation of antifungal compounds from Gardenia jasminoides. Pak J Biol Sci 12: 949–956. Li X, Luo R, Chen R, Song L, Zhang S, Hua W, et al. (2014). Cleavage of IkappaBalpha by calpain induces myocardial NF-kappaB activation, TNF-alpha expression, and cardiac dysfunction in septic mice. Am J Physiol Heart Circ Physiol 306: H833–H843. Lin CW, Lo S, Hsu C, Hsieh CH, Chang YF, Hou BS, et al. (2014). T-cell autophagy deficiency increases mortality and suppresses immune responses after sepsis. PLoS One 9: e102066. Lo S, Yuan SS, Hsu C, Cheng YJ, Chang YF, Hsueh HW, et al. (2013). Lc3 over-expression improves survival and attenuates lung injury through increasing autophagosomal clearance in septic mice. Ann Surg 257: 352–363.
Kang R, Zeh HJ, Lotze MT, Tang D (2011). The beclin 1 network regulates autophagy and apoptosis. Cell Death Differ 18: 571–580.
McGrath JC, Lilley E (2015). Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP. Br J Pharmacol 172: 3189–3193.
Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG (2010). NC3Rs Reporting Guidelines Working Group. Br J Pharmacol 160: 1577–1579.
Noda T, Klionsky DJ (2008). The quantitative Pho8Delta60 assay of nonspecific autophagy. Methods Enzymol 451: 33–42.
Kim ES, Jeong CS, Moon A (2012a). Genipin, a constituent of Gardenia jasminoides Ellis, induces apoptosis and inhibits invasion in MDA-MB-231 breast cancer cells. Oncol Rep 27: 567–572. Kim J, Kim HY, Lee SM (2013). Protective effects of geniposide and genipin against hepatic ischemia/reperfusion injury in mice. Biomol Ther (Seoul) 21: 132–137. Kim JS, Nitta T, Mohuczy D, O’Malley KA, Moldawer LL, Dunn WA Jr, et al. (2008). Impaired autophagy: a mechanism of mitochondrial dysfunction in anoxic rat hepatocytes. Hepatology 47: 1725–1736. Kim SJ, Kim JK, Lee DU, Kwak JH, Lee SM (2010). Genipin protects lipopolysaccharide-induced apoptotic liver damage in D-galactosaminesensitized mice. Eur J Pharmacol 635: 188–193. Kim TH, Yoon SJ, Lee SM (2012b). Genipin attenuates sepsis by inhibiting toll-like receptor signaling. Mol Med 18: 455–465. Kimura T, Watanabe E, Sakamoto T, Takasu O, Ikeda T, Ikeda K, et al. (2014). Autophagy-related IRGM polymorphism is associated with mortality of patients with severe sepsis. PLoS One 9: e91522. Kirkegaard K, Taylor MP, Jackson WT (2004). Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nat Rev Microbiol 2: 301–314. Koo HJ, Song YS, Kim HJ, Lee YH, Hong SM, Kim SJ, et al. (2004). Antiinflammatory effects of genipin, an active principle of Gardenia. Eur J Pharmacol 495: 201–208. Kramer L, Jordan B, Druml W, Bauer P, Metnitz PG, Austrian Epidemiologic Study on Intensive Care ASG (2007). Incidence and prognosis of early hepatic dysfunction in critically ill patients—a prospective multicenter study. Crit Care Med 35: 1099–1104. Lamoureux F, Thomas C, Crafter C, Kumano M, Zhang F, Davies BR, et al. (2013). Blocked autophagy using lysosomotropic agents sensitizes resistant prostate tumor cells to the novel Akt inhibitor AZD5363. Clin Cancer Res 19: 833–844. Laufenberg LJ, Pruznak AM, Navaratnarajah M, Lang CH (2014). Sepsis-induced changes in amino acid transporters and leucine signaling via mTOR in skeletal muscle. Amino Acids 46: 2787–2798. Lee HS, Daniels BH, Salas E, Bollen AW, Debnath J, Margeta M (2012). Clinical utility of LC3 and p62 immunohistochemistry in diagnosis of drug-induced autophagic vacuolar myopathies: a case–control study. PLoS One 7: e36221.
990
British Journal of Pharmacology (2016) 173 980–991
Pawson AJ, Sharman JL, Benson HE, Faccenda E, Alexander SP, Buneman OP et al., NC-IUPHAR (2014). The IUPHAR/BPS guide to PHARMACOLOGY: an expert-driven knowledge base of drug targets and their ligands. Nucleic Acids Res 42: D1098–D1106. Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, et al. (2007). p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 282: 24131–24145. Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, et al. (2004). Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 36: 585–595. Recknagel P, Gonnert FA, Westermann M, Lambeck S, Lupp A, Rudiger A, et al. (2012). Liver dysfunction and phosphatidylinositol-3-kinase signalling in early sepsis: experimental studies in rodent models of peritonitis. PLoS Med 9: e1001338. Russo R, Berliocchi L, Adornetto A, Varano GP, Cavaliere F, Nucci C, et al. (2011). Calpain-mediated cleavage of beclin-1 and autophagy deregulation following retinal ischemic injury in vivo. Cell Death Dis 2: e144. Savva A, Plantinga TS, Kotanidou A, Farcas M, Baziaka F, Raftogiannis M, et al. (2014). Association of autophagy-related 16-like 1 (ATG16L1) gene polymorphism with sepsis severity in patients with sepsis and ventilator-associated pneumonia. Eur J Clin Microbiol Infect Dis 33: 1609–1614. Takahashi W, Watanabe E, Fujimura L, Watanabe-Takano H, Yoshidome H, Swanson PE, et al. (2013). Kinetics and protective role of autophagy in a mouse cecal ligation and puncture-induced sepsis. Crit Care 17: R160. Takeuchi S, Goto T, Mikami K, Miura K, Ohshima S, Yoneyama K, et al. (2005). Genipin prevents fulminant hepatic failure resulting in reduction of lethality through the suppression of TNF-alpha production. Hepatol Res 33: 298–305. Tang Z, Ni L, Javidiparsijani S, Hu F, Gatto LA, Cooney R, et al. (2013). Enhanced liver autophagic activity improves survival of septic mice lacking surfactant proteins A and D. Tohoku J Exp Med 231: 127–138. Taylor DE, Ghio AJ, Piantadosi CA (1995). Reactive oxygen species produced by liver mitochondria of rats in sepsis. Arch Biochem Biophys 316: 70–76. Valdor R, Macian F (2012). Autophagy and the regulation of the immune response. Pharmacol Res 66: 475–483.
