INTIMP-03592; No of Pages 6 International Immunopharmacology xxx (2015) xxx–xxx
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
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Chen-wei Pan a,1, Guang-yao Zhou a,1, Wei-lai Chen b, Lu Zhuge a, Ling-xiang Jin a, Yi Zheng a, Wei Lin a, Zhen-zhen Pan c,⁎
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Article history: Received 11 December 2014 Received in revised form 12 February 2015 Accepted 10 March 2015 Available online xxxx
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Keywords: Forsythiaside Lipopolysaccharide (LPS) Nrf2 NF-κB Acute liver injury
Department of Infectious Disease, The Second Affiliated Hospital & Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, ZheJiang 325027, China Department of Neurology, Wenzhou People's Hospital, Wenzhou, ZheJiang 325027, China Department of Infectious Disease, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, ZheJiang 325000, China
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Forsythiaside A, an active constituent isolated from air-dried fruits of Forsythia suspensa, has been reported to have multiple pharmacological activities including anti-inflammatory, anti-oxidant, and antioxidant activities. In the present study, the hepatoprotective effect of forsythiaside A was investigated in lipopolysaccharide (LPS)/D-galactosamine (GalN)-induced acute liver injury in mice. Mice acute liver injury model was induced by LPS (50 μg/kg)/GalN (800 mg/kg). Forsythiaside A was administrated 1 h prior to LPS/GalN exposure. The results showed that forsythiaside A attenuated hepatic pathological damage, malondialdehyde (MDA) content, and serum ALT, and AST levels induced by LPS/GalN. Moreover, forsythiaside A inhibited NF-κB activation, serum TNF-α and hepatic TNF-α levels induced by LPS/GalN. Furthermore, we found that forsythiaside A upregulated the expression of Nrf2 and heme oxygenase-1. Our results showed that forsythiaside A protected against LPS/GalN-induced liver injury through activation of Nrf2 and inhibition of NF-κB activation. © 2015 Published by Elsevier B.V.
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1. Introduction
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Endotoxemia and sepsis occur frequently in cases of liver failure, which has a high mortality due to the lock of effective drugs [1,2]. Therefore, the development of definitive and targeted drug therapies for hepatic injury is urgently needed. LPS and GalN-induced acute hepatic injury in mice is a widely used model for human hepatic injury [3,4]. It has been used for preliminary pharmacological studies of potential therapeutic drugs and agents [5]. GalN is a hepatotoxic agent and the hepatotoxicity is mainly through inhibiting hepatocyte RNA and protein synthesis [6]. LPS is an endotoxin that could induce inflammatory cytokine production [7]. These pro-inflammatory mediators lead to liver tissue injury [8]. Forsythia suspensa is one of the most famous Chinese herbal medicines that has been widely used to treat various infective diseases, such as nephritis, ulcers, and erysipelas [9,10]. F. suspensa is the main component of traditional Chinese formula Shuang–Huang–Lian [11]. Forsythiaside, an active constituent isolated from air-dried fruits of F. suspensa, has been reported to have anti-inflammatory, antioxidant, and antioxidant activities [12]. Previous studies showed that
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Protective effect of forsythiaside A on lipopolysaccharide/D-galactosamine-induced liver injury
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⁎ Corresponding author. E-mail address: [email protected] (Z. Pan). 1 Chen-wei Pan and Guang-yao Zhou have equal contribution to the paper.
forsythiaside had the ability to protect against hydrogen peroxideinduced apoptosis and oxidative stress in PC12 cell [13]. Furthermore, forsythiaside was found to inhibit LPS-induced acute lung injury in mice [14] and LPS-induced inflammatory responses in the bursa of chickens [15]. In addition, forsythiaside was also found to have neuroprotective effects in senescence-accelerated mouse prone mice [12]. However, the effect of forsythiaside A on LPS/GalN-induced liver injury remains unclear. In this study, we sought to assess the effects of forsythiaside A on LPS/GalN-induced liver injury in mice.
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2. Materials and methods
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2.1. Materials
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Forsythiaside A was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Mouse TNF-α ELISA kit was purchased from R&D Systems (Minneapolis, MN). LPS (Escherichia coli, O55:B5) and D-galactosamine were purchased from Sigma (St. Louis, MO, USA). Anti-pNF-κB p65, anti-NF-κB p65, anti-HO-1, anti-nrf2, and anti-β-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The malondialdehyde (MDA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) detection kits were provided by
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http://dx.doi.org/10.1016/j.intimp.2015.03.009 1567-5769/© 2015 Published by Elsevier B.V.
Please cite this article as: C. Pan, et al., Protective effect of forsythiaside A on lipopolysaccharide/D-galactosamine-induced liver injury, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.03.009
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the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). All other reagents were of analytical grade.
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2.2. Animals
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Male BALB/c mice, weighing approximately 18 to 22 g, were purchased from the Center of Experimental Animals of Zhengzhou University (Zhengzhou, China). The mice were housed in an environmentally controlled room (24 ± 1 °C, 40%–80% humidity). They were allowed free access to food and water. All experimental protocols described in this study were performed in accordance with the guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
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2.3. Experimental design
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The liver injury model was induced by intraperitoneal injection of GalN (800 mg/kg) and LPS (50 μg/kg) dissolved in PBS. To investigate the protective effect of forsythiaside A on LPS/GalN-induced acute liver injury, mice were given with an intraperitoneal injection (i.p.) of forsythiaside A (15, 30, 60 mg/kg) 1 h before LPS/GalN treatment. Seventy-five mice were randomly divided into five groups and each group contained 15 mice: Group I, normal control, the mice received equal volume of PBS. Group II, model control, the mice received GalN (800 mg/kg) and LPS (50 μg/kg). Group III–V, the mice received forsythiaside A (15, 30, 60 mg/kg) 1 h before LPS/GalN treatment. The mice were after LPS/GalN treatment. Then the blood and liver tissues were collected for subsequent analysis.
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2.4. Cytokine assays
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Two hours after LPS/GalN treatment, serum and liver tissues were collected for TNF-α detection. Levels of TNF-α in serum and hepatic samples were measured using ELISA kits (R&D) according to the manufacturer's instructions.
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2.5. Analysis of liver enzymes
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Hepatocyte damage was assessed by detecting plasma enzyme activities of ALT and AST. To analyze plasma enzyme activities of ALT and AST, serum was collected 8 h after LPS/GalN injection. ALT and AST levels were measured using test kits purchased from Jiancheng Bioengineering Institute of Nanjing according to the instructions.
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2.6. MDA content assay
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MDA, an end product of lipid peroxidation, was used to assess lipid peroxidation in the liver. Liver tissues were collected 8 h after LPS/ GalN injection and liver MDA content was measured by MDA test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the instructions.
