BASIC
AND
EXPERIMENTAL RESEARCH
Dioscin Attenuates Hepatic Ischemia-Reperfusion Injury in Rats Through Inhibition of Oxidative-Nitrative Stress, Inflammation and Apoptosis Xufeng Tao,1 Xianyao Wan,2 Youwei Xu,1 Lina Xu,1 Yan Qi,1 Lianhong Yin,1 Xu Han,1 Yuan Lin,1 and Jinyong Peng1,3,4 Background. Dioscin shows potent effects against liver damage in our previous studies; however, the action of it on hepatic ischemia-reperfusion (I/R) injury is still unknown. In the present article, the effects and possible mechanisms of dioscin against hepatic I/R injury were investigated. Methods. Seventy percent partial hepatic warm ischemia was induced in Wistar rats for 60 min followed by succedent reperfusion. In the prophylactic test, dioscin was administered intragastrically to the rats at doses of 20, 40, and 60 mg/kg once daily for seven consecutive days before I/R. In the therapeutic test, the rats received dioscin intragastrically at a dose of 60 mg/kg once 2 hr before I/R. Results. We found that dioscin significantly decreased serum alanine aminotransferase and aspartate aminotransferase activities, increased survival rate of rats, and improved I/R-induced hepatocyte abnormality. In addition, dioscin obviously increased the levels of SOD, CAT, GSH-Px, GSH, decreased the levels of MDA, TNOS, iNOS, NO, and prevented DNA fragmentation caused by I/R injury. Further research indicated that dioscin markedly decreased the gene expressions of interleukin-1A, interleukin-6, tumor necrosis factor->, intercellular adhesion molecule-1, MIP-1>, MIP-2, Fas, FasL, decreased the protein expressions of NF-JB, AP-1, COX-2, HMGB-1, CYP2E1, Bak, caspase-3, p53, PARP, Caspase-9, decreased the levels of JNK, ERK and p38 MAPKs phosphorylation, and upregulated the levels of Bcl-2 and Bcl-x. Conclusion. Our results suggest that dioscin has potent actions against hepatic I/R injury through suppression of inflammation, oxidative-nitrative stress, and apoptosis, which should be developed as a new drug to treat hepatic I/R injury in the future. Keywords: Apoptosis, Dioscin, Hepatic I/R injury, Inflammation, Natural product. (Transplantation 2014;98: 604Y611)
schemia-reperfusion (I/R) is an important cause of tissue damage in pathologic conditions including surgical procedures, stroke, vascular surgery, and shocks (1, 2). Orthotopic liver transplantation has been considered as the well-acknowledged curative approach for patients with endstage liver disease (3). However, hepatic I/R injury causes up to 10% of early failures of liver transplantation and higher incidence of acute and chronic rejections (4).
I
This work was financially supported by National Natural Science Foundation of China (no. 81274195), the Program for Liaoning Innovative Research Team in University (LT2013019) and the Program for New Century Excellent Talents in University (NCET-11-1007). The authors declare no conflicts of interest. 1 College of Pharmacy, Dalian Medical University, Liaoning Province, China. 2 Department of Critical Care Medicine of the First Affiliated Hospital of Dalian Medical University, Dalian, China. 3 Research Institute of Integrated Traditional and Western Medicine of Dalian Medical University, Dalian, China. 4 Address correspondence to: Jinyong Peng, M.D., College of Pharmacy, Dalian Medical University, Dalian, China. E-mail: [email protected] X.T. participated in the research design, performance of the research, data analysis, and the article. X.W. participated in the research design and
604
www.transplantjournal.com
The procedure of ischemia results from temporarily blood flow deprivation, which can lead to oxygen and glycogen supporting cellular homeostasis to be insufficient (5). Kupffer cells can produce a large number of reactive oxygen species (ROS), reactive nitrogen species (RNS), and proinflammatory cytokines when they are activated during I/R injury (6). These substances can cause a chain of deleterious cellular responses, resulting in increased inflammatory mediators and elevations of apoptotic molecules, which eventually cause organ failure (7, 8). commented on the draft. Y.X. and L.X. performed the research and commented on the draft. Y.Q., L.Y., and X.H. designed and supervised the research; Y.L. is the discussant and advisor. J.P. is the overall adviser, facilitated the final article version, and sponsored the project. Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com). Received 13 March 2014. Revision requested 28 March 2014. Accepted 22 April 2014. Copyright * 2014 by Lippincott Williams & Wilkins ISSN: 0041-1337/14/9806-604 DOI: 10.1097/TP.0000000000000262
Transplantation
& Volume 98, Number 6, September 27, 2014
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tao et al.
* 2014 Lippincott Williams & Wilkins
Up to now, there are no ideal drugs to treat I/R injury owing to the perplexing pathologic process, and thus it is urgent to develop new and efficient drugs for the treatment of this disease. Some active components including riboflavin (9), green tea polyphenols (10), and ligustrazine (11) from medicinal plants have shown excellent activities against I/R injury. Thus, it is reasonable to exploit effective natural products from herbs for the treatment of hepatic I/R injury. Dioscin, a natural product, is derived from some medicinal plants including Discorea nipponica Makino and Dioscorea zingiberensis C.H. Wright. (12, 13). Our previous investigations have demonstrated that dioscin has potent effects against acetaminophen-induced, CCl4induced, and ethanol-induced liver damages (14Y16). However, to the best of our knowledge, there have been no articles to report the effect of dioscin against hepatic I/R injury. The aim of the present article was to clarify the effect and possible mechanism of dioscin against hepatic I/R injury by using an in vivo lethal hepatic failure rat model. Our findings showed that dioscin has a potential activity for prevention and treatment of hepatic I/R injury, which should be developed as a new drug to treat this disease in the future.
605
RESULTS Effects of Dioscin Against I/R-Induced Liver Injury Hematoxylin-eosinYstained sections are shown in Figure 1(A). The I/R-induced coagulation necrosis with massive inflammatory cells infiltration in liver was clearly observed, which was dramatically attenuated by dioscin with a dose-dependent manner. Furthermore, the increased serum alanine aminotransferase (ALT) (818T64 U/L) and aspartate aminotransferase (AST) (561T83 U/L) activities caused by I/R, shown in Figure 1(B) , were significantly prevented by 40 and 60 mg/kg of dioscin pretreatment. Effects of Dioscin on Survival of Rats and Continuous ALT-AST Activities As shown in Figure 1(C), after 1-hr ischemia and 4 days reperfusion, only did 10% of rats survive in the model group, and treatment with dioscin at the dose of 60 mg/kg markedly improved the survival rate of the animals to 50%. Furthermore, the actions of dioscin on serum ALT and AST activities are shown in Figure 1(D). After 1 hr of ischemia and different times of reperfusion, severe liver damage was significantly attenuated by dioscin.