Genipin alleviates liver injury by restoring autophagy
Villalpando Rodriguez GE, Torriglia A (2013). Calpain 1 induce lysosomal permeabilization by cleavage of lysosomal associated membrane protein 2. Biochim Biophys Acta 1833: 2244–2253. Wang B, Yang Q, Sun YY, Xing YF, Wang YB, Lu XT, et al. (2014). Resveratrol-enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice. J Cell Mol Med 18: 1599–1611. Watanabe E, Muenzer JT, Hawkins WG, Davis CG, Dixon DJ, McDunn JE, et al. (2009). Sepsis induces extensive autophagic vacuolization in hepatocytes: a clinical and laboratory-based study. Lab Invest 89: 549–561. Yen YT, Yang HR, Lo HC, Hsieh YC, Tsai SC, Hong CW, et al. (2013). Enhancing autophagy with activated protein C and rapamycin protects against sepsis-induced acute lung injury. Surgery 153: 689–698. Yu L, McPhee CK, Zheng L, Mardones GA, Rong Y, Peng J, et al. (2010). Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature 465: 942–946. Zafrani L, Gerotziafas G, Byrnes C, Hu X, Perez J, Levi C, et al. (2012). Calpastatin controls polymicrobial sepsis by limiting
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procoagulant microparticle release. Am J Respir Crit Care Med 185: 744–755. Zhu X, Messer JS, Wang Y, Lin F, Cham CM, Chang J, et al. (2015). Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation. J Clin Invest 125: 1098–1110.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: http://dx.doi.org/10.1111/bph.13397 Figure S1 Effect of chloroquine (CQ) on LC3-II and p62 protein expressions in the liver (A), sepsis-induced lethality (B) and serum ALT and AST (C) levels. Mice were intravenously administered vehicle (saline) or CQ (60 mg·kg 1) 1 h prior to CLP (n = 10 for survival test and n = 8 for biochemical assay). * denotes significant difference (P < 0.05) versus sham group; + denotes significant difference (P < 0.05) versus CLP group.
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RESEARCH PAPER Genipin alleviates sepsisinduced liver injury by restoring autophagy
Correspondence Sun-Mee Lee, School of Pharmacy, Sungkyunkwan University, Suwon 440–746, Korea. E-mail: [email protected] ---------------------------------------------------------
Received 10 October 2015
Revised 18 November 2015
Accepted
Hong-Ik Cho, So-Jin Kim, Joo-Wan Choi and Sun-Mee Lee
30 November 2015
School of Pharmacy, Sungkyunkwan University, Suwon 440–746, South Korea
BACKGROUND AND PURPOSE Autophagy is an essential cytoprotective system that is rapidly activated in response to various stimuli including inflammation and microbial infection. Genipin, an aglycon of geniposide found in gardenia fruit, is well known to have anti-inflammatory, antibacterial and antioxidative properties. This study examined the protective mechanisms of genipin against sepsis, with particular focus on the autophagic signalling pathway.
EXPERIMENTAL APPROACH Mice were subjected to sepsis by caecal ligation and puncture (CLP). Genipin (1, 2.5 and 5 mg·kg 1) or vehicle (saline) was injected i.v. immediately (0 h) after CLP, and chloroquine (60 mg·kg 1), an autophagy inhibitor, was injected i.p. 1 h before CLP. Blood and liver tissues were isolated 6 h after CLP.
KEY RESULTS Genipin improved survival rate and decreased serum levels of aminotransferases and pro-inflammatory cytokines after CLP; effects abolished by chloroquine. The liver expression of autophagy-related protein (Atg)12-Atg5 conjugate increased after CLP, and this increase was enhanced by genipin. CLP decreased Atg3 protein liver expression, and genipin attenuated this decrease. CLP impaired autophagic flux, as indicated by increased liver expression of microtubule-associated protein-1 light chain 3-II and sequestosome-1/p62 protein; this impaired autophagic flux was restored by genipin, and chloroquine abolished this effect. Genipin also attenuated the decreased expression of lysosome-associated membrane protein-2 and Rab7 protein and increased expression of calpain 1 protein induced by CLP in the liver.
CONCLUSIONS AND IMPLICATIONS Our findings suggest that genipin protects against septic injury by restoring impaired autophagic flux. Therefore, genipin might be a potential therapeutic agent for the treatment of sepsis.
Abbreviations ALT, serum alanine aminotransferase; AST, serum aspartate aminotransferase; Atg, autophagy-related protein; CLP, caecal ligation and puncture; LAMP, lysosome-associated membrane protein; LC3, microtubule-associated protein 1 light chain 3; mTOR, mammalian target of rapamycin; PE, phosphatidylethanolamine; phospho, phosphorylated; p62, sequestosome1/p62; p70S6K, p70S6 kinase; TBS/T, 0.1% Tween 20 in 1× Tris-buffered saline; TEM, transmission electron microscopy; 4E-BP1, eukaryotic translation initiation factor 4E binding protein 1
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Genipin alleviates liver injury by restoring autophagy
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Tables of Links TARGETS Calpain 1 Calpain 2
LIGANDS Cathepsin B
Chloroquine
mTOR
IL-6
TNF-α
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology. org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).
Introduction Sepsis is a massive systemic inflammatory syndrome that is triggered by severe infection, trauma or toxin and leads to multiple organ failure and death. During the development of sepsis, bacterial components lead to excessive secretion of pro-inflammatory cytokines, which induce endothelial and epithelial injury, vasodilatation and oedema, subsequently causing the development of multiple organ dysfunction syndromes (Taylor et al. 1995). Despite advances in therapeutic care and an increased understanding of the pathophysiology of sepsis, it remains a leading cause of mortality in intensive care units. There is a substantial need for an effective therapy that will decrease the morbidity and mortality associated with sepsis. The liver is a critical organ for host survival in response to sepsis through its role in scavenging bacteria and producing inflammatory mediators; however, it might also become damaged in the pathogenesis of sepsis as the result of a dysregulated inflammatory response (Recknagel et al. 2012; Gonnert et al. 2013). Clinical evidence has shown that hepatic dysfunction is an early event in sepsis and is a specific risk factor for poor outcome (Kramer et al. 2007). Autophagy is an evolutionarily conserved self-eating–recycling process in which cytoplasmic proteins or organelles are sequestered by a double-membraned autophagosome and delivered to lysosomes for proteolytic degradation. Autophagy has recently been recognized as a crucial modulator of immune responses as it participates in pathogen killing or antigen processing and presentation by immune cells, thus highlighting its potential as a therapeutic target in infectious diseases, including sepsis (Valdor and Macian 2012). Several studies have demonstrated the critical association between autophagy and sepsis. Autophagy was activated in patients with sepsis and in caecal ligation and puncture (CLP) animal models of sepsis (Hsieh et al. 2011; Lo et al. 2013). Moreover, polymorphisms in the autophagy-related protein (Atg) 16 L1 or autophagy-related human immunity-related GTPase gene affect the clinical outcome of patients with severe sepsis (Kimura et al. 2014; Savva et al. 2014). Accumulating evidence from human and animal studies indicates that increased autophagic vacuolization in the liver, particularly in the hepatocytes, is possibly associated with mitochondrial injury (Watanabe et al. 2009). In the liver of the CLP animal model, the number of autophagosomes was increased, as demonstrated by microtubule-associated protein-1 light chain 3 (LC3)-II accumulation and electron microscopic examination, and its inhibition resulted in elevated serum transaminase levels (Takahashi et al. 2013). However, there is
limited information on the core molecular machinery of autophagic flux and its regulatory signalling in sepsis. Genipin, an aglycone derived from an iridoid glycoside called geniposide, is a major component of the fruit of Gardenia jasminoides, which has been widely used for the treatment of inflammatory diseases and hepatic disorders in traditional medicine (Koo et al. 2004). A plethora of studies also showed that genipin has various pharmacological effects including antioxidative, anticancer and antifungal activities (Lelono et al. 2009; Kim et al. 2012a; Kim et al. 2013). Genipin reduced mortality induced by D-galactosamine/LPS-induced acute liver injury by preventing apoptotic cell death and the inflammatory response (Takeuchi et al. 2005). Recently, our group reported that genipin attenuates mortality and multiple organ injury in sepsis through inhibition of toll-like receptor-dependent inflammatory signalling pathways (Kim et al. 2012b). Therefore, we investigated the hepatoprotective mechanisms of genipin in septic injury, in particular focusing on the autophagy machinery. Our present study may provide a novel strategy to counteract sepsis, which is currently the most challenging problem in intensive care.