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2.7. Histopathologic evaluation
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Liver tissues were harvested at 8 h after LPS/GalN injection. Liver tissues were fixed with 10% buffered formalin, imbedded in paraffin and sliced. After hematoxylin and eosin (H&E) staining, pathological changes of liver tissues were observed under a light microscope.
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2.8. Western blot analysis
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Liver tissues were collected 8 h after LPS/GalN injection. Proteins were extracted from the liver using T-PER Tissue Protein Extraction Reagent Kit (Thermo) according to the manufacturer's instructions. Nuclear and cytoplasmic proteins were extracted from the liver using NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo)
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3.1. Forsythiaside A inhibited serum ALT and AST levels induced by LPS/GalN 141
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Serum ALT and AST levels were detected 8 h after LPS/GalN treatment. As shown in Fig. 1, ALT and AST levels increased significantly after LPS/GalN treatment. These increases were attenuated by administration of forsythiaside.
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3.2. Effects of forsythiaside A on LPS/GalN-mediated liver histopathologic changes To test protective effect of forsythiaside A on LPS/GalN-induced liver injury in mice, we observed the liver histopathologic changes under a light microscope. As shown in Fig. 2, liver sections of normal control group showed normal lobular architecture and cellular structure. Liver sections obtained from LPS/GalN group showed significant pathologic changes, including extensive areas of portal inflammation, cellular necrosis, and inflammatory cell infiltration. However, these pathological changes were significantly attenuated by forsythiaside.
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3. Results
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All values are expressed as means ± S.E.M. Differences between mean values of normally distributed data were analyzed using oneway ANOVA (Dunnett's t-test) and two-tailed Student's t-test. Statistical significance was accepted p b 0.05 or p b 0.01.
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according to the manufacturer's protocol. Protein concentrations were determined by BCA protein assay kit. Equal amounts of protein were fractionated on 12% polyacrylamide-SDS gel. Then proteins were transferred to polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk for 2 h at room temperature followed by primary antibody (1:1000) overnight at 4 °C. Subsequently, the membrane was treated with horseradish peroxidase (HRP)-conjugated secondary antibody. The immunoactive proteins were detected by using an enhanced chemiluminescence (ECL) Western blotting detection kit.
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Fig. 1. Effects of forsythiaside A on serum ALT and AST levels. The values presented are the Q1 mean ± SEM (n = 15 in each group). #p b 0.01 vs. control group, *p b 0.05, **p b 0.01 vs. LPS/GalN group.
Please cite this article as: C. Pan, et al., Protective effect of forsythiaside A on lipopolysaccharide/D-galactosamine-induced liver injury, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.03.009
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Fig. 2. Effects of forsythiaside A on histopathological changes in liver tissues. Representative histological changes of liver obtained from mice of different groups. A: Control group, B: LPS/ GalN group, C: LPS/GalN + forsythiaside A (15 mg/kg) group, D: LPS/GalN + forsythiaside A (30 mg/kg) group E: LPS/GalN + forsythiaside A (60 mg/kg) group (hematoxylin and eosin staining, magnification 200×).
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3.3. Effects of forsythiaside A on TNF-α production
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Levels of TNF-α in serum and hepatic samples were detected by ELISA. The results showed that TNF-α level in serum and hepatic samples was increased significantly after LPS/GalN injection. However, forsythiaside A inhibited TNF-α production induced by LPS/GalN in a dose dependent manner (Fig. 3).
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Malondialdehyde (MDA), the end product of lipid peroxidation, is known as a marker of oxidative stress. Liver MDA level was detected 8 h after LPS/GalN injection. As shown in Fig. 4, LPS/GalN challenge produced a significant increase of liver MDA level. However, administrations of forsythiaside A significantly reduced the increase of liver MDA level (Fig. 4).
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3.5. Effect of forsythiaside A on NF-κB activation
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NF-κB has been reported to be a dominant transcription factor re- 170 sponsible for inflammation [16]. Activation of NF-κB by LPS could in- 171 duce the production of cytokine [17]. In this study we detected the 172 effects of forsythiaside A on NF-κB activation. LPS/GalN-treated mice 173 displayed that p65 subunit of NF-κB increased in nucleus and decreased 174 in cytoplasm, and the tendency was reversed by forsythiaside A (Fig. 5). 175
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3.4. Effects of forsythiaside A on lipid peroxidation
Fig. 3. Effects of forsythiaside A on serum and hepatic TNF-α levels. The values presented are the means ± SEM (n = 15 in each group). #p b 0.01 vs. control group, *p b 0.05 and **p b 0.01 vs. LPS/GalN group.
Fig. 4. Effects of forsythiaside A on liver MDA level. The values presented are the mean ± Q2 SEM (n = 15 in each group). #p b 0.01 vs. control group, *p b 0.05, **p b 0.01 vs. LPS/GalN group.
Please cite this article as: C. Pan, et al., Protective effect of forsythiaside A on lipopolysaccharide/D-galactosamine-induced liver injury, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.03.009
C. Pan et al. / International Immunopharmacology xxx (2015) xxx–xxx
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Fig. 5. Forsythiaside A inhibited LPS/GalN-induced NF-κB activation with Western blotting. The values presented are the mean ± SEM (n = 15 in each group). #p b 0.01 vs. control group, *p b 0.05 and **p b 0.01 group vs. LPS group.
3.6. Forsythiaside A promoted the expression of Nrf2 and HO-1
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Nrf-2 has been reported to play an important role in orchestrating cellular antioxidant defenses. In this study, we detected the effects of
forsythiaside A on Nrf2 and HO-1 expression. The results showed that Nrf2 in nuclear and HO-1 in cytosolic were upregulated at 8 h after LPS/GalN injection. These increases in Nrf2 and HO-1 expression were augmented by forsythiaside A (Fig. 6).
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Fig. 6. Effects of forsythiaside A on the expression of Nrf2 and HO-1. The values presented are the mean ± SEM (n = 15 in each group). #p b 0.01 vs. control group, *p b 0.05 and **p b 0.01 group vs. LPS group.