FIGURE 1. (A) H&E staining of representative liver sections (magnification, 100). (B) Serum ALT, AST activities of rats in the prophylactic test. (C) Rat survival rate. (D) Serum ALT, AST activities of rats in the therapeutic test. H&E staining showed the injured areas. Values expressed as meanTSD (nQ6). *PG0.05 and **PG0.01 compared with the model group. H&E, hematoxylin-eosin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; SD, standard deviation.
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
606
www.transplantjournal.com
Dioscin Attenuated I/R-Induced Oxidative and Nitrative Stress In I/R-treated group, the levels of superoxide dismutase (SOD) (181T20 U/mg prot), catalase (CAT) (458T56 U/g prot), glutathione peroxidase (GSH-Px) (845T89 U/mg prot), and glutathione (GSH) (0.57T0.08 U/mg prot) significantly decreased compared with sham rats. However, dioscin significantly increased their levels in a dose-dependent manner (Fig. 2A). In addition, the increased levels of malondialdehyde (MDA) (3.4T1.1 nmol/mg prot), total nitric oxide synthase (TNOS) (0.83T0.26 U/mg prot), inducible nitric oxide synthase (iNOS) (0.74T0.13 U/mg prot), and nitrogen monoxide (NO) (0.20T0.06 Kmol/g prot) were observed in I/R rats, which were significantly decreased by dioscin (Fig. 2B). Effects of Dioscin on Nuclear Injury As shown in Figure 2(C), the hepatocyte nuclei in the normal group were round with uniform chromatin and emitted even blue fluorescence based on 4¶,6-diamidino-2phenylindole (DAPI) assay while they were condensed, and a lot of particulate matters were observed in I/R-treated
Transplantation
& Volume 98, Number 6, September 27, 2014
group, which were lightened by diocsin at a dose of 60 mg/kg. In addition, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining was conducted to identify apoptotic cells among the injured hepatocytes, and more TUNEL-positive cells showed green fluorescence and brown were observed in I/R-treated group than those in the dioscin-treated group (60 mg/kg). These results indicated that dioscin had the potential activity to attenuate I/R-induced apoptosis. Effects of Dioscin on the Expressions of I/R-Induced Inflammatory Mediators In the present article, the messenger RNA (mRNA) expressions of cytokines interleukin (IL)-1A, IL-6, tumor necrosis factor (TNF)->, adhesion molecule intercellular adhesion molecule (ICAM)-1, and chemokines macrophage inflammatory protein-1> (MIP-1>) and MIP-2 (Fig. 3A), and the expressions of the proteins including nuclear factor JB (NF-JB), AP-1, COX-2, HMGB-1 (Fig. 3B) in I/R-induced liver tissues were significantly increased compared with the sham group. However, dioscin pretreatment markedly attenuated the
FIGURE 2. A and B, effects of the dioscin on the levels of SOD, CAT, GSH-Px, GSH, MDA, TNOS, iNOS, and NO in rat livers. C, representative micrographs of DAPI stained nuclei and TUNEL-stained liver sections (magnification, 200). Fragmentation or condensation of nucleus in the liver tissues was shown in DAPI-stained sections. In fluorescent images, the green fluorescence indicated the positive cells. In DAB&H-stained images, the brown staining represented the positive areas of TUNEL-stained sections. Values expressed as meanTSD (nQ6). *PG0.05 and **PG0.01 compared with the model group. DAB&H, 3,3¶-diaminobenzidine tetrahydrochloride and hematoxylin; TUNEL, deoxynucleotide transferase-mediated dUTP nick-end labeling; SD, standard deviation; DAPI, 4¶,6-diamidino-2-phenylindole; SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; GSH, glutathione; MDA, malondi-aldehyde; TNOS, total nitric oxide synthase; iNOS, inducible nitric oxide synthase; NO, nitrogen monoxide.
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
* 2014 Lippincott Williams & Wilkins
Tao et al.
607
FIGURE 3. A, effects of dioscin on the gene expressions of IL-1, IL-6, TNF->, ICAM-1, MIP-1> and MIP-2. B, effects of dioscin on the protein expressions of NF-JB, AP-1, COX2 and HMGB-1. C, effects of dioscin on the expressions of HMGB-1, PARP and caspase-9 with the immunohistochemical and Immunofluorescent method (magnification, 200). Values expressed as meanTSD (nQ6). *PG0.05 and **PG0.01 compared with the model group. IL, interleukin; SD, standard deviation; TNF, tumor necrosis factor; ICAM-1, intercellular adhesion molecule 1; MIP-1>, macrophage inflammatory protein-1>; MIP2, macrophage inflammatory protein-2; NF-JB, nuclear factor JB; PARP, Poly(adenosine diphosphate-ribose) polymerase.
elevations of these gene and protein expressions. Furthermore, the protein expression of HMGB-1 (Fig. 3C) was considerably upregulated in the I/R group, which was significantly decreased by dioscin. Effects of Dioscin on the Expressions of Apoptotic Molecules and CYP2E1 In the present work, the expressions of Poly(adenosine diphosphate-ribose) polymerase (PARP) and caspase-9 (Fig. 3C) were considerably increased in the I/R group, which were significantly decreased by dioscin at the dose of 60 mg/kg. As shown in Figure 4(A) and (B), compared with model group, the expressions of CYP2E1, Bak, caspase-3, and p53 were obviously decreased, whereas the expressions Bcl-2 and Bcl-x were significantly upregulated by dioscin. In addition, I/R injury obviously increased the mRNA expressions of Fas and Fas ligand (FasL) (Fig. 4C) compared with the sham group, which were significantly inhibited by dioscin. Effects of Dioscin on the Levels of MAPKs Phosphorylation The effects of dioscin on the levels of mitogen-activated protein kinases (MAPKs) phosphorylation were tested in the present work. As shown in Figure 5(A), the increased levels of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK phosphorylation in the model group were found, which were all significantly attenuated by dioscin.
DISCUSSION Hepatic I/R injury is a frequent complication implicated in liver failure associated with liver transplantation, shock,
stroke, and vascular surgery (1, 2). Dioscin is a natural product that shows anti-apoptosis and anti-inflammatory effects in our previous tests (14Y16). In the present work, rat hepatic I/R, a well-established experimental model, was confirmed by the increased serum ALT and AST activities as well as the histopathologic examination, and dioscin considerably reversed the alternations and protected hepatocytes through attenuating the inflammatory cells infiltration and coagulation necrosis in the liver. These results showed that dioscin has potent effects for the prevention and treatment of hepatic I/R damage in rats. Hepatic injury occurs during the ischemic phase, and much damage arises in reperfusion phase, which is characterized by a state of oxidative-nitrative stress (17). In detail, the oxidative-nitrative stress will happen when the endogenous antioxidant defense system fails to effectively counteract the toxic effects of ROS and RNS (18). Furthermore, in the pathogenesis of I/R liver injury, producing proinflammatory cytokines can lead to intense inflammatory reaction (19), and complex cellular events can cause necrosis and apoptosis of liver cells (20). Antioxidant defense system aims to support endogenous antioxidants and inhibit ROS generation in liver I/R process. High levels of the enzymes including SOD, CAT, GSH, and GSH-Px can protect hepatic I/R injury (21). In addition, excessive production of oxygen-free radicals can increase the level of MDA, which is one production of lipid peroxidation and a well-known indicator of ROS (22). NO, a well-known indicator of the formation of RNS, is a toxic substance that can induce nitration of some proteins (23) and can be produced by iNOS-TNOS. The present study
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
608
www.transplantjournal.com
Transplantation
& Volume 98, Number 6, September 27, 2014
FIGURE 4. A and B, Effects of dioscin on the protein expressions of CYP2E1, Bak, Caspase -3, p53, Bcl-2, Bcl-x; (C) Effects of dioscin on gene expressions of Fas, FasL. Values expressed as meanTSD (n=6). *PG0.05 and **PG0.01 compared with the model group. SD, standard deviation.