Methods Group sizes Ten mice were used per each group for the survival test and eight mice per each group for biochemical assay.
Randomization Randomization was conducted by an individual other than the operator. The animals with similar degrees of body weight were selected randomly from the pool of all cages eligible for inclusion in the study and divided into groups.
Statistical comparison Results are presented as mean ± SEM. Survival data were analysed by Kaplan–Meier curves and the log-rank test. The overall significance of results was analysed by one-way ANOVA. Differences between compared groups were considered statistically significant at P < 0.05 with the appropriate Bonferroni correction for multiple comparisons. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). British Journal of Pharmacology (2016) 173 980–991
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Validity of animal species or model selection CLP in rodents has become the most widely used and clinically relevant model for experimental sepsis and is currently considered as the gold standard in sepsis research. The CLP model has been considered to be a realistic model for the induction of polymicrobial sepsis in experimental settings to study the underlying mechanisms of sepsis.
Ethical statement All experiments were approved by the Animal Care Committee of the Sungkyunkwan University School of Pharmacy (SUSP14-27) and performed in accordance with the guidelines of the National Institutes of Health (NIH publication No. 86–23, revised 1985) with adherence to the 3Rs (Replacement, Refinement and Reduction). Animal studies follow the ARRIVE guidelines (Kilkenny et al., 2010; McGrath & Lilley, 2015). Animals. Male imprinting control region mice weighing 27–29 g were obtained from Orient Bio Inc. (Seongnam, Korea).
Experimental procedures Polymicrobial sepsis was induced by CLP as described previously by Chaudry et al. (1979. Mice were anaesthetized by i.m. injection of ketamine (55 mg·kg 1, Yuhan Corporation, Seoul, Korea) and xylazine (7 mg·kg 1, Bayer, Germany), a midline abdominal incision was then made and the caecum was carefully exposed avoiding damage to the blood vessels. The caecum was ligated just distal to the ileocecal valve without intestinal obstruction, punctured twice using a 20-gauge needle and squeezed to expel a small amount of faecal material to ensure patency of the puncture sites. The caecum was returned to its normal intra-abdominal position, and the abdominal incision was closed with two layers of running sutures. Shamoperated animals were subjected to laparotomy and intestinal manipulation without ligation and puncture. All animals received 1 mL of normal saline s.c. immediately after the operation for fluid resuscitation. For the survival experiment, mice received an i.v. injection of genipin (Wako Pure Chemical Industries, Ltd., Osaka, Japan; 1, 2.5, and 5 mg·kg 1) or saline (vehicle) via the tail vein immediately (0 h) after CLP. The dose and timing of genipin treatment was selected based on our previous study (Kim et al. 2012b). Mortality was monitored up to 10 days after the operation, and the mice were followed for 3 weeks to ensure that no late mortalities occurred. On the basis of a survival test, 2.5 mg·kg 1 genipin was selected as the optimally effective dose for further biochemical studies. Animals were randomly divided into five groups as follows: (1) vehicle-treated sham-operated (sham), (2) 2.5 mg·kg 1 genipin-treated sham-operated (genipin), (3) vehicle-treated CLP (CLP), (4) 2.5 mg·kg 1 genipin-treated CLP (CLP + genipin), (5) 60 mg·kg 1 chloroquine (CQ) and 2.5 mg·kg 1 genipintreated CLP (CQ + CLP + genipin). Chloroquine was dissolved in saline and administered i.p. (60 mg·kg 1) 1 h before the CLP for confirming the involvement of autophagy in the protective effects of genipin on sepsis. Under ketamine (55 mg·kg 1) and xylazine (7 mg·kg 1) anaesthesia, mice were killed at 6 h after CLP. Blood was collected from the inferior vena cava 6 h after CLP, centrifuged (10 000 rpm, 10 min, 4°C) to obtain a serum sample and stored at 75°C until assayed. Liver tissue was 982
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simultaneously isolated at 6 h after CLP and stored at until assayed.
75°C
Housing and husbandry Mice (n = 5 animals per cage) were kept in a temperaturecontrolled and humidity-controlled room (25 ± 1°C and 55 ± 5%, respectively) under a 12 h light–dark cycle, with water and food provided ad libitum. Mice were given 7 days to acclimatize to the housing conditions and reverse light cycle and habituated to handling before starting the experiments. Mice were monitored for confirming abnormal symptoms or wound that would be reasons for removal of the animal from the experiment prematurely.