Please cite this article as: C. Pan, et al., Protective effect of forsythiaside A on lipopolysaccharide/D-galactosamine-induced liver injury, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.03.009
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[1] B.G. Harbrecht, T.R. Billiar, J. Stadler, A.J. Demetris, J.B. Ochoa, R.D. Curran, et al., Nitric oxide synthesis serves to reduce hepatic damage during acute murine endotoxemia, Crit. Care Med. 20 (1992) 1568–1574. [2] J. Belghiti, K. Hiramatsu, S. Benoist, P. Massault, A. Sauvanet, O. Farges, Seven hundred forty-seven hepatectomies in the 1990s: an update to evaluate the actual risk of liver resection, J. Am. Coll. Surg. 191 (2000) 38–46. [3] L. Zhang, H.Z. Li, X. Gong, F.L. Luo, B. Wang, N. Hu, et al., Protective effects of Asiaticoside on acute liver injury induced by lipopolysaccharide/D-galactosamine in mice, Phytomedicine: Int. J. Phytother. Phytopharmacology 17 (2010) 811–819. [4] T. Nakama, S. Hirono, A. Moriuchi, S. Hasuike, K. Nagata, T. Hori, et al., Etoposide prevents apoptosis in mouse liver with D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure resulting in reduction of lethality, Hepatology 33 (2001) 1441–1450. [5] B. Feng, S. Wu, S. Lv, F. Liu, H. Chen, X. Yan, et al., Metabolic profiling analysis of a Dgalactosamine/lipopolysaccharide-induced mouse model of fulminant hepatic failure, J. Proteome Res. 6 (2007) 2161–2167. [6] A. Sathivel, Balavinayagamani, B.R. Hanumantha Rao, T. Devaki, Sulfated polysaccharide isolated from Ulva lactuca attenuates d-galactosamine induced DNA fragmentation and necrosis during liver damage in rats, Pharm. Biol. (2013). [7] L. Haversen, B.G. Ohlsson, M. Hahn-Zoric, L.A. Hanson, I. Mattsby-Baltzer, Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NFkappa B, Cell. Immunol. 220 (2002) 83–95. [8] T. Masaki, S. Chiba, H. Tatsukawa, T. Yasuda, H. Noguchi, M. Seike, et al., Adiponectin protects LPS-induced liver injury through modulation of TNF-alpha in KK-Ay obese mice, Hepatology 40 (2004) 177–184. [9] H. Qu, Y. Zhang, Y. Wang, B. Li, W. Sun, Antioxidant and antibacterial activity of two compounds (forsythiaside and forsythin) isolated from Forsythia suspensa, J. Pharm. Pharmacol. 60 (2008) 261–266. [10] Y. Ozaki, J. Rui, Y.T. Tang, Antiinflammatory effect of Forsythia suspensa V(AHL) and its active principle, Biol. Pharm. Bull. 23 (2000) 365–367. [11] W. Zhou, L.Q. Di, J.J. Shan, X.L. Bi, L.T. Chen, L.C. Wang, et al., In vitro metabolism in Sprague–Dawley rat liver microsomes of forsythoside A in different compositions of Shuang–Huang–Lian, Fitoterapia 82 (2011) 1222–1230. [12] H.M. Wang, L.W. Wang, X.M. Liu, C.L. Li, S.P. Xu, A.D. Farooq, Neuroprotective effects of forsythiaside on learning and memory deficits in senescence-accelerated mouse prone (SAMP8) mice, Pharmacol. Biochem. Behav. 105 (2013) 134–141. [13] C. Huang, Y. Lin, H. Su, D. Ye, Forsythiaside protects against hydrogen peroxideinduced oxidative stress and apoptosis in PC12 cell, Neurochem. Res. (2014). [14] L. Zhou, H. Yang, Y. Ai, Y. Xie, Y. Fu, Protective effect of forsythiaside A on acute lung injure induced by lipopolysaccharide in mice, Cell. Mol. Immunol. (Xi bao yu fen zi mian yi xue za zhi), 30 (2014) 151–154. [15] G. Cheng, Y. Zhao, H. Li, Y. Wu, X. Li, Q. Han, et al., Forsythiaside attenuates lipopolysaccharide-induced inflammatory responses in the bursa of Fabricius of chickens by downregulating the NF-kappaB signaling pathway, Exp. Ther. Med. 7 (2014) 179–184. [16] N.D. Perkins, Integrating cell-signalling pathways with NF-kappaB and IKK function, Nat. Rev. Mol. Cell Biol. 8 (2007) 49–62. [17] L. Haversen, B.G. Ohlsson, M. Hahn-Zoric, L.A. Hanson, I. Mattsby-Baltzer, Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NFkappa B, Cell. Immunol. 220 (2002) 83–95. [18] A.J. Hanley, K. Williams, A. Festa, L.E. Wagenknecht, R.B. D'Agostino Jr., J. Kempf, et al., Elevations in markers of liver injury and risk of type 2 diabetes: the insulin resistance atherosclerosis study, Diabetes 53 (2004) 2623–2632. [19] R.F. Schwabe, D.A. Brenner, Mechanisms of liver injury. I. TNF-alpha-induced liver injury: role of IKK, JNK, and ROS pathways, Am. J. Physiol. Gastrointest. Liver Physiol. 290 (2006) G583–G589. [20] W.X. Ding, X.M. Yin, Dissection of the multiple mechanisms of TNF-alpha-induced apoptosis in liver injury, J. Cell. Mol. Med. 8 (2004) 445–454. [21] G. Ramesh, W.B. Reeves, TNF-alpha mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity, J. Clin. Invest. 110 (2002) 835–842. [22] C. Ji, Q. Deng, N. Kaplowitz, Role of TNF-alpha in ethanol-induced hyperhomocysteinemia and murine alcoholic liver injury, Hepatology 40 (2004) 442–451. [23] N. Enomoto, Y. Takei, M. Hirose, K. Ikejima, H. Miwa, T. Kitamura, et al., Thalidomide prevents alcoholic liver injury in rats through suppression of Kupffer cell sensitization and TNF-alpha production, Gastroenterology 123 (2002) 291–300. [24] Q. Li, I.M. Verma, NF-kappaB regulation in the immune system, Nat. Rev. Immunol. 2 (2002) 725–734. [25] D. Avni, Y. Glucksam, T. Zor, The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 modulates cytokine expression in macrophages via p50 nuclear factor kappaB inhibition, in a PI3K-independent mechanism, Biochem. Pharmacol. 83 (2012) 106–114. [26] E.H. Joh, I.A. Lee, I.H. Jung, D.H. Kim, Ginsenoside Rb1 and its metabolite compound K inhibit IRAK-1 activation—the key step of inflammation, Biochem. Pharmacol. 82 (2011) 278–286. [27] T.D. Gilmore, Introduction to NF-kappaB: players, pathways, perspectives, Oncogene 25 (2006) 6680–6684.