showed that the levels of SOD, CAT, GSH-Px, and GSH were significantly increased in the liver after dioscin pretreatment compared with the model group, and the levels of MDA, iNOS, TNOS, and NO were dramatically reduced. All of these indicated that the dioscin showed prominent antioxidant activity against I/R-induced oxidative-nitrative liver damage. CYP2E1, one P450 enzyme, plays an important role in the generation of free radicals (24). Our data showed that pretreatment with dioscin significantly decreased CYP2E1 expression in rat, which was an important aspect of the hepatoprotective effect of dioscin against I/R-induced liver damage. After reperfusion, inflammatory reaction has happened after the release of proinflammatory chemokines and cytokines including interleukin (IL)-1A, IL-6, tumor necrosis factor (TNF)->, MIP-1>, and MIP-2 (7, 8). Subsequently, cell surface
adhesion molecule, ICAM-1, regulated by the proinflammatory transcription factor NF-JB, can be expressed in hepatocytes and neutrophils (25). Furthermore, COX-2, one highly inducible enzyme and a known inflammatory early-response protein (26), is activated by a specific subset of proinflammatory cytokines, which can also be secreted through stimulating HMGB-1 (27). In the current study, we found that dioscin dramatically decreased the mRNA levels of IL-1A, IL-6, TNF->, MIP-1>, MIP-2 and ICAM-1, and the protein expressions of COX-2 and HMGB-1. Further exploration showed that dioscin reduced the activations of NF-JB and AP-1 which are also the key regulators of some genes involved in inflammation (28). These findings indicated that the activity of dioscin against hepatic I/R damage may be through attenuating inflammation. Complex cellular events can cause necrosis and apoptosis of liver cells in acute liver injury induced by I/R,
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tao et al.
* 2014 Lippincott Williams & Wilkins
609
FIGURE 5. A, effects of dioscin on the levels of MAPKs phosphorylation. B, experimental protocol for the prophylactic test. C, experimental protocol for the therapeutic test. Values expressed as meanTSD (n=6). *PG0.05 and **PG0.01 compared with the model group. SD, standard deviation; MAPKs, mitogen-activated protein kinases.
which can make hepatocyte sensitized to apoptosis mediated by Fas-FasL (29). Fas-FasL is the upstream regulator of caspase-3, and the activation of caspase-3 can promote the upregulation of PARP (30). Activation of p53 plays a crucial role in I/R-induced hepatocyte apoptosis, and p53 is known to promote apoptosis by upregulating the expression of apoptotic proteins including caspase-9, caspase-3, PARP, Bax, and decreasing the antiapoptotic proteins including Bcl-2 and Bcl-x (31). In the present article, we found that dioscin was helpful to maintain the normal cellular structure and function against I/R-induced hepatocyte apoptosis. Furthermore, dioscin significantly decreased the protein expressions of PARP, Bak, caspase-3, p53, and caspase-9, and the mRNA expressions of Fas and FasL, and also increased the levels of Bcl-2 and Bcl-x. These findings indicated that the protection of dioscin against hepatic I/R damage may be accomplished through reducing apoptosis. MAPKs signaling pathway is closely related with inflammation and apoptosis during hepatic I/R injury (8). Concretely, because of a variety of physiologic and pathophysiologic stresses, phosphorylation levels of p38, JNK, and ERK are activated. Furthermore, activations of phosphorylated p38, JNK, and ERK advance stress-induced cell death and regulate downstream signaling events relevant to inflammation and apoptosis (32Y35). In the present study, we found that administration of dioscin suppressed the levels of phosphorylated p38, JNK, and ERK. These findings suggested that the activity of dioscin against I/R-induced hepatic damage may be through affecting MAPKs pathway. In conclusion, dioscin has a good protective effect against hepatic I/R injury in rats through attenuating oxidative-nitrative stress, inflammation, and apoptosis, which should be developed as a new and potent candidate for treatment of I/R-induced liver injury in the future. Of
course, mechanisms, drug-target, and clinical applications of dioscin are needed further investigations.
MATERIALS AND METHODS Chemicals and Materials Dioscin with a purity greater than 98% was purchased from the National Institutes of Food and Drug Control of China (Beijing, China). ALT, AST, CAT, SOD, GSH, GSH-Px, MDA, TNOS, iNOS, and NO kits were obtained from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Tissue Protein Extraction Kit was obtained from KEYGEN Biotech. Co., Ltd. (Nanjing, China). Bicinchoninic acid protein assay kit was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). 3,3¶Diaminobenzidine (DAB) substrate kit was purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). The DAPI was purchased from Sigma Chemical Co. (St. Louis, MO). In situ cell death detection kit was provided by Roche Diagnostics (Germany). RNAiso Plus, PrimeScript RT reagent Kit with gDNA Eraser (perfect real time) and SYBR Premix Ex Taq II (Tli RNaseH Plus) were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China).
Experimental Animals Male Wistar rats (180Y220 g) were purchased from the Experimental Animal Center of Dalian Medical University (Dalian, China) (SCXK: 2008-0002). All experimental procedures were approved by the Animal Care and Use Committee of Dalian Medical University and performed in strict accordance with the PR China Legislation Regarding the Use and Care of Laboratory Animals.
Experimental Design The animals were allowed to adapt to the environment for 1 week before the experiments. As shown in Figure 5(B), in the prophylactic test, dioscin was administered intragastrically to the animals at the doses of 20, 40, and 60 mg/kg once daily for seven consecutive days. The rats in the sham operation and model groups were administered with vehicle. On the eighth day, the murine model of 70% partial hepatic ischemia was established as
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
610
www.transplantjournal.com
described previously (21, 36). Briefly, the rats were anesthetized, and the livers were exposed by midline laparotomy, then the inflow of the left lateral and median lobes of the livers was choked by placing a bulldog clamp, whereas the right lobes were remainedly perfused to prevent intestinal congestion occlusion. After 60 min of hepatic ischemic, the bulldog clamp was removed, and the liver was reperfused for 6 hr. At the end of the surgery, the animals were anesthetized for collecting the blood and then killed to collect the left lateral and middle lobes of the livers. As shown in Figure 5(C), in the therapeutic test, the rats were randomized into three groups, of which the animals in sham and model groups were given vehicle, and the rats in dioscin group received dioscin intragastrically at a dose 60 mg/kg once 2 hr before I/R. After 60 min of hepatic ischemic, the bulldog clamp was removed, and the orbital blood samples were collected at 0.5, 1, 2, 4, 6, 12, 24, 48, and 96 hr after reperfusion (34). In addition, the survival rate of the animals was assayed.