Interpretation This study thoroughly considered the 3Rs. Assessment of hepatic damage. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, the representative biomarkers of liver damage, were assayed at 37°C by monitoring decrease in absorbance at 340 nm for 1 min because of the disappearance of NADH with ChemiLab ALT and AST assay kit (IVDLab Co., Uiwang, Korea), respectively, using a Hitachi 7600 automatic analyzer (Hitachi, Tokyo, Japan). Serum cytokine levels. Serum TNF-α and IL-6 levels were quantified using commercial mouse ELISA kits (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Western blot immunoassay. Fresh liver tissue was isolated and homogenized in PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology In., Seongnam, Korea) for whole protein samples according to the manufacturer’s instructions. After being kept in an ice-cold bath for a period of 30 min for cell lysis, the whole homogenate was centrifuged at 13 000 x g for 5 min. Protein concentration was determined using a BCA Protein Assay kit (Pierce Biotechnology Inc., Rockford, IL, USA). Protein samples (20 μg) were separated by 10–15% SDS-PAGE and transferred to PVDF membranes using the Semi-Dry Trans-Blot Cell (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were washed with 0.1% Tween 20 in 1× Tris-buffered saline (TBS/T) and blocked for 1 h at room temperature with 5% (w/v 1) skimmed milk powder or 5% BSA in TBS/T. Blocked membranes were incubated overnight at 4°C with primary antibodies, washed five times for 7 min each in TBS/T and incubated with the appropriate secondary antibodies for 1 h at room temperature, followed by detection using an ECL detection system (iNtRON Biotechnology Inc.) according to the manufacturer’s instructions. The intensity of the immunoreactive bands was determined using TOTALLAB TL 120 software (Nonlinear Dynamics Ltd., Newcastle, UK). The following primary antibodies were used: Atg3, Atg12–5 complex, Atg7, phosphorylated (phospho)-mammalian target of rapamycin (mTOR), mTOR, phospho-eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), 4E-BP1, phospho-p70S6 kinase (p70S6K), and 70S6K (all from Cell Signaling Technology, Beverly, MA, USA); sequestosome-1/p62 (p62) (Abcam, Cambridge, MA, USA); beclin-1, cleaved Atg5, calpain 1, calpain 2, Rab7, and
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cathepsin B (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); LC3 and lysosome-associated membrane protein (LAMP)2 (Novus Biologicals, Littleton, CO, USA); and β-actin (Sigma-Aldrich, St. Louis, MO, USA). Protein densities were standardised to those of β-actin, and phosphorylated protein densities were standardised to those of total protein for total lysates. Transmission electron microscopy (TEM). Liver tissues were fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in 100 mM sodium phosphate (pH 7.2). Samples were washed with 100 mM Na cacodylate (pH 7.4), postfixed in 2% osmium tetroxide and then washed again. The samples were dehydrated in a graded series of ethanol and propylene oxide and embedded in epoxy resin (TAAB 812 Resin; Marivac Industries, Montreal, QC, Canada). Ultrathin (60–70 nm) sections were counterstained with uranyl acetate and lead citrate and viewed using a Hitachi 7600 TEM (Hitachi High-Technologies America, Inc., Schaumburg, IL, USA) equipped with a Macrofire monochrome progressive scan CCD camera (Optronics, Inc., Muskogee, OK, USA) and AMTV image capture software (Advanced Microscopy Techniques, Corp., Danvers, MA, USA). Immunohistochemistry. Formalin-fixed, paraffin-embedded liver tissues were prepared for immunohistochemistry assay. Each sample was fixed by immersion in 10% neutralbuffered formalin. The liver sections were deparaffinised
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with xylene, hydrated in serial dilutions of alcohol and microwaved in citrate buffer for antigen retrieval. The sections were immersed in 3% hydrogen peroxide solution to inhibit endogenous peroxidase activity and then incubated with monoclonal antibodies against LC3 (Novus Biologicals; 1:200) or LAMP-2 (Santa Cruz Biotechnology; 1:1000) followed by Polink-2 Plus HRP anti-mouse or rabbit DAB detection kit (Golden Bridge International, Inc., Bothell, WA, USA) according to the manufacturer’s instructions. The sections were stained with diaminobenzidine (Dako, Glostrup, Denmark) and counterstained with Mayer haematoxylin (ScyTek Laboratories, Inc., West Logan, UT, USA), washed again, dehydrated in alcohol, cleared in xylene, mounted with MM-24 (Leica Microsystems, Wetzlar, Germany) and coverslipped. Histological changes were evaluated in randomly chosen histological fields at 400× magnification.
Results Effect of genipin on CLP-induced lethality and hepatocellular damage In the CLP group, the survival rate was 70% on the first day after CLP and stabilized at 20% on the fifth day of observation. Treatment with genipin at a dose of 2.5 or 5 mg·kg 1 immediately
Figure 1 Effect of genipin on sepsis-induced lethality (A) and serum ALT and AST (B) levels. (A) Mice were i.v. administered vehicle or various doses of 1 genipin (1, 2.5 or 5 mg) immediately after CLP (n = 10). (B) Mice were administered 2.5 mg·kg genipin i.v. immediately after CLP (n = 8). Serum + was obtained and assayed for ALT and AST levels at 6 h after CLP. *P < 0.05 versus sham group; P < 0.05 versus CLP group. British Journal of Pharmacology (2016) 173 980–991
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after CLP significantly improved the survival rate compared with the CLP only group (P = 0.0131 and 0.0492, respectively; Figure 1A). The levels of serum ALT and AST in the sham group were 29.8 ± 2.7 and 84.9 ± 5.1 U·L 1, respectively. At 6 h after CLP, the levels of serum ALT and AST significantly increased and these effects were attenuated by treatment with genipin at 2.5 mg·kg 1 (Figure 1B).
Effect of genipin on CLP-induced release of serum cytokines The levels of serum TNF-α and IL-6 in the sham group were 31.6 ± 3.3 and 27.6 ± 5.5 pg.·mL 1, respectively. At 6 h after CLP, the levels of serum TNF-α and IL-6 dramatically increased and these effects were were attenuated by treatment with genipin at 2.5 mg·kg 1 (Figure 2).
Effect of genipin on autophagic flux The protein expression levels of LC3-II, a reliable marker of the autophagosome, and p62, a selective substrate for autophagy, significantly increased 2.2- and 2.6-fold, respectively, compared with those of the sham group at 6 h after CLP. Genipin enhanced the increase in LC3-II and attenuated the increase in p62 (Figure 3A). Moreover, we utilized autophagic flux inhibitor chloroquine to confirm the autophagy activation by genipin and its possible contribution to attenuation of liver damage triggered by sepsis. Treatment with chloroquine enhanced the increased level of LC3-II and reversed the attenuated level of p62 by genipin (Figure3B). Furthermore, chloroquine reversed the protective effects of genipin against sepsis-induced mortality and hepatocellular damage as indicated by decreased survival rate and increased ALT and AST activities compared with genipintreated CLP animals (Figure 3C and D). To confirm our Western blot results, we observed autophagic vacuoles, including autophagosomes and autolysosomes, by TEM. Compared with the basal level of autophagic vacuoles in the sham group, the number of autophagic vacuoles was markedly increased in the CLP group and was further increased by genipin (Figure 4).