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References
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C
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R
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Forsythiaside, an active constituent isolated from air-dried fruits of F. suspensa, has been reported to have anti-inflammatory, antioxidant, and antioxidant activities [12]. In the present study, we observed the effects of forsythiaside A on LPS/GalN-induced acute liver injury in mice. The results showed that forsythiaside A attenuated hepatic pathological damage, MDA content, and serum ALT, and AST levels induced by LPS/GalN. Moreover, forsythiaside A inhibited NF-κB activation, serum and hepatic TNF-α levels induced by LPS/ GalN. Furthermore, we found that forsythiaside A up-regulated the expression of Nrf2 and heme oxygenase-1. Forsythiaside A may be a promising potential therapeutic reagent for acute liver injury treatment. Serum ALT and AST were used as biochemical indicator of hepatic injury [18]. In the present study, serum ALT, AST and histological analysis of liver tissues were used to assess the protective effect of forsythiaside A on LPS/GalN-induced acute liver injury. The results showed that forsythiaside A significantly decreased serum ALT and AST levels. Meanwhile, histological analysis demonstrated that forsythiaside A could attenuate cellular necrosis, and inflammatory cell infiltration. Forsythiaside A significantly attenuated liver injury. These results suggested that forsythiaside A had a protective effect on LPS/GalN-induced hepatic injury. TNF-α has been demonstrated to play vital role in the pathogenesis of liver injury [19]. It could induce hepatocyte necrosis and organ failure and contribute to the severity of liver injury [20]. Meanwhile, TNF-α can induce other cytokine production [21]. These cytokines amplify and perpetuate the inflammatory response. Elevated TNF-α level was observed in serum and liver tissues [22]. Furthermore, previous reports demonstrated that inhibition of TNF-α could alleviate liver injury [23]. Thus, serum and liver TNF-α levels were detected in this study. The results showed that serum and liver TNF-α levels were dramatically inhibited by forsythiaside. MDA, the last product of lipid breakdown caused by oxidative stress, is used as a marker of oxidative stress. The results of this study showed that forsythiaside A significantly inhibited liver MDA level induced by LPS/GalN. The results suggested that forsythiaside A protected against LPS/GalN-induced liver injury by reducing inflammatory and oxidative response. NF-κB is a pleiotropic regulator of various genes which is involved in the inflammatory responses [24,25]. Normally, NF-κB is sequestered in the cytoplasm by a family of inhibitory proteins known as inhibitors of NF-κB (IκBs) [26]. Once stimulated with LPS, IκB is phosphorylated and degraded leading to the release of free NF-κB. Then NF-κB enters the nucleus and regulates the expression of inflammatory cytokines [27,28]. To characterize the inhibitory effect of forsythiaside A on cytokines production, we examined the effects of forsythiaside A on NF-κB activation and IκB-α degradation. The results showed that forsythiaside A inhibited NF-κB activation in a dose-dependent manner. Nrf2 is an important transcription factor that plays an important role in orchestrating cellular antioxidant defenses by regulating antioxidant enzymes expression [29,30]. Nrf2 controls the coordinated expression of important antioxidant and detoxification genes such as HO-1 [31, 32]. HO-1 is a ubiquitous and redox-sensitive inducible stress protein that has been reported to have antioxidant activity [33,34]. Our results showed that Nrf2 in nuclear and HO-1 in cytosolic were upregulated at 1 h after LPS/GalN injection. However, the increases in Nrf2 and HO-1 expression were augmented by forsythiaside. The results suggested that forsythiaside A could decrease oxidative stress induced by LPS/GalN by inducing oxidative defense system. Meanwhile, previous studies indicated that there was an interaction between Nrf2 and NFκB [35]. Activation of Nrf2 could inhibit NF-κB signaling pathway [36] and other studies demonstrated that Nrf2-deficient mice showed increased NF-κB activation in response to LPS stimulation [37]. Thus, the inhibition of NF-κB activation by forsythiaside A may be due to the activation of Nrf2 by forsythiaside.
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In conclusion, our results demonstrated that forsythiaside A had a protective effect on LPS/GalN-induced liver injury. The mechanisms underlying this protective effect may be related to inhibition of NF-κB activation and activation of Nrf2. Forsythiaside A may be an agent for preventing and treating LPS/GalN-induced liver injury.
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[33] S. Turkseven, A. Kruger, C.J. Mingone, P. Kaminski, M. Inaba, L.F. Rodella, et al., Antioxidant mechanism of heme oxygenase-1 involves an increase in superoxide dismutase and catalase in experimental diabetes, Am. J. Physiol. Heart Circ. Physiol. 289 (2005) H701–H707. [34] C.F. Chang, X.M. Liu, K.J. Peyton, W. Durante, Heme oxygenase-1 counteracts contrast media-induced endothelial cell dysfunction, Biochem. Pharmacol. 87 (2014) 303–311. [35] J. Rao, X. Qian, G. Li, X. Pan, C. Zhang, F. Zhang, et al., ATF3-mediated NRF2/HO-1 signaling regulates TLR4 innate immune responses in mouse liver ischemia/reperfusion injury, Am. J. Transplant. 15 (2015) 76–87. [36] C. Zeng, P. Zhong, Y. Zhao, K. Kanchana, Y. Zhang, Z.A. Khan, et al., Curcumin protects hearts from FFA-induced injury by activating Nrf2 and inactivating NF-kappaB both in vitro and in vivo, J. Mol. Cell. Cardiol. 79 (2015) 1–12. [37] R.K. Thimmulappa, H. Lee, T. Rangasamy, S.P. Reddy, M. Yamamoto, T.W. Kensler, et al., Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis, J. Clin. Invest. 116 (2006) 984–995.
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C
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[28] T. Yu, J. Shim, Y. Yang, S.E. Byeon, J.H. Kim, H.S. Rho, et al., 3-(4-(Tertoctyl)phenoxy)propane-1,2-diol suppresses inflammatory responses via inhibition of multiple kinases, Biochem. Pharmacol. 83 (2012) 1540–1551. [29] K. Ueda, T. Ueyama, K.I. Yoshida, H. Kimura, T. Ito, Y. Shimizu, et al., Adaptive HNE– Nrf2–HO-1 pathway against oxidative stress is associated with acute gastric mucosal lesions, Am. J. Physiol. Gastrointest. Liver Physiol. 295 (2008) G460–G469. [30] C.C. Chen, C.B. Chu, K.J. Liu, C.Y. Huang, J.Y. Chang, W.Y. Pan, et al., Gene expression profiling for analysis acquired oxaliplatin resistant factors in human gastric carcinoma TSGH-S3 cells: the role of IL-6 signaling and Nrf2/AKR1C axis identification, Biochem. Pharmacol. 86 (2013) 872–887. [31] K. Srisook, C. Kim, Y.N. Cha, Molecular mechanisms involved in enhancing HO-1 expression: de-repression by heme and activation by Nrf2, the “one-two” punch, Antioxid. Redox Signal. 7 (2005) 1674–1687. [32] J.L. Ehren, P. Maher, Concurrent regulation of the transcription factors Nrf2 and ATF4 mediates the enhancement of glutathione levels by the flavonoid fisetin, Biochem. Pharmacol. 85 (2013) 1816–1826.