Histopathologic Examination Formalin-fixed liver samples were embedded in paraffin and cut for 5-Km slices and then stained with hematoxylin-eosin. The staining was visualized, and the images were acquired using a microscope (Leica DM4000B, Germany) with 100 magnification.
Biochemical Assay The activities of ALT, AST, and the contents of CAT, SOD, GSH, GSH-Px, MDA, TNOS, iNOS, and NO were measured by using the commercial kits according to the manufacturer’s instructions.
DAPI Staining The paraffin sections were deparaffinized with xylene (two times, once every 15 min) and rehydrated with descending concentrations of alcohol (100%, 90%, 80%, 70%, and 60%) once for 3 min. Then the sections were incubated at 37-C with DAPI (1 Kg/mL) for 8 min, then washed with phosphate-buffered saline and examined by a fluorescence microscope (Nikon Eclipse TE2000-U; NIKON, Japan) with 200 magnification.
TUNEL Assay Paraffin sections were dewaxed as described above, and the assay of apoptosis in hepatic tissues was performed by TUNEL assay according to the kit. Briefly, the fluorescein (green)-labeled deoxyuridine triphosphate solution was added on the sections and incubated at 37-C for 1 hr, then washed with phosphate-buffered saline, and the images were photographed using fluorescence microscopy (Nikon Eclipse TE2000-U) with 200 magnification. After photographing, the slides were stained with converter-POD, then counter stained with DAB solution and hematoxylin. The images were obtained using microscope (Leica DM4000B) with 200 magnification.
Quantitative Real-Time Polymerase Chain Reaction Assay Total RNA samples from the left lateral lobe of livers were extracted by using RNAiso Plus reagent following the manufacturer’s protocol. Reverse transcription polymerase chain reaction (PCR) was performed using PrimeScript RT reagent kit following the manufacturer’s instructions with a TC-512 PCR system (TECHNE, UK). The levels of mRNA expression were quantified by real-time PCR with SYBR PremixEx TaqII (Tli RNaseH Plus) and ABI 7500 Real Time PCR System (Applied Biosystems, Grand Island, NY). The sequences of the primers for rats are shown in (Table S1, SDC, http://links.lww.com/TP/B10). A no-template control was analyzed in parallel for each gene, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was selected as the housekeeping gene in our study. Finally, the unknown template was calculated through the standard curve for quantitative analysis.
Transplantation
with 5% dried skim milk in triethanolamine buffered saline solution + Tween (TTBS) at room temperature, the membrane was individually incubated for overnight at 4-C with primary antibodies (Table S2, SDC, http://links.lww.com/TP/B10). Then the membrane was incubated at room temperature for 2 hr with horseradish peroxidase-conjugated antibodies at a 1:2,000 dilution (Beyotime Institute of Biotechnology, China). Protein expression was detected by an enhanced chemiluminescence method and imaged using Bio-Spectrum Gel Imaging System (UVP, Upland, CA). To eliminate the variations of protein expression, the data were adjusted to GAPDH expression (integral optical density value of target protein vs. integral optical density of GAPDH).
Immunohistochemical Assay Deparaffinized sections were incubated in 3% hydrogen peroxide (H2O2) for 30 min and normal goat serum to block nonspecific protein binding for 30 min. The sections were then incubated overnight at 4-C with rabbit anti-PARP and anti-HMGB-1 antibodies at a 1:100 dilution, followed by incubation in biotin-labeled goat anti-rabbit immunoglobulin G and horseradish peroxidase-conjugated streptavidin for 15 min, respectively. The slides were incubated with DAB solution for 10 min at room temperature, counterstained by hematoxylin and mounted with a permanent mounting medium. Image was taken by a light microscope (Leica DM4000B) with 200 magnification.
Immunofluorescent Assay The sections were incubated in a moist box for overnight at 4-C with the rabbit anti-caspase-9 antibody (1:100, dilution) after the blocking of nonspecific protein binding using normal goat serum for 30 min, then incubated with tetramethyl rhodamine isothiocynate conjugated goat antirabbit immunoglobulin G for 3 hr. The photographs were captured by a fluorescent microscopy (Olympus BX63, Japan) with 200 magnification.
Statistical Analysis All values for each group were given as mean and standard deviation. The data were analyzed by one-way analysis of variance coupled with leastsignificant difference in Post Hoc Multiple Comparisons using the SPSS Statistics 13.0 (IBM, New York). Differences were considered significant when P is less than 0.05 or 0.01.
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8.
Western Blot Assay Total protein was extracted from the livers of different groups using appropriate cold lysis buffer containing 1-mM phenylmethylsulfonyl fluoride based on the manufacturer’s instructions. Sample was loaded onto the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (10%Y15%), separated electrophoretically and transferred onto a PVDF membrane (Millipore, Billerica, MA). After blocking nonspecific binding sites for 3 hr
& Volume 98, Number 6, September 27, 2014
9. 10.
Arab HA, Sasani F, Rafiee MH, et al. Histological and biochemical alterations in early-stage lobar ischemia-reperfusion in rat liver. World J Gastroenterol 2009; 15: 1951. Kapan M, Gumus M, Onder A, et al. The effects of ellagic acid on the liver and remote organs’ oxidative stress and structure after hepatic ischemia reperfusion injury caused by pringle maneuver in rats. Bratisl Lek Listy 2012; 113: 274. Kim SJ, Park JG, Lee SM. Protective effect of heme oxygenase-1 induction against hepatic injury in alcoholic steatotic liver exposed to cold ischemia/reperfusion. Life Sci 2012; 90: 169. Conti A, Scala S, D’Agostino P, et al. Wide gene expression profiling of ischemia reperfusion injury in human liver transplantation. Liver Transpl 2007; 13: 99. Matsuda A, Jacob A, Wu R, et al. Milk fat globulee EGF factor VIII ameliorates liver injury after hepatic ischemia-reperfusion. J Surg Res 2013; 180: 37. Caban A, Oczkowicz G, Abdel-Samad O, et al. Influence of Kupffer cells on the early phase of liver reperfusion. Transplant Proc 2002; 34: 694. Hines IN, Grisham MB. Divergent roles of superoxide and nitric oxide in liver ischemia and reperfusion injury. J Clin Biochem Nutr 2011; 48: 50. Mukhopadhyay P, Rajesh M, Horva´th B, et al. Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death. Free Radic Biol Med 2011; 50: 1368. Iwanaga K, Hasegawa T, Hultquist DE, et al. Riboflavin-mediated reduction of oxidant injury, rejection, and vasculopathy after cardiac allotransplantation. Transplantation 2007; 83: 747. Rah DK, Han DW, Baek HS, et al. Protection of rabbit kidney from ischemia/reperfusion injury by green tea polyphenol pretreatment. Arch Pharm Res 2007; 30: 1447.