Effect of genipin on autophagosome formation Beclin-1 complex formation leads to nucleation of a phagophore, which is initially elongated by the Atg12–516 L1 complex and completed by LC3-II to yield the mature
autophagosome. Protein expression of the Atg12–5 complex increased to 1.6-fold that of the sham group at 6 h after CLP, and this increase was enhanced by genipin treatment. However, the level of beclin-1 protein expression was not affected by sepsis or treatment with genipin (Figure 5A). Next, we assessed two hallmarks of LC3 lipidation, Atg3 and Atg7. The level of Atg3 protein expression decreased to approximately 53% that of the sham group, and this decrease was attenuated by genipin treatment. The level of Atg7 protein expression was not affected by sepsis, but was increased by genipin treatment (Figure 5B).
Effect of genipin on autophagosome–lysosome fusion and degradation Fusion of the autophagosome with a lysosome to form the autolysosome is a prerequisite for complete degradation of the cargo contents. We investigated the expression of the lysosomal membrane proteins LAMP-2 and Rab7 and the lysosomal protease cathepsin B. As shown in Figure 6A, in the CLP group, the expression levels of LAMP-2 and Rab7 protein significantly decreased to approximately 74% and 67% that of the sham group, respectively, and these decreases were attenuated by genipin. There were no significant changes in cathepsin B protein expression in any of the experimental groups. To further confirm that genipin restores CLP-induced impairment of autophagosome–lysosome fusion, we analysed the distribution of respective LC3 and LAMP-2 by immunohistochemistry assay. CLP group exhibited the increase in the distribution of LC3 and the decrease that of LAMP-2 compared with the sham group. Genipin treatment enhanced the increase in LC3 distribution and attenuated the decrease in LAMP-2 distribution (Figure 6B).
Effect of genipin on CLP-induced mTOR and calpain activation To elucidate the mechanisms by which genipin affects autophagy activation in sepsis, we investigated the involvement of the mTOR-dependent pathway and calpain system, which is independent of mTOR. The phosphorylation levels of mTOR and its downstream substrates, 4E-BP1 (Ser65) and p70S6K (Thr389), significantly increased 2.1-, 1.4-, and 1.6fold, respectively, compared with those of the sham group after CLP. Genipin did not affect these phosphorylations (Figure 7A). Calpain 1 protein expression significantly increased 2.6-fold compared with that of the sham group after
Figure 2 1
Effect of genipin on serum TNF-α and IL-6 levels at 6 h after CLP. Mice were administered 2.5 mg·kg genipin i.v. immediately after CLP (n = 8). + Serum was obtained and assayed for TNF-α and IL-6 levels at 6 h after CLP. * P < 0.05 versus sham group; P < 0.05 versus CLP group. 984
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Figure 3 Effect of genipin on LC3-II and p62 protein expressions in the liver (A), effect of chloroquine (CQ) in the presence of genipin on LC3-II and p62 protein expressions in the liver (B), sepsis-induced lethality (C) and serum ALT and AST levels (D) at 6 h after CLP. Mice were administered 1 2.5 mg·kg genipin i.v. immediately after CLP (n = 8). (B–D) Mice were pretreated with chloroquine i.p. 1 h before CLP. * P < 0.05 versus + # sham group; P < 0.05 versus CLP group; P < 0.05 versus CLP + genipin group.
Figure 4 Effect of genipin on autophagic vacuoles in the liver at 6 h after CLP. Autophagic vacuoles (arrows indicated) were observed from TEM images. * + P < 0.05 versus sham group; P < 0.05 versus CLP group. British Journal of Pharmacology (2016) 173 980–991
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Figure 5 Effect of genipin on beclin-1 and Atg12–5 (A), Atg3 and Atg7 (B) protein expressions in the liver at 6 h after CLP. Mice were administered 1 2.5 mg·kg genipin i.v. immediately after CLP (n = 8). The protein expressions were measured by Western blot analysis at 6 h after CLP. + * P < 0.05 versus sham group; P < 0.05 versus CLP group.
CLP. Genipin attenuated the increase in calpain 1; however, the level of calpain 2 protein expression was not affected by sepsis or genipin (Figure 7B). The protein expression level of cleaved Atg5, which is cleaved by the calpain system, significantly increased to 1.6-fold that of the sham group at 6 h after CLP, and this increase was attenuated by genipin treatment (Figure 7B).
Discussion The participation of autophagy in the dysfunction of multiple organs during sepsis makes autophagy an attractive target for therapeutic manipulation after sepsis. Previous studies reported that complete activation of autophagy by rapamycin exerts a cardioprotective effect by restoring depressed cardiac performance and decreasing inflammatory responses (Hsieh et al. 2011). More recently, it has been shown that activation of autophagy inhibits hepatocyte death and ultimately prevents liver injury (Takahashi et al. 2013; Tang et al. 2013). Genipin is the hydrolytic product of geniposide that has potent antioxidant, anti-inflammatory and anti-apoptotic properties. We previously showed that genipin prevents oxidative stress and subsequent apoptotic cell death in a mouse model of fulminant hepatic failure (Kim et al. 2010). Genipin exhibits anti-inflammatory and antibacterial effects, which protect against LPS-induced acute systemic inflammation and CLP-induced sepsis (Kim et al. 2012b). Furthermore, 986
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Dando et al. (2013 demonstrated that genipin has anticancer activity through induction of autophagic cell death in a pancreatic adenocarcinoma cell line. However, there is limited information on the effect of genipin on autophagy in in vivo animal studies. In the present study, we found that genipin enhanced autophagic flux, which may have contributed to increased survival in murine sepsis. Earlier reports indicated that autophagy is transiently increased in response to septic insult, based on findings of increased LC3-II accumulation and the autophagic vacuoles observed in hepatocytes by TEM (Watanabe et al. 2009; Takahashi et al. 2013). However, it is not clear whether increased levels of LC3-II can be translated as acceleration of autophagy in this situation or as partial or complete impairment of autophagic flux by blockage of lysosomal fusion and degradation. LC3II is known to only exist on mature autophagosomes. An increased level of LC3-II is interpreted as evidence for autophagy activation, although it could also indicate autophagy suppression caused by decreased autolysosomal degradation of LC3. For example, in a rat model of pancreatitis, LC3-II levels significantly increased after LPS administration; however, this was not the result of induction of autophagy but rather a blockade of fusion between autophagosomes and lysosomes (Fortunato et al. 2009). The autophagic substrate p62 is an ubiquitinbinding protein that recognizes ubiquitinated substrates and recruits phagophores through direct interaction with LC3-II (Pankiv et al. 2007). Because LC3-II and p62 are both degraded with the autophagic cargo in the autolysosome, the accumulation of LC3-II and p62 aggregates is regarded as a robust marker
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Figure 6 Effect of genipin on LAMP-2, Rab7 and cathepsin B protein expressions (A) and immunohistochemistry with LC3 and LAMP-2 (B) in the liver at 6 h 1 after CLP. Mice were administered 2.5 mg·kg genipin i.v. immediately after CLP (n = 8). (A) The protein expressions were measured by Western blot analysis at 6 h after CLP. (B) Liver sections were fixed and immunostained with anti-LC3 (1:200; brown) and anti-LAMP-2 (1:1000; brown) + antibodies. Original magnification 400×. * P < 0.05 versus sham group; P < 0.05 versus CLP group.