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Please cite this article as: C. Pan, et al., Protective effect of forsythiaside A on lipopolysaccharide/D-galactosamine-induced liver injury, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.03.009
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Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
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Chen-wei Pan a,1, Guang-yao Zhou a,1, Wei-lai Chen b, Lu Zhuge a, Ling-xiang Jin a, Yi Zheng a, Wei Lin a, Zhen-zhen Pan c,⁎
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Article history: Received 11 December 2014 Received in revised form 12 February 2015 Accepted 10 March 2015 Available online xxxx
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Keywords: Forsythiaside Lipopolysaccharide (LPS) Nrf2 NF-κB Acute liver injury
Department of Infectious Disease, The Second Affiliated Hospital & Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, ZheJiang 325027, China Department of Neurology, Wenzhou People's Hospital, Wenzhou, ZheJiang 325027, China Department of Infectious Disease, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, ZheJiang 325000, China
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Forsythiaside A, an active constituent isolated from air-dried fruits of Forsythia suspensa, has been reported to have multiple pharmacological activities including anti-inflammatory, anti-oxidant, and antioxidant activities. In the present study, the hepatoprotective effect of forsythiaside A was investigated in lipopolysaccharide (LPS)/D-galactosamine (GalN)-induced acute liver injury in mice. Mice acute liver injury model was induced by LPS (50 μg/kg)/GalN (800 mg/kg). Forsythiaside A was administrated 1 h prior to LPS/GalN exposure. The results showed that forsythiaside A attenuated hepatic pathological damage, malondialdehyde (MDA) content, and serum ALT, and AST levels induced by LPS/GalN. Moreover, forsythiaside A inhibited NF-κB activation, serum TNF-α and hepatic TNF-α levels induced by LPS/GalN. Furthermore, we found that forsythiaside A upregulated the expression of Nrf2 and heme oxygenase-1. Our results showed that forsythiaside A protected against LPS/GalN-induced liver injury through activation of Nrf2 and inhibition of NF-κB activation. © 2015 Published by Elsevier B.V.
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1. Introduction
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Endotoxemia and sepsis occur frequently in cases of liver failure, which has a high mortality due to the lock of effective drugs [1,2]. Therefore, the development of definitive and targeted drug therapies for hepatic injury is urgently needed. LPS and GalN-induced acute hepatic injury in mice is a widely used model for human hepatic injury [3,4]. It has been used for preliminary pharmacological studies of potential therapeutic drugs and agents [5]. GalN is a hepatotoxic agent and the hepatotoxicity is mainly through inhibiting hepatocyte RNA and protein synthesis [6]. LPS is an endotoxin that could induce inflammatory cytokine production [7]. These pro-inflammatory mediators lead to liver tissue injury [8]. Forsythia suspensa is one of the most famous Chinese herbal medicines that has been widely used to treat various infective diseases, such as nephritis, ulcers, and erysipelas [9,10]. F. suspensa is the main component of traditional Chinese formula Shuang–Huang–Lian [11]. Forsythiaside, an active constituent isolated from air-dried fruits of F. suspensa, has been reported to have anti-inflammatory, antioxidant, and antioxidant activities [12]. Previous studies showed that
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Protective effect of forsythiaside A on lipopolysaccharide/D-galactosamine-induced liver injury
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⁎ Corresponding author. E-mail address: [email protected] (Z. Pan). 1 Chen-wei Pan and Guang-yao Zhou have equal contribution to the paper.
forsythiaside had the ability to protect against hydrogen peroxideinduced apoptosis and oxidative stress in PC12 cell [13]. Furthermore, forsythiaside was found to inhibit LPS-induced acute lung injury in mice [14] and LPS-induced inflammatory responses in the bursa of chickens [15]. In addition, forsythiaside was also found to have neuroprotective effects in senescence-accelerated mouse prone mice [12]. However, the effect of forsythiaside A on LPS/GalN-induced liver injury remains unclear. In this study, we sought to assess the effects of forsythiaside A on LPS/GalN-induced liver injury in mice.
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2. Materials and methods
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Forsythiaside A was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Mouse TNF-α ELISA kit was purchased from R&D Systems (Minneapolis, MN). LPS (Escherichia coli, O55:B5) and D-galactosamine were purchased from Sigma (St. Louis, MO, USA). Anti-pNF-κB p65, anti-NF-κB p65, anti-HO-1, anti-nrf2, and anti-β-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The malondialdehyde (MDA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) detection kits were provided by
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http://dx.doi.org/10.1016/j.intimp.2015.03.009 1567-5769/© 2015 Published by Elsevier B.V.
Please cite this article as: C. Pan, et al., Protective effect of forsythiaside A on lipopolysaccharide/D-galactosamine-induced liver injury, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.03.009
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the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). All other reagents were of analytical grade.
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Male BALB/c mice, weighing approximately 18 to 22 g, were purchased from the Center of Experimental Animals of Zhengzhou University (Zhengzhou, China). The mice were housed in an environmentally controlled room (24 ± 1 °C, 40%–80% humidity). They were allowed free access to food and water. All experimental protocols described in this study were performed in accordance with the guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
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2.3. Experimental design
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The liver injury model was induced by intraperitoneal injection of GalN (800 mg/kg) and LPS (50 μg/kg) dissolved in PBS. To investigate the protective effect of forsythiaside A on LPS/GalN-induced acute liver injury, mice were given with an intraperitoneal injection (i.p.) of forsythiaside A (15, 30, 60 mg/kg) 1 h before LPS/GalN treatment. Seventy-five mice were randomly divided into five groups and each group contained 15 mice: Group I, normal control, the mice received equal volume of PBS. Group II, model control, the mice received GalN (800 mg/kg) and LPS (50 μg/kg). Group III–V, the mice received forsythiaside A (15, 30, 60 mg/kg) 1 h before LPS/GalN treatment. The mice were after LPS/GalN treatment. Then the blood and liver tissues were collected for subsequent analysis.
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Two hours after LPS/GalN treatment, serum and liver tissues were collected for TNF-α detection. Levels of TNF-α in serum and hepatic samples were measured using ELISA kits (R&D) according to the manufacturer's instructions.
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2.5. Analysis of liver enzymes
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Hepatocyte damage was assessed by detecting plasma enzyme activities of ALT and AST. To analyze plasma enzyme activities of ALT and AST, serum was collected 8 h after LPS/GalN injection. ALT and AST levels were measured using test kits purchased from Jiancheng Bioengineering Institute of Nanjing according to the instructions.
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MDA, an end product of lipid peroxidation, was used to assess lipid peroxidation in the liver. Liver tissues were collected 8 h after LPS/ GalN injection and liver MDA content was measured by MDA test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the instructions.