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tao et al.
* 2014 Lippincott Williams & Wilkins
11. 12. 13. 14. 15. 16. 17.
18.
19. 20. 21. 22. 23.
Lv L, Jiang SS, Xu J, et al. Protective effect of ligustrazine against myocardial ischaemia reperfusion in rats: the role of endothelial nitric oxide synthase. Clin Exp Pharmacol Physiol 2012; 39: 20. Liu CZ, Zhou HY, Yan Q. Fingerprint analysis of Dioscorea nipponica by high-performance liquid chromatography with evaporative light scattering detection. Anal Chim Acta 2007; 582: 61. Li H, Huang W, Wen Y, et al. Anti-thrombotic activity and chemical characterization of steroidal saponins from Dioscorea zingiberensis C.H. Wright. Fitoterapia 2010; 81: 1147. Zhao X, Cong X, Zheng L, et al. Dioscin, a natural steroid saponin, shows remarkable protective effect against acetaminophen-induced liver damage in vitro and in vivo. Toxicol Lett 2012; 214: 69. Lu BN, Xu YS, Xu LN, et al. Mechanism investigation of dioscin against CCl4-induced acute liver damage in mice. Environ Toxicol Pharmacol 2012; 34: 127. Xu TT, Zheng LL, Xu LN, et al. Protective effects of dioscin against alcohol-induced liver injury. Arch Toxicol 2014; 88: 739. Mukhopadhyay P, Horva´th B, Zsengelle˙r Z, et al. Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemiaYreperfusion: therapeutic potential of mitochondrially targeted antioxidants. Free Radic Biol Med 2012; 53: 1123. Van Golen RF, Van Gulik TM, Heger M. Mechanistic overview of reactive species -induced degradation of the endothelial glycocalyx during hepatic ischemia/reperfusion injury. Free Radic Biol Med 2012; 52: 1382. Kim SJ, Moon YJ, Lee SM. Protective effects of baicalin against ischemia/reperfusion injury in rat liver. J Nat Prod 2010; 73: 2003. Fouad AA, Jresat I. Therapeutic potential of cannabidiol against ischemia/reperfusion liver injury in rats. Eur J Pharmacol 2011; 670: 216. Bahde R, Spiegel HU. Hepatic ischaemia-reperfusion injury from bench to bedside. Br J Surg 2010; 97: 1461. Bilzer M, Baron A, Schauer R, et al. Glutathione treatment protects the rat liver against injury after warm ischemia and Kupffer cell activation. Digestion 2002; 66: 49. Liu Q, Rehman H, Krishnasamy Y, et al. Role of inducible nitric oxide synthase in mitochondrial depolarization and graft injury after transplantation of fatty livers. Free Radic Biol Med 2012; 53: 250.
24. 25.
26. 27. 28.
29. 30. 31. 32. 33. 34. 35. 36.
611
Zhang S, Lu BN, Han X, et al. Protection of the flavonoid fraction from Rosa laevigata Michx fruit against carbon tetrachloride-induced acute liver injury in mice. Food Chem Toxicol 2013; 55: 60. Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophilinduced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions. Am J Physiol Gastrointest Liver Physiol 2006; 290: 1083. Liu SF, Newton R, Evans TW, et al. Differential regulation of cyclooxygenase-1 and cyclooxygenase-2 gene expression by lipopolysaccharide treatment in vivo in the rat. Clin Sci (Lond) 1996; 90: 301. Watanabe T, Kubota S, Nagaya M, et al. The role of HMGB-1 on the development of necrosis during hepatic ischemia and hepatic ischemia/reperfusion injury in mice. J Surg Res 2005; 124: 59. Taki-Eldin A, Zhou L, Xie HY, et al. Triiodothyronine attenuates hepatic ischemia/reperfusion injury in a partial hepatectomy model through inhibition of proinflammatory cytokines, transcription factors, and adhesion molecules. J Surg Res 2012; 178: 646. Kim J, Kim M, Song JH, et al. Endogenous A1 adenosine receptors protect against hepatic ischemia reperfusion injury in mice. Liver Transpl 2008; 14: 845. Yin XM, Ding WX. Death receptor activation-induced hepatocyte apoptosis and liver injury. Curr Mol Med 2003; 3: 491. Weng SY, Yang CY, Li CC, et al. Synergism between p53 and Mcl-1 in protecting from hepatic injury, fibrosis and cancer. J Hepatol 2011; 54: 685. Ikeda R, Che XF, Ushiyama M, et al. 2-Deoxy-d-ribose inhibits hypoxia-induced apoptosis by suppressing the phosphorylation of p38 MAPK. Biochem Biophys Res Commun 2006; 342: 280. Petrache I, Choi ME, Otterbein LE, et al. Mitogen-activated protein kinase pathway mediates hyperoxia induced apoptosis in cultured macrophage cells. Am J Physiol 1999; 277: 589. Uehara T, Bennett B, Sakata ST, et al. JNK mediates hepatic ischemia reperfusion injury. J Hepatol 2005; 42: 850. Wang Y, Singh R, Lefkowitch JH, et al. Tumor necrosis factor-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway. J Biol Chem 2006; 281: 15258. Zhang M, Ueki S, Kimura S, et al. Roles of dendritic cells in murine hepatic warm and liver transplantation-induced cold ischemia/reperfusion injury. Hepatology 2013; 57: 1585.