of impaired autophagic flux (Lee et al. 2012). In this study, we initially observed crosstalk between autophagy and liver injury during sepsis. The hepatic levels of LC3-II and p62 protein expression significantly increased 6 h after CLP. Treatment with the autophagy inhibitor chloroquine further increased the levels of LC3-II and p62 protein expression. In addition, chloroquine exacerbated the liver injury, as evidenced by a decreased
survival rate and increased levels of serum ALT and AST (Supporting Information Figure SS1). These results indicate that impaired autophagic flux is responsible for sepsis-induced liver injury. Genipin further increased LC3-II protein expression and reduced p62 accumulation, attenuated the liver injury by decreasing serum ALT, AST and inflammatory cytokine levels and improved the survival rate. TEM images revealed that British Journal of Pharmacology (2016) 173 980–991
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Figure 7 Effect of genipin on phospho-mTOR, phospho-4E-BP1 and phospho-p70S6K (A), calpain 1, calpain 2 and cleaved Atg5 (B) protein expressions in 1 the liver at 6 h after CLP. Mice were administered 2.5 mg·kg genipin i.v. immediately after CLP (n = 8). The protein expressions were measured + by Western blot analysis at 6 h after CLP. *P < 0.05 versus sham group; P < 0.05 versus CLP group.
genipin further increased the number of autophagic vacuoles in the livers of mice during sepsis, which correlated with our Western blot results. In order to further examine the autophagy activation by genipin and its possible contribution to attenuation of liver damage triggered by sepsis, we utilized autophagy inhibitor chloroquine. Treatment with chloroquine blocked the autophagic flux restoration by genipin and reversed the hepatoprotective effects of genipin during sepsis. Collectively, our data suggest that genipin restores the autophagic flux that is impaired by sepsis and septic liver injury. Correct interactions between the autophagy machinery and autophagy receptors are important for the execution of the complete autophagy process. The process of autophagy involves some complex key steps: membrane isolation, phagophore elongation, autophagosome formation, autophagosome–lysosome fusion and degradation (Kang et al. 2011). After the beclin-1/Vps34 complex initiates membrane isolation from varied intracellular origins and gathers autophagic proteins essential for pre-autophagosomal structure, two conjugation systems play essential roles in the formation of the autophagosome: an Atg12-Atg5 conjugate and membrane-associated LC3-II. The Atg12-Atg5 conjugate binds to the outer membrane of the phagophore and dissociates after mature autophagosome formation, and LC3-II subsequently binds to the Atg12–5-associated phagophore and forms the mature autophagosome. The cytosolic pre-LC3 form is cleaved by Atg4 to LC3-I, which binds covalently to PE by the action of Atg7 and Atg3 enzymes and finally becomes LC3-II. Several reports have shown that gene silencing of Atg5, Atg3 or Atg7 leads to impairment of autophagosome formation and causes cellular damage in several in vitro and in vivo tests (Cutting et al. 2014; Diakopoulos et al. 2014; Lamoureux et al. 2013; Lin et al. 2014). 988
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In the present study, sepsis significantly increased the level of the Atg12–5 complex and decreased the level of Atg3; however, it did not affect the level of Atg7. Genipin enhanced the Atg12–5 complex level, attenuated the decreased Atg3 level and increased the Atg7 levels, indicating that genipin enhances the phagophore elongation and increases mature autophagosome formation during sepsis. The level of beclin-1 protein expression was not affected by sepsis. Considering that beclin-1 modulates autophagy through assembly and disassembly with a variety of activator and/or inhibitor proteins, such as Vps34, Bcl-2 Vps15, HMGB1, Ambra1 and rubicon, the precise effect of sepsis on the formation of beclin-1 complex should be studied further. Fusion of the lysosome with a mature autophagosome in the late stage of the autophagy process is a critical step for degradation of its contents (Yu et al. 2010). Mature autophagosomes initially fuse with endosomal vesicles and acquire LAMP-1 and LAMP-2, thus gaining the ability to combine with lysosomes. These structures then fuse with lysosomes (autolysosomes) and acquire the lysosomal proteases cathespins and acid phosphatases required for substrate degradation (Kirkegaard et al. 2004). Gene silencing of LAMP-2 is characterized by a disturbance in lysosomal positioning, which leads to dysfunctional fusion and accumulation of autophagosomes (Noda and Klionsky 2008). Furthermore, recent studies demonstrated that Rab7, a member of the small GTPase family, is also required for the fusion of autophagosomes and lysosomes (Ganley et al. 2011; Wang et al. 2014). In sepsis-induced kidney injury, down-regulation of the expression of Rab7 leads to incomplete activation of the autophagic process (Yen et al. 2013). In this study, sepsis significantly decreased the levels of LAMP-2 and Rab7 protein expression, whereas it did not
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affect the level of cathepsin B protein. Genipin treatment attenuated the decrease in LAMP-2 and Rab7 protein levels. Furthermore, genipin enhanced the distribution of LC3 protein and attenuated the decreased distribution of LAMP-2 after CLP in liver tissue. These results suggested that genipin restores autophagosome–lysosome fusion in the liver during sepsis. Numerous components, such as mTOR, intracellular Ca2+ and oxidative stress, appear to be involved in the regulation of autophagy in mammalian cells (Hoyer-Hansen et al. 2007). Among them, mTOR, a serine/threonine protein kinase that regulates diverse cellular functions, is the most studied negative modulator of autophagy (Ravikumar et al. 2004). In the normal state, mTOR directly interacts with the Atg1-Atg13 complex and mediates phosphorylation-dependent inhibition of its kinase activities. Laufenberg et al. (2014 showed that sepsis decreased basal protein synthesis, in association with a reduction in mTOR activation, in skeletal muscle. Furthermore, the mTOR inhibitor temsirolimus induced autophagy and protected against endotoxaemia-induced acute kidney injury (Howell et al. 2013). Calpain is a Ca2+-dependent cysteine protease that plays a crucial role in a variety of cellular processes, including different signal transduction pathways, the regulation of apoptosis and autophagic degradation. The two main isoforms of calpain—micromolar Ca2+-requiring calpain (μ-calpain, calpain 1) and millimolar Ca2+-requiring calpain (m-calpain, calpain 2)—differ primarily in their calcium requirements, and deregulation of calpain activity following loss of Ca2+ homeostasis leads to tissue damage in various pathophysiological events including myocardial infarcts and stroke (Goll et al. 2003). Recent studies have suggested that the calpain system could inhibit the autophagy process by directly cleaving several Atg proteins involved in all steps of autophagy from autophagosome formation to autolysosome degradation (Kim et al. 2008; Russo et al. 2011; Villalpando Rodriguez and Torriglia 2013). Among the Atg proteins, it has been recognized that the Atg5 is the specific target: full-length Atg5 is cleaved into the truncated Atg5 by activated calpains, which play important roles in the crosstalk between autophagy and apoptosis. Actually, several studies have shown the cleaved Atg5 levels with consistent calpain activation as the evidence for calpainmediated autophagy impairment (Zhu et al. 2015; Cha et al. 2014). Moreover, calpain activity increased in endotoxaemiainduced or CLP-induced sepsis animal models, suggesting that this increased protease activity could be a negative autophagy regulatory mechanism during sepsis (Zafrani et al. 2012; Li et al. 2014). In our study, the levels of mTOR, calpain 1 and cleaved Atg5 protein expression significantly increased in sepsis, and genipin attenuated the levels of calpain 1 and cleaved Atg5, but not mTOR. Collectively, our data suggest that genipin activates autophagic flux through the downregulation of calpain.