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Liver tissues were harvested at 8 h after LPS/GalN injection. Liver tissues were fixed with 10% buffered formalin, imbedded in paraffin and sliced. After hematoxylin and eosin (H&E) staining, pathological changes of liver tissues were observed under a light microscope.
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Liver tissues were collected 8 h after LPS/GalN injection. Proteins were extracted from the liver using T-PER Tissue Protein Extraction Reagent Kit (Thermo) according to the manufacturer's instructions. Nuclear and cytoplasmic proteins were extracted from the liver using NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo)
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Serum ALT and AST levels were detected 8 h after LPS/GalN treatment. As shown in Fig. 1, ALT and AST levels increased significantly after LPS/GalN treatment. These increases were attenuated by administration of forsythiaside.
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3.2. Effects of forsythiaside A on LPS/GalN-mediated liver histopathologic changes To test protective effect of forsythiaside A on LPS/GalN-induced liver injury in mice, we observed the liver histopathologic changes under a light microscope. As shown in Fig. 2, liver sections of normal control group showed normal lobular architecture and cellular structure. Liver sections obtained from LPS/GalN group showed significant pathologic changes, including extensive areas of portal inflammation, cellular necrosis, and inflammatory cell infiltration. However, these pathological changes were significantly attenuated by forsythiaside.
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All values are expressed as means ± S.E.M. Differences between mean values of normally distributed data were analyzed using oneway ANOVA (Dunnett's t-test) and two-tailed Student's t-test. Statistical significance was accepted p b 0.05 or p b 0.01.
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according to the manufacturer's protocol. Protein concentrations were determined by BCA protein assay kit. Equal amounts of protein were fractionated on 12% polyacrylamide-SDS gel. Then proteins were transferred to polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk for 2 h at room temperature followed by primary antibody (1:1000) overnight at 4 °C. Subsequently, the membrane was treated with horseradish peroxidase (HRP)-conjugated secondary antibody. The immunoactive proteins were detected by using an enhanced chemiluminescence (ECL) Western blotting detection kit.
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Fig. 1. Effects of forsythiaside A on serum ALT and AST levels. The values presented are the Q1 mean ± SEM (n = 15 in each group). #p b 0.01 vs. control group, *p b 0.05, **p b 0.01 vs. LPS/GalN group.
Please cite this article as: C. Pan, et al., Protective effect of forsythiaside A on lipopolysaccharide/D-galactosamine-induced liver injury, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.03.009
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Fig. 2. Effects of forsythiaside A on histopathological changes in liver tissues. Representative histological changes of liver obtained from mice of different groups. A: Control group, B: LPS/ GalN group, C: LPS/GalN + forsythiaside A (15 mg/kg) group, D: LPS/GalN + forsythiaside A (30 mg/kg) group E: LPS/GalN + forsythiaside A (60 mg/kg) group (hematoxylin and eosin staining, magnification 200×).
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Levels of TNF-α in serum and hepatic samples were detected by ELISA. The results showed that TNF-α level in serum and hepatic samples was increased significantly after LPS/GalN injection. However, forsythiaside A inhibited TNF-α production induced by LPS/GalN in a dose dependent manner (Fig. 3).
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Malondialdehyde (MDA), the end product of lipid peroxidation, is known as a marker of oxidative stress. Liver MDA level was detected 8 h after LPS/GalN injection. As shown in Fig. 4, LPS/GalN challenge produced a significant increase of liver MDA level. However, administrations of forsythiaside A significantly reduced the increase of liver MDA level (Fig. 4).
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3.5. Effect of forsythiaside A on NF-κB activation
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NF-κB has been reported to be a dominant transcription factor re- 170 sponsible for inflammation [16]. Activation of NF-κB by LPS could in- 171 duce the production of cytokine [17]. In this study we detected the 172 effects of forsythiaside A on NF-κB activation. LPS/GalN-treated mice 173 displayed that p65 subunit of NF-κB increased in nucleus and decreased 174 in cytoplasm, and the tendency was reversed by forsythiaside A (Fig. 5). 175
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3.4. Effects of forsythiaside A on lipid peroxidation
Fig. 3. Effects of forsythiaside A on serum and hepatic TNF-α levels. The values presented are the means ± SEM (n = 15 in each group). #p b 0.01 vs. control group, *p b 0.05 and **p b 0.01 vs. LPS/GalN group.
Fig. 4. Effects of forsythiaside A on liver MDA level. The values presented are the mean ± Q2 SEM (n = 15 in each group). #p b 0.01 vs. control group, *p b 0.05, **p b 0.01 vs. LPS/GalN group.
Please cite this article as: C. Pan, et al., Protective effect of forsythiaside A on lipopolysaccharide/D-galactosamine-induced liver injury, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.03.009
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Fig. 5. Forsythiaside A inhibited LPS/GalN-induced NF-κB activation with Western blotting. The values presented are the mean ± SEM (n = 15 in each group). #p b 0.01 vs. control group, *p b 0.05 and **p b 0.01 group vs. LPS group.
3.6. Forsythiaside A promoted the expression of Nrf2 and HO-1
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Nrf-2 has been reported to play an important role in orchestrating cellular antioxidant defenses. In this study, we detected the effects of
forsythiaside A on Nrf2 and HO-1 expression. The results showed that Nrf2 in nuclear and HO-1 in cytosolic were upregulated at 8 h after LPS/GalN injection. These increases in Nrf2 and HO-1 expression were augmented by forsythiaside A (Fig. 6).
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Fig. 6. Effects of forsythiaside A on the expression of Nrf2 and HO-1. The values presented are the mean ± SEM (n = 15 in each group). #p b 0.01 vs. control group, *p b 0.05 and **p b 0.01 group vs. LPS group.