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
AND
EXPERIMENTAL RESEARCH
Dioscin Attenuates Hepatic Ischemia-Reperfusion Injury in Rats Through Inhibition of Oxidative-Nitrative Stress, Inflammation and Apoptosis Xufeng Tao,1 Xianyao Wan,2 Youwei Xu,1 Lina Xu,1 Yan Qi,1 Lianhong Yin,1 Xu Han,1 Yuan Lin,1 and Jinyong Peng1,3,4 Background. Dioscin shows potent effects against liver damage in our previous studies; however, the action of it on hepatic ischemia-reperfusion (I/R) injury is still unknown. In the present article, the effects and possible mechanisms of dioscin against hepatic I/R injury were investigated. Methods. Seventy percent partial hepatic warm ischemia was induced in Wistar rats for 60 min followed by succedent reperfusion. In the prophylactic test, dioscin was administered intragastrically to the rats at doses of 20, 40, and 60 mg/kg once daily for seven consecutive days before I/R. In the therapeutic test, the rats received dioscin intragastrically at a dose of 60 mg/kg once 2 hr before I/R. Results. We found that dioscin significantly decreased serum alanine aminotransferase and aspartate aminotransferase activities, increased survival rate of rats, and improved I/R-induced hepatocyte abnormality. In addition, dioscin obviously increased the levels of SOD, CAT, GSH-Px, GSH, decreased the levels of MDA, TNOS, iNOS, NO, and prevented DNA fragmentation caused by I/R injury. Further research indicated that dioscin markedly decreased the gene expressions of interleukin-1A, interleukin-6, tumor necrosis factor->, intercellular adhesion molecule-1, MIP-1>, MIP-2, Fas, FasL, decreased the protein expressions of NF-JB, AP-1, COX-2, HMGB-1, CYP2E1, Bak, caspase-3, p53, PARP, Caspase-9, decreased the levels of JNK, ERK and p38 MAPKs phosphorylation, and upregulated the levels of Bcl-2 and Bcl-x. Conclusion. Our results suggest that dioscin has potent actions against hepatic I/R injury through suppression of inflammation, oxidative-nitrative stress, and apoptosis, which should be developed as a new drug to treat hepatic I/R injury in the future. Keywords: Apoptosis, Dioscin, Hepatic I/R injury, Inflammation, Natural product. (Transplantation 2014;98: 604Y611)
schemia-reperfusion (I/R) is an important cause of tissue damage in pathologic conditions including surgical procedures, stroke, vascular surgery, and shocks (1, 2). Orthotopic liver transplantation has been considered as the well-acknowledged curative approach for patients with endstage liver disease (3). However, hepatic I/R injury causes up to 10% of early failures of liver transplantation and higher incidence of acute and chronic rejections (4).
I
This work was financially supported by National Natural Science Foundation of China (no. 81274195), the Program for Liaoning Innovative Research Team in University (LT2013019) and the Program for New Century Excellent Talents in University (NCET-11-1007). The authors declare no conflicts of interest. 1 College of Pharmacy, Dalian Medical University, Liaoning Province, China. 2 Department of Critical Care Medicine of the First Affiliated Hospital of Dalian Medical University, Dalian, China. 3 Research Institute of Integrated Traditional and Western Medicine of Dalian Medical University, Dalian, China. 4 Address correspondence to: Jinyong Peng, M.D., College of Pharmacy, Dalian Medical University, Dalian, China. E-mail: [email protected] X.T. participated in the research design, performance of the research, data analysis, and the article. X.W. participated in the research design and
604
www.transplantjournal.com
The procedure of ischemia results from temporarily blood flow deprivation, which can lead to oxygen and glycogen supporting cellular homeostasis to be insufficient (5). Kupffer cells can produce a large number of reactive oxygen species (ROS), reactive nitrogen species (RNS), and proinflammatory cytokines when they are activated during I/R injury (6). These substances can cause a chain of deleterious cellular responses, resulting in increased inflammatory mediators and elevations of apoptotic molecules, which eventually cause organ failure (7, 8). commented on the draft. Y.X. and L.X. performed the research and commented on the draft. Y.Q., L.Y., and X.H. designed and supervised the research; Y.L. is the discussant and advisor. J.P. is the overall adviser, facilitated the final article version, and sponsored the project. Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com). Received 13 March 2014. Revision requested 28 March 2014. Accepted 22 April 2014. Copyright * 2014 by Lippincott Williams & Wilkins ISSN: 0041-1337/14/9806-604 DOI: 10.1097/TP.0000000000000262
Transplantation
& Volume 98, Number 6, September 27, 2014
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tao et al.
* 2014 Lippincott Williams & Wilkins
Up to now, there are no ideal drugs to treat I/R injury owing to the perplexing pathologic process, and thus it is urgent to develop new and efficient drugs for the treatment of this disease. Some active components including riboflavin (9), green tea polyphenols (10), and ligustrazine (11) from medicinal plants have shown excellent activities against I/R injury. Thus, it is reasonable to exploit effective natural products from herbs for the treatment of hepatic I/R injury. Dioscin, a natural product, is derived from some medicinal plants including Discorea nipponica Makino and Dioscorea zingiberensis C.H. Wright. (12, 13). Our previous investigations have demonstrated that dioscin has potent effects against acetaminophen-induced, CCl4induced, and ethanol-induced liver damages (14Y16). However, to the best of our knowledge, there have been no articles to report the effect of dioscin against hepatic I/R injury. The aim of the present article was to clarify the effect and possible mechanism of dioscin against hepatic I/R injury by using an in vivo lethal hepatic failure rat model. Our findings showed that dioscin has a potential activity for prevention and treatment of hepatic I/R injury, which should be developed as a new drug to treat this disease in the future.
605
RESULTS Effects of Dioscin Against I/R-Induced Liver Injury Hematoxylin-eosinYstained sections are shown in Figure 1(A). The I/R-induced coagulation necrosis with massive inflammatory cells infiltration in liver was clearly observed, which was dramatically attenuated by dioscin with a dose-dependent manner. Furthermore, the increased serum alanine aminotransferase (ALT) (818T64 U/L) and aspartate aminotransferase (AST) (561T83 U/L) activities caused by I/R, shown in Figure 1(B) , were significantly prevented by 40 and 60 mg/kg of dioscin pretreatment. Effects of Dioscin on Survival of Rats and Continuous ALT-AST Activities As shown in Figure 1(C), after 1-hr ischemia and 4 days reperfusion, only did 10% of rats survive in the model group, and treatment with dioscin at the dose of 60 mg/kg markedly improved the survival rate of the animals to 50%. Furthermore, the actions of dioscin on serum ALT and AST activities are shown in Figure 1(D). After 1 hr of ischemia and different times of reperfusion, severe liver damage was significantly attenuated by dioscin.
FIGURE 1. (A) H&E staining of representative liver sections (magnification, 100). (B) Serum ALT, AST activities of rats in the prophylactic test. (C) Rat survival rate. (D) Serum ALT, AST activities of rats in the therapeutic test. H&E staining showed the injured areas. Values expressed as meanTSD (nQ6). *PG0.05 and **PG0.01 compared with the model group. H&E, hematoxylin-eosin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; SD, standard deviation.