Conclusion In conclusion, these findings suggest that genipin protects against septic injury by restoring the impaired autophagic flux via inhibition of the calpain system. Thus, we propose that genipin may provide new a pharmacological intervention strategy for septic injury.
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Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2013R1A1A3008145). H-I. C. (NRF-2012H1A2A1016419) and So-Jin Kim (NRF-2013H1A2A1034472) received ‘Global Ph.D. Fellowship Program’ support from the NRF funded by the Ministry of Education, Science, and Technology (MEST) in Korea.
Author contributions H-I. C., S. J. K. and J. W. C. performed the research. H- I. C., S J. K. and S. M. L. designed the research. H-I. C. and S. J. K. analysed the data. H-I. C. and S. M. L. wrote the paper.
Conflicts of interest None to declare.
References Alexander SPH, Fabbro D, Kelly E, Marrion N, Peters JA, Benson HE, et al. (2016). The Concise Guide to PHARMACOLOGY 2015/16: Enzymes. Br J Pharmacol 172: 6024–6109. Cha HH, Hwang JR, Kim HY, Choi SJ, Oh SY, Roh CR (2014). Autophagy induced by tumor necrosis factor α mediates intrinsic apoptosis in trophoblastic cells. Reprod Sci 21: 612–622. Chaudry IH, Wichterman KA, Baue AE (1979). Effect of sepsis on tissue adenine nucleotide levels. Surgery 85: 205–211. Curtis MJ, Bond RA, Spina D, Ahluwalia A, Alexander SP, Giembycz MA, et al. (2015). Experimental design and analysis and their reporting: new guidance for publication in BJP. Br J Pharmacol 172: 3461–3471. Cutting AS, Del Rosario Y, Mu R, Rodriguez A, Till A, Subramani S, et al. (2014). The role of autophagy during group B streptococcus infection of blood–brain barrier endothelium. J Biol Chem 289: 35711–35723. Dando I, Fiorini C, Pozza ED, Padroni C, Costanzo C, Palmieri M, et al. (2013). UCP2 inhibition triggers ROS-dependent nuclear translocation of GAPDH and autophagic cell death in pancreatic adenocarcinoma cells. Biochim Biophys Acta 1833: 672–679. Diakopoulos KN, Lesina M, Wormann S, Song L, Aichler M, Schild L, et al. (2014). Impaired autophagy induces chronic atrophic pancreatitis in mice via sex- and nutrition-dependent processes. Gastroenterology 148: 626–638. Fortunato F, Burgers H, Bergmann F, Rieger P, Buchler MW, Kroemer G, et al. (2009). Impaired autolysosome formation correlates with Lamp2 depletion: role of apoptosis, autophagy, and necrosis in pancreatitis. Gastroenterology 137: 350–360. Ganley IG, Wong PM, Gammoh N, Jiang X (2011). Distinct autophagosomal–lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest. Mol Cell 42: 731–743. Goll DE, Thompson VF, Li H, Wei W, Cong J (2003). The calpain system. Physiol Rev 83: 731–801.
British Journal of Pharmacology (2016) 173 980–991
989
BJP
H-I Cho et al.
Gonnert FA, Recknagel P, Hilger I, Claus RA, Bauer M, Kortgen A (2013). Hepatic excretory function in sepsis: implications from biophotonic analysis of transcellular xenobiotic transport in a rodent model. Crit Care 17: R67. Howell GM, Gomez H, Collage RD, Loughran P, Zhang X, Escobar DA, et al. (2013). Augmenting autophagy to treat acute kidney injury during endotoxemia in mice. PLoS One 8: e69520. Hoyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, et al. (2007). Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell 25: 193–205. Hsieh CH, Pai PY, Hsueh HW, Yuan SS, Hsieh YC (2011). Complete induction of autophagy is essential for cardioprotection in sepsis. Ann Surg 253: 1190–1200.
Lelono RA, Tachibana S, Itoh K (2009). Isolation of antifungal compounds from Gardenia jasminoides. Pak J Biol Sci 12: 949–956. Li X, Luo R, Chen R, Song L, Zhang S, Hua W, et al. (2014). Cleavage of IkappaBalpha by calpain induces myocardial NF-kappaB activation, TNF-alpha expression, and cardiac dysfunction in septic mice. Am J Physiol Heart Circ Physiol 306: H833–H843. Lin CW, Lo S, Hsu C, Hsieh CH, Chang YF, Hou BS, et al. (2014). T-cell autophagy deficiency increases mortality and suppresses immune responses after sepsis. PLoS One 9: e102066. Lo S, Yuan SS, Hsu C, Cheng YJ, Chang YF, Hsueh HW, et al. (2013). Lc3 over-expression improves survival and attenuates lung injury through increasing autophagosomal clearance in septic mice. Ann Surg 257: 352–363.
Kang R, Zeh HJ, Lotze MT, Tang D (2011). The beclin 1 network regulates autophagy and apoptosis. Cell Death Differ 18: 571–580.
McGrath JC, Lilley E (2015). Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP. Br J Pharmacol 172: 3189–3193.
Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG (2010). NC3Rs Reporting Guidelines Working Group. Br J Pharmacol 160: 1577–1579.
Noda T, Klionsky DJ (2008). The quantitative Pho8Delta60 assay of nonspecific autophagy. Methods Enzymol 451: 33–42.