Please cite this article as: C. Pan, et al., Protective effect of forsythiaside A on lipopolysaccharide/D-galactosamine-induced liver injury, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.03.009
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[1] B.G. Harbrecht, T.R. Billiar, J. Stadler, A.J. Demetris, J.B. Ochoa, R.D. Curran, et al., Nitric oxide synthesis serves to reduce hepatic damage during acute murine endotoxemia, Crit. Care Med. 20 (1992) 1568–1574. [2] J. Belghiti, K. Hiramatsu, S. Benoist, P. Massault, A. Sauvanet, O. Farges, Seven hundred forty-seven hepatectomies in the 1990s: an update to evaluate the actual risk of liver resection, J. Am. Coll. Surg. 191 (2000) 38–46. [3] L. Zhang, H.Z. Li, X. Gong, F.L. Luo, B. Wang, N. Hu, et al., Protective effects of Asiaticoside on acute liver injury induced by lipopolysaccharide/D-galactosamine in mice, Phytomedicine: Int. J. Phytother. Phytopharmacology 17 (2010) 811–819. [4] T. Nakama, S. Hirono, A. Moriuchi, S. Hasuike, K. Nagata, T. Hori, et al., Etoposide prevents apoptosis in mouse liver with D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure resulting in reduction of lethality, Hepatology 33 (2001) 1441–1450. [5] B. Feng, S. Wu, S. Lv, F. Liu, H. Chen, X. Yan, et al., Metabolic profiling analysis of a Dgalactosamine/lipopolysaccharide-induced mouse model of fulminant hepatic failure, J. Proteome Res. 6 (2007) 2161–2167. [6] A. Sathivel, Balavinayagamani, B.R. Hanumantha Rao, T. Devaki, Sulfated polysaccharide isolated from Ulva lactuca attenuates d-galactosamine induced DNA fragmentation and necrosis during liver damage in rats, Pharm. Biol. (2013). [7] L. Haversen, B.G. Ohlsson, M. Hahn-Zoric, L.A. Hanson, I. Mattsby-Baltzer, Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NFkappa B, Cell. Immunol. 220 (2002) 83–95. [8] T. Masaki, S. Chiba, H. Tatsukawa, T. Yasuda, H. Noguchi, M. Seike, et al., Adiponectin protects LPS-induced liver injury through modulation of TNF-alpha in KK-Ay obese mice, Hepatology 40 (2004) 177–184. [9] H. Qu, Y. Zhang, Y. Wang, B. Li, W. Sun, Antioxidant and antibacterial activity of two compounds (forsythiaside and forsythin) isolated from Forsythia suspensa, J. Pharm. Pharmacol. 60 (2008) 261–266. [10] Y. Ozaki, J. Rui, Y.T. Tang, Antiinflammatory effect of Forsythia suspensa V(AHL) and its active principle, Biol. Pharm. Bull. 23 (2000) 365–367. [11] W. Zhou, L.Q. Di, J.J. Shan, X.L. Bi, L.T. Chen, L.C. Wang, et al., In vitro metabolism in Sprague–Dawley rat liver microsomes of forsythoside A in different compositions of Shuang–Huang–Lian, Fitoterapia 82 (2011) 1222–1230. [12] H.M. Wang, L.W. Wang, X.M. Liu, C.L. Li, S.P. Xu, A.D. Farooq, Neuroprotective effects of forsythiaside on learning and memory deficits in senescence-accelerated mouse prone (SAMP8) mice, Pharmacol. Biochem. Behav. 105 (2013) 134–141. [13] C. Huang, Y. Lin, H. Su, D. Ye, Forsythiaside protects against hydrogen peroxideinduced oxidative stress and apoptosis in PC12 cell, Neurochem. Res. (2014). [14] L. Zhou, H. Yang, Y. Ai, Y. Xie, Y. Fu, Protective effect of forsythiaside A on acute lung injure induced by lipopolysaccharide in mice, Cell. Mol. Immunol. (Xi bao yu fen zi mian yi xue za zhi), 30 (2014) 151–154. [15] G. Cheng, Y. Zhao, H. Li, Y. Wu, X. Li, Q. Han, et al., Forsythiaside attenuates lipopolysaccharide-induced inflammatory responses in the bursa of Fabricius of chickens by downregulating the NF-kappaB signaling pathway, Exp. Ther. Med. 7 (2014) 179–184. [16] N.D. Perkins, Integrating cell-signalling pathways with NF-kappaB and IKK function, Nat. Rev. Mol. Cell Biol. 8 (2007) 49–62. [17] L. Haversen, B.G. Ohlsson, M. Hahn-Zoric, L.A. Hanson, I. Mattsby-Baltzer, Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NFkappa B, Cell. Immunol. 220 (2002) 83–95. [18] A.J. Hanley, K. Williams, A. Festa, L.E. Wagenknecht, R.B. D'Agostino Jr., J. Kempf, et al., Elevations in markers of liver injury and risk of type 2 diabetes: the insulin resistance atherosclerosis study, Diabetes 53 (2004) 2623–2632. [19] R.F. Schwabe, D.A. Brenner, Mechanisms of liver injury. I. TNF-alpha-induced liver injury: role of IKK, JNK, and ROS pathways, Am. J. Physiol. Gastrointest. Liver Physiol. 290 (2006) G583–G589. [20] W.X. Ding, X.M. Yin, Dissection of the multiple mechanisms of TNF-alpha-induced apoptosis in liver injury, J. Cell. Mol. Med. 8 (2004) 445–454. [21] G. Ramesh, W.B. Reeves, TNF-alpha mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity, J. Clin. Invest. 110 (2002) 835–842. [22] C. Ji, Q. Deng, N. Kaplowitz, Role of TNF-alpha in ethanol-induced hyperhomocysteinemia and murine alcoholic liver injury, Hepatology 40 (2004) 442–451. [23] N. Enomoto, Y. Takei, M. Hirose, K. Ikejima, H. Miwa, T. Kitamura, et al., Thalidomide prevents alcoholic liver injury in rats through suppression of Kupffer cell sensitization and TNF-alpha production, Gastroenterology 123 (2002) 291–300. [24] Q. Li, I.M. Verma, NF-kappaB regulation in the immune system, Nat. Rev. Immunol. 2 (2002) 725–734. [25] D. Avni, Y. Glucksam, T. Zor, The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 modulates cytokine expression in macrophages via p50 nuclear factor kappaB inhibition, in a PI3K-independent mechanism, Biochem. Pharmacol. 83 (2012) 106–114. [26] E.H. Joh, I.A. Lee, I.H. Jung, D.H. Kim, Ginsenoside Rb1 and its metabolite compound K inhibit IRAK-1 activation—the key step of inflammation, Biochem. Pharmacol. 82 (2011) 278–286. [27] T.D. Gilmore, Introduction to NF-kappaB: players, pathways, perspectives, Oncogene 25 (2006) 6680–6684.