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
606
www.transplantjournal.com
Dioscin Attenuated I/R-Induced Oxidative and Nitrative Stress In I/R-treated group, the levels of superoxide dismutase (SOD) (181T20 U/mg prot), catalase (CAT) (458T56 U/g prot), glutathione peroxidase (GSH-Px) (845T89 U/mg prot), and glutathione (GSH) (0.57T0.08 U/mg prot) significantly decreased compared with sham rats. However, dioscin significantly increased their levels in a dose-dependent manner (Fig. 2A). In addition, the increased levels of malondialdehyde (MDA) (3.4T1.1 nmol/mg prot), total nitric oxide synthase (TNOS) (0.83T0.26 U/mg prot), inducible nitric oxide synthase (iNOS) (0.74T0.13 U/mg prot), and nitrogen monoxide (NO) (0.20T0.06 Kmol/g prot) were observed in I/R rats, which were significantly decreased by dioscin (Fig. 2B). Effects of Dioscin on Nuclear Injury As shown in Figure 2(C), the hepatocyte nuclei in the normal group were round with uniform chromatin and emitted even blue fluorescence based on 4¶,6-diamidino-2phenylindole (DAPI) assay while they were condensed, and a lot of particulate matters were observed in I/R-treated
Transplantation
& Volume 98, Number 6, September 27, 2014
group, which were lightened by diocsin at a dose of 60 mg/kg. In addition, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining was conducted to identify apoptotic cells among the injured hepatocytes, and more TUNEL-positive cells showed green fluorescence and brown were observed in I/R-treated group than those in the dioscin-treated group (60 mg/kg). These results indicated that dioscin had the potential activity to attenuate I/R-induced apoptosis. Effects of Dioscin on the Expressions of I/R-Induced Inflammatory Mediators In the present article, the messenger RNA (mRNA) expressions of cytokines interleukin (IL)-1A, IL-6, tumor necrosis factor (TNF)->, adhesion molecule intercellular adhesion molecule (ICAM)-1, and chemokines macrophage inflammatory protein-1> (MIP-1>) and MIP-2 (Fig. 3A), and the expressions of the proteins including nuclear factor JB (NF-JB), AP-1, COX-2, HMGB-1 (Fig. 3B) in I/R-induced liver tissues were significantly increased compared with the sham group. However, dioscin pretreatment markedly attenuated the
FIGURE 2. A and B, effects of the dioscin on the levels of SOD, CAT, GSH-Px, GSH, MDA, TNOS, iNOS, and NO in rat livers. C, representative micrographs of DAPI stained nuclei and TUNEL-stained liver sections (magnification, 200). Fragmentation or condensation of nucleus in the liver tissues was shown in DAPI-stained sections. In fluorescent images, the green fluorescence indicated the positive cells. In DAB&H-stained images, the brown staining represented the positive areas of TUNEL-stained sections. Values expressed as meanTSD (nQ6). *PG0.05 and **PG0.01 compared with the model group. DAB&H, 3,3¶-diaminobenzidine tetrahydrochloride and hematoxylin; TUNEL, deoxynucleotide transferase-mediated dUTP nick-end labeling; SD, standard deviation; DAPI, 4¶,6-diamidino-2-phenylindole; SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; GSH, glutathione; MDA, malondi-aldehyde; TNOS, total nitric oxide synthase; iNOS, inducible nitric oxide synthase; NO, nitrogen monoxide.
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
* 2014 Lippincott Williams & Wilkins
Tao et al.
607
FIGURE 3. A, effects of dioscin on the gene expressions of IL-1, IL-6, TNF->, ICAM-1, MIP-1> and MIP-2. B, effects of dioscin on the protein expressions of NF-JB, AP-1, COX2 and HMGB-1. C, effects of dioscin on the expressions of HMGB-1, PARP and caspase-9 with the immunohistochemical and Immunofluorescent method (magnification, 200). Values expressed as meanTSD (nQ6). *PG0.05 and **PG0.01 compared with the model group. IL, interleukin; SD, standard deviation; TNF, tumor necrosis factor; ICAM-1, intercellular adhesion molecule 1; MIP-1>, macrophage inflammatory protein-1>; MIP2, macrophage inflammatory protein-2; NF-JB, nuclear factor JB; PARP, Poly(adenosine diphosphate-ribose) polymerase.
elevations of these gene and protein expressions. Furthermore, the protein expression of HMGB-1 (Fig. 3C) was considerably upregulated in the I/R group, which was significantly decreased by dioscin. Effects of Dioscin on the Expressions of Apoptotic Molecules and CYP2E1 In the present work, the expressions of Poly(adenosine diphosphate-ribose) polymerase (PARP) and caspase-9 (Fig. 3C) were considerably increased in the I/R group, which were significantly decreased by dioscin at the dose of 60 mg/kg. As shown in Figure 4(A) and (B), compared with model group, the expressions of CYP2E1, Bak, caspase-3, and p53 were obviously decreased, whereas the expressions Bcl-2 and Bcl-x were significantly upregulated by dioscin. In addition, I/R injury obviously increased the mRNA expressions of Fas and Fas ligand (FasL) (Fig. 4C) compared with the sham group, which were significantly inhibited by dioscin. Effects of Dioscin on the Levels of MAPKs Phosphorylation The effects of dioscin on the levels of mitogen-activated protein kinases (MAPKs) phosphorylation were tested in the present work. As shown in Figure 5(A), the increased levels of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK phosphorylation in the model group were found, which were all significantly attenuated by dioscin.
DISCUSSION Hepatic I/R injury is a frequent complication implicated in liver failure associated with liver transplantation, shock,
stroke, and vascular surgery (1, 2). Dioscin is a natural product that shows anti-apoptosis and anti-inflammatory effects in our previous tests (14Y16). In the present work, rat hepatic I/R, a well-established experimental model, was confirmed by the increased serum ALT and AST activities as well as the histopathologic examination, and dioscin considerably reversed the alternations and protected hepatocytes through attenuating the inflammatory cells infiltration and coagulation necrosis in the liver. These results showed that dioscin has potent effects for the prevention and treatment of hepatic I/R damage in rats. Hepatic injury occurs during the ischemic phase, and much damage arises in reperfusion phase, which is characterized by a state of oxidative-nitrative stress (17). In detail, the oxidative-nitrative stress will happen when the endogenous antioxidant defense system fails to effectively counteract the toxic effects of ROS and RNS (18). Furthermore, in the pathogenesis of I/R liver injury, producing proinflammatory cytokines can lead to intense inflammatory reaction (19), and complex cellular events can cause necrosis and apoptosis of liver cells (20). Antioxidant defense system aims to support endogenous antioxidants and inhibit ROS generation in liver I/R process. High levels of the enzymes including SOD, CAT, GSH, and GSH-Px can protect hepatic I/R injury (21). In addition, excessive production of oxygen-free radicals can increase the level of MDA, which is one production of lipid peroxidation and a well-known indicator of ROS (22). NO, a well-known indicator of the formation of RNS, is a toxic substance that can induce nitration of some proteins (23) and can be produced by iNOS-TNOS. The present study
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
608
www.transplantjournal.com
Transplantation
& Volume 98, Number 6, September 27, 2014
FIGURE 4. A and B, Effects of dioscin on the protein expressions of CYP2E1, Bak, Caspase -3, p53, Bcl-2, Bcl-x; (C) Effects of dioscin on gene expressions of Fas, FasL. Values expressed as meanTSD (n=6). *PG0.05 and **PG0.01 compared with the model group. SD, standard deviation.
showed that the levels of SOD, CAT, GSH-Px, and GSH were significantly increased in the liver after dioscin pretreatment compared with the model group, and the levels of MDA, iNOS, TNOS, and NO were dramatically reduced. All of these indicated that the dioscin showed prominent antioxidant activity against I/R-induced oxidative-nitrative liver damage. CYP2E1, one P450 enzyme, plays an important role in the generation of free radicals (24). Our data showed that pretreatment with dioscin significantly decreased CYP2E1 expression in rat, which was an important aspect of the hepatoprotective effect of dioscin against I/R-induced liver damage. After reperfusion, inflammatory reaction has happened after the release of proinflammatory chemokines and cytokines including interleukin (IL)-1A, IL-6, tumor necrosis factor (TNF)->, MIP-1>, and MIP-2 (7, 8). Subsequently, cell surface
adhesion molecule, ICAM-1, regulated by the proinflammatory transcription factor NF-JB, can be expressed in hepatocytes and neutrophils (25). Furthermore, COX-2, one highly inducible enzyme and a known inflammatory early-response protein (26), is activated by a specific subset of proinflammatory cytokines, which can also be secreted through stimulating HMGB-1 (27). In the current study, we found that dioscin dramatically decreased the mRNA levels of IL-1A, IL-6, TNF->, MIP-1>, MIP-2 and ICAM-1, and the protein expressions of COX-2 and HMGB-1. Further exploration showed that dioscin reduced the activations of NF-JB and AP-1 which are also the key regulators of some genes involved in inflammation (28). These findings indicated that the activity of dioscin against hepatic I/R damage may be through attenuating inflammation. Complex cellular events can cause necrosis and apoptosis of liver cells in acute liver injury induced by I/R,
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tao et al.
* 2014 Lippincott Williams & Wilkins
609
FIGURE 5. A, effects of dioscin on the levels of MAPKs phosphorylation. B, experimental protocol for the prophylactic test. C, experimental protocol for the therapeutic test. Values expressed as meanTSD (n=6). *PG0.05 and **PG0.01 compared with the model group. SD, standard deviation; MAPKs, mitogen-activated protein kinases.
which can make hepatocyte sensitized to apoptosis mediated by Fas-FasL (29). Fas-FasL is the upstream regulator of caspase-3, and the activation of caspase-3 can promote the upregulation of PARP (30). Activation of p53 plays a crucial role in I/R-induced hepatocyte apoptosis, and p53 is known to promote apoptosis by upregulating the expression of apoptotic proteins including caspase-9, caspase-3, PARP, Bax, and decreasing the antiapoptotic proteins including Bcl-2 and Bcl-x (31). In the present article, we found that dioscin was helpful to maintain the normal cellular structure and function against I/R-induced hepatocyte apoptosis. Furthermore, dioscin significantly decreased the protein expressions of PARP, Bak, caspase-3, p53, and caspase-9, and the mRNA expressions of Fas and FasL, and also increased the levels of Bcl-2 and Bcl-x. These findings indicated that the protection of dioscin against hepatic I/R damage may be accomplished through reducing apoptosis. MAPKs signaling pathway is closely related with inflammation and apoptosis during hepatic I/R injury (8). Concretely, because of a variety of physiologic and pathophysiologic stresses, phosphorylation levels of p38, JNK, and ERK are activated. Furthermore, activations of phosphorylated p38, JNK, and ERK advance stress-induced cell death and regulate downstream signaling events relevant to inflammation and apoptosis (32Y35). In the present study, we found that administration of dioscin suppressed the levels of phosphorylated p38, JNK, and ERK. These findings suggested that the activity of dioscin against I/R-induced hepatic damage may be through affecting MAPKs pathway. In conclusion, dioscin has a good protective effect against hepatic I/R injury in rats through attenuating oxidative-nitrative stress, inflammation, and apoptosis, which should be developed as a new and potent candidate for treatment of I/R-induced liver injury in the future. Of
course, mechanisms, drug-target, and clinical applications of dioscin are needed further investigations.
MATERIALS AND METHODS Chemicals and Materials Dioscin with a purity greater than 98% was purchased from the National Institutes of Food and Drug Control of China (Beijing, China). ALT, AST, CAT, SOD, GSH, GSH-Px, MDA, TNOS, iNOS, and NO kits were obtained from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Tissue Protein Extraction Kit was obtained from KEYGEN Biotech. Co., Ltd. (Nanjing, China). Bicinchoninic acid protein assay kit was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). 3,3¶Diaminobenzidine (DAB) substrate kit was purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). The DAPI was purchased from Sigma Chemical Co. (St. Louis, MO). In situ cell death detection kit was provided by Roche Diagnostics (Germany). RNAiso Plus, PrimeScript RT reagent Kit with gDNA Eraser (perfect real time) and SYBR Premix Ex Taq II (Tli RNaseH Plus) were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China).
Experimental Animals Male Wistar rats (180Y220 g) were purchased from the Experimental Animal Center of Dalian Medical University (Dalian, China) (SCXK: 2008-0002). All experimental procedures were approved by the Animal Care and Use Committee of Dalian Medical University and performed in strict accordance with the PR China Legislation Regarding the Use and Care of Laboratory Animals.
Experimental Design The animals were allowed to adapt to the environment for 1 week before the experiments. As shown in Figure 5(B), in the prophylactic test, dioscin was administered intragastrically to the animals at the doses of 20, 40, and 60 mg/kg once daily for seven consecutive days. The rats in the sham operation and model groups were administered with vehicle. On the eighth day, the murine model of 70% partial hepatic ischemia was established as
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
610
www.transplantjournal.com
described previously (21, 36). Briefly, the rats were anesthetized, and the livers were exposed by midline laparotomy, then the inflow of the left lateral and median lobes of the livers was choked by placing a bulldog clamp, whereas the right lobes were remainedly perfused to prevent intestinal congestion occlusion. After 60 min of hepatic ischemic, the bulldog clamp was removed, and the liver was reperfused for 6 hr. At the end of the surgery, the animals were anesthetized for collecting the blood and then killed to collect the left lateral and middle lobes of the livers. As shown in Figure 5(C), in the therapeutic test, the rats were randomized into three groups, of which the animals in sham and model groups were given vehicle, and the rats in dioscin group received dioscin intragastrically at a dose 60 mg/kg once 2 hr before I/R. After 60 min of hepatic ischemic, the bulldog clamp was removed, and the orbital blood samples were collected at 0.5, 1, 2, 4, 6, 12, 24, 48, and 96 hr after reperfusion (34). In addition, the survival rate of the animals was assayed.
Histopathologic Examination Formalin-fixed liver samples were embedded in paraffin and cut for 5-Km slices and then stained with hematoxylin-eosin. The staining was visualized, and the images were acquired using a microscope (Leica DM4000B, Germany) with 100 magnification.
Biochemical Assay The activities of ALT, AST, and the contents of CAT, SOD, GSH, GSH-Px, MDA, TNOS, iNOS, and NO were measured by using the commercial kits according to the manufacturer’s instructions.
DAPI Staining The paraffin sections were deparaffinized with xylene (two times, once every 15 min) and rehydrated with descending concentrations of alcohol (100%, 90%, 80%, 70%, and 60%) once for 3 min. Then the sections were incubated at 37-C with DAPI