Kim ES, Jeong CS, Moon A (2012a). Genipin, a constituent of Gardenia jasminoides Ellis, induces apoptosis and inhibits invasion in MDA-MB-231 breast cancer cells. Oncol Rep 27: 567–572. Kim J, Kim HY, Lee SM (2013). Protective effects of geniposide and genipin against hepatic ischemia/reperfusion injury in mice. Biomol Ther (Seoul) 21: 132–137. Kim JS, Nitta T, Mohuczy D, O’Malley KA, Moldawer LL, Dunn WA Jr, et al. (2008). Impaired autophagy: a mechanism of mitochondrial dysfunction in anoxic rat hepatocytes. Hepatology 47: 1725–1736. Kim SJ, Kim JK, Lee DU, Kwak JH, Lee SM (2010). Genipin protects lipopolysaccharide-induced apoptotic liver damage in D-galactosaminesensitized mice. Eur J Pharmacol 635: 188–193. Kim TH, Yoon SJ, Lee SM (2012b). Genipin attenuates sepsis by inhibiting toll-like receptor signaling. Mol Med 18: 455–465. Kimura T, Watanabe E, Sakamoto T, Takasu O, Ikeda T, Ikeda K, et al. (2014). Autophagy-related IRGM polymorphism is associated with mortality of patients with severe sepsis. PLoS One 9: e91522. Kirkegaard K, Taylor MP, Jackson WT (2004). Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nat Rev Microbiol 2: 301–314. Koo HJ, Song YS, Kim HJ, Lee YH, Hong SM, Kim SJ, et al. (2004). Antiinflammatory effects of genipin, an active principle of Gardenia. Eur J Pharmacol 495: 201–208. Kramer L, Jordan B, Druml W, Bauer P, Metnitz PG, Austrian Epidemiologic Study on Intensive Care ASG (2007). Incidence and prognosis of early hepatic dysfunction in critically ill patients—a prospective multicenter study. Crit Care Med 35: 1099–1104. Lamoureux F, Thomas C, Crafter C, Kumano M, Zhang F, Davies BR, et al. (2013). Blocked autophagy using lysosomotropic agents sensitizes resistant prostate tumor cells to the novel Akt inhibitor AZD5363. Clin Cancer Res 19: 833–844. Laufenberg LJ, Pruznak AM, Navaratnarajah M, Lang CH (2014). Sepsis-induced changes in amino acid transporters and leucine signaling via mTOR in skeletal muscle. Amino Acids 46: 2787–2798. Lee HS, Daniels BH, Salas E, Bollen AW, Debnath J, Margeta M (2012). Clinical utility of LC3 and p62 immunohistochemistry in diagnosis of drug-induced autophagic vacuolar myopathies: a case–control study. PLoS One 7: e36221.
990
British Journal of Pharmacology (2016) 173 980–991
Pawson AJ, Sharman JL, Benson HE, Faccenda E, Alexander SP, Buneman OP et al., NC-IUPHAR (2014). The IUPHAR/BPS guide to PHARMACOLOGY: an expert-driven knowledge base of drug targets and their ligands. Nucleic Acids Res 42: D1098–D1106. Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, et al. (2007). p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 282: 24131–24145. Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, et al. (2004). Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 36: 585–595. Recknagel P, Gonnert FA, Westermann M, Lambeck S, Lupp A, Rudiger A, et al. (2012). Liver dysfunction and phosphatidylinositol-3-kinase signalling in early sepsis: experimental studies in rodent models of peritonitis. PLoS Med 9: e1001338. Russo R, Berliocchi L, Adornetto A, Varano GP, Cavaliere F, Nucci C, et al. (2011). Calpain-mediated cleavage of beclin-1 and autophagy deregulation following retinal ischemic injury in vivo. Cell Death Dis 2: e144. Savva A, Plantinga TS, Kotanidou A, Farcas M, Baziaka F, Raftogiannis M, et al. (2014). Association of autophagy-related 16-like 1 (ATG16L1) gene polymorphism with sepsis severity in patients with sepsis and ventilator-associated pneumonia. Eur J Clin Microbiol Infect Dis 33: 1609–1614. Takahashi W, Watanabe E, Fujimura L, Watanabe-Takano H, Yoshidome H, Swanson PE, et al. (2013). Kinetics and protective role of autophagy in a mouse cecal ligation and puncture-induced sepsis. Crit Care 17: R160. Takeuchi S, Goto T, Mikami K, Miura K, Ohshima S, Yoneyama K, et al. (2005). Genipin prevents fulminant hepatic failure resulting in reduction of lethality through the suppression of TNF-alpha production. Hepatol Res 33: 298–305. Tang Z, Ni L, Javidiparsijani S, Hu F, Gatto LA, Cooney R, et al. (2013). Enhanced liver autophagic activity improves survival of septic mice lacking surfactant proteins A and D. Tohoku J Exp Med 231: 127–138. Taylor DE, Ghio AJ, Piantadosi CA (1995). Reactive oxygen species produced by liver mitochondria of rats in sepsis. Arch Biochem Biophys 316: 70–76. Valdor R, Macian F (2012). Autophagy and the regulation of the immune response. Pharmacol Res 66: 475–483.
Genipin alleviates liver injury by restoring autophagy
Villalpando Rodriguez GE, Torriglia A (2013). Calpain 1 induce lysosomal permeabilization by cleavage of lysosomal associated membrane protein 2. Biochim Biophys Acta 1833: 2244–2253. Wang B, Yang Q, Sun YY, Xing YF, Wang YB, Lu XT, et al. (2014). Resveratrol-enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice. J Cell Mol Med 18: 1599–1611. Watanabe E, Muenzer JT, Hawkins WG, Davis CG, Dixon DJ, McDunn JE, et al. (2009). Sepsis induces extensive autophagic vacuolization in hepatocytes: a clinical and laboratory-based study. Lab Invest 89: 549–561. Yen YT, Yang HR, Lo HC, Hsieh YC, Tsai SC, Hong CW, et al. (2013). Enhancing autophagy with activated protein C and rapamycin protects against sepsis-induced acute lung injury. Surgery 153: 689–698. Yu L, McPhee CK, Zheng L, Mardones GA, Rong Y, Peng J, et al. (2010). Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature 465: 942–946. Zafrani L, Gerotziafas G, Byrnes C, Hu X, Perez J, Levi C, et al. (2012). Calpastatin controls polymicrobial sepsis by limiting
BJP
procoagulant microparticle release. Am J Respir Crit Care Med 185: 744–755. Zhu X, Messer JS, Wang Y, Lin F, Cham CM, Chang J, et al. (2015). Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation. J Clin Invest 125: 1098–1110.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: http://dx.doi.org/10.1111/bph.13397 Figure S1 Effect of chloroquine (CQ) on LC3-II and p62 protein expressions in the liver (A), sepsis-induced lethality (B) and serum ALT and AST (C) levels. Mice were intravenously administered vehicle (saline) or CQ (60 mg·kg 1) 1 h prior to CLP (n = 10 for survival test and n = 8 for biochemical assay). * denotes significant difference (P < 0.05) versus sham group; + denotes significant difference (P < 0.05) versus CLP group.
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