254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 Q8 271 272 Q9 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 Q10 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329
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References
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C
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Forsythiaside, an active constituent isolated from air-dried fruits of F. suspensa, has been reported to have anti-inflammatory, antioxidant, and antioxidant activities [12]. In the present study, we observed the effects of forsythiaside A on LPS/GalN-induced acute liver injury in mice. The results showed that forsythiaside A attenuated hepatic pathological damage, MDA content, and serum ALT, and AST levels induced by LPS/GalN. Moreover, forsythiaside A inhibited NF-κB activation, serum and hepatic TNF-α levels induced by LPS/ GalN. Furthermore, we found that forsythiaside A up-regulated the expression of Nrf2 and heme oxygenase-1. Forsythiaside A may be a promising potential therapeutic reagent for acute liver injury treatment. Serum ALT and AST were used as biochemical indicator of hepatic injury [18]. In the present study, serum ALT, AST and histological analysis of liver tissues were used to assess the protective effect of forsythiaside A on LPS/GalN-induced acute liver injury. The results showed that forsythiaside A significantly decreased serum ALT and AST levels. Meanwhile, histological analysis demonstrated that forsythiaside A could attenuate cellular necrosis, and inflammatory cell infiltration. Forsythiaside A significantly attenuated liver injury. These results suggested that forsythiaside A had a protective effect on LPS/GalN-induced hepatic injury. TNF-α has been demonstrated to play vital role in the pathogenesis of liver injury [19]. It could induce hepatocyte necrosis and organ failure and contribute to the severity of liver injury [20]. Meanwhile, TNF-α can induce other cytokine production [21]. These cytokines amplify and perpetuate the inflammatory response. Elevated TNF-α level was observed in serum and liver tissues [22]. Furthermore, previous reports demonstrated that inhibition of TNF-α could alleviate liver injury [23]. Thus, serum and liver TNF-α levels were detected in this study. The results showed that serum and liver TNF-α levels were dramatically inhibited by forsythiaside. MDA, the last product of lipid breakdown caused by oxidative stress, is used as a marker of oxidative stress. The results of this study showed that forsythiaside A significantly inhibited liver MDA level induced by LPS/GalN. The results suggested that forsythiaside A protected against LPS/GalN-induced liver injury by reducing inflammatory and oxidative response. NF-κB is a pleiotropic regulator of various genes which is involved in the inflammatory responses [24,25]. Normally, NF-κB is sequestered in the cytoplasm by a family of inhibitory proteins known as inhibitors of NF-κB (IκBs) [26]. Once stimulated with LPS, IκB is phosphorylated and degraded leading to the release of free NF-κB. Then NF-κB enters the nucleus and regulates the expression of inflammatory cytokines [27,28]. To characterize the inhibitory effect of forsythiaside A on cytokines production, we examined the effects of forsythiaside A on NF-κB activation and IκB-α degradation. The results showed that forsythiaside A inhibited NF-κB activation in a dose-dependent manner. Nrf2 is an important transcription factor that plays an important role in orchestrating cellular antioxidant defenses by regulating antioxidant enzymes expression [29,30]. Nrf2 controls the coordinated expression of important antioxidant and detoxification genes such as HO-1 [31, 32]. HO-1 is a ubiquitous and redox-sensitive inducible stress protein that has been reported to have antioxidant activity [33,34]. Our results showed that Nrf2 in nuclear and HO-1 in cytosolic were upregulated at 1 h after LPS/GalN injection. However, the increases in Nrf2 and HO-1 expression were augmented by forsythiaside. The results suggested that forsythiaside A could decrease oxidative stress induced by LPS/GalN by inducing oxidative defense system. Meanwhile, previous studies indicated that there was an interaction between Nrf2 and NFκB [35]. Activation of Nrf2 could inhibit NF-κB signaling pathway [36] and other studies demonstrated that Nrf2-deficient mice showed increased NF-κB activation in response to LPS stimulation [37]. Thus, the inhibition of NF-κB activation by forsythiaside A may be due to the activation of Nrf2 by forsythiaside.
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In conclusion, our results demonstrated that forsythiaside A had a protective effect on LPS/GalN-induced liver injury. The mechanisms underlying this protective effect may be related to inhibition of NF-κB activation and activation of Nrf2. Forsythiaside A may be an agent for preventing and treating LPS/GalN-induced liver injury.
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[33] S. Turkseven, A. Kruger, C.J. Mingone, P. Kaminski, M. Inaba, L.F. Rodella, et al., Antioxidant mechanism of heme oxygenase-1 involves an increase in superoxide dismutase and catalase in experimental diabetes, Am. J. Physiol. Heart Circ. Physiol. 289 (2005) H701–H707. [34] C.F. Chang, X.M. Liu, K.J. Peyton, W. Durante, Heme oxygenase-1 counteracts contrast media-induced endothelial cell dysfunction, Biochem. Pharmacol. 87 (2014) 303–311. [35] J. Rao, X. Qian, G. Li, X. Pan, C. Zhang, F. Zhang, et al., ATF3-mediated NRF2/HO-1 signaling regulates TLR4 innate immune responses in mouse liver ischemia/reperfusion injury, Am. J. Transplant. 15 (2015) 76–87. [36] C. Zeng, P. Zhong, Y. Zhao, K. Kanchana, Y. Zhang, Z.A. Khan, et al., Curcumin protects hearts from FFA-induced injury by activating Nrf2 and inactivating NF-kappaB both in vitro and in vivo, J. Mol. Cell. Cardiol. 79 (2015) 1–12. [37] R.K. Thimmulappa, H. Lee, T. Rangasamy, S.P. Reddy, M. Yamamoto, T.W. Kensler, et al., Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis, J. Clin. Invest. 116 (2006) 984–995.
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[28] T. Yu, J. Shim, Y. Yang, S.E. Byeon, J.H. Kim, H.S. Rho, et al., 3-(4-(Tertoctyl)phenoxy)propane-1,2-diol suppresses inflammatory responses via inhibition of multiple kinases, Biochem. Pharmacol. 83 (2012) 1540–1551. [29] K. Ueda, T. Ueyama, K.I. Yoshida, H. Kimura, T. Ito, Y. Shimizu, et al., Adaptive HNE– Nrf2–HO-1 pathway against oxidative stress is associated with acute gastric mucosal lesions, Am. J. Physiol. Gastrointest. Liver Physiol. 295 (2008) G460–G469. [30] C.C. Chen, C.B. Chu, K.J. Liu, C.Y. Huang, J.Y. Chang, W.Y. Pan, et al., Gene expression profiling for analysis acquired oxaliplatin resistant factors in human gastric carcinoma TSGH-S3 cells: the role of IL-6 signaling and Nrf2/AKR1C axis identification, Biochem. Pharmacol. 86 (2013) 872–887. [31] K. Srisook, C. Kim, Y.N. Cha, Molecular mechanisms involved in enhancing HO-1 expression: de-repression by heme and activation by Nrf2, the “one-two” punch, Antioxid. Redox Signal. 7 (2005) 1674–1687. [32] J.L. Ehren, P. Maher, Concurrent regulation of the transcription factors Nrf2 and ATF4 mediates the enhancement of glutathione levels by the flavonoid fisetin, Biochem. Pharmacol. 85 (2013) 1816–1826.
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Please cite this article as: C. Pan, et al., Protective effect of forsythiaside A on lipopolysaccharide/D-galactosamine-induced liver injury, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.03.009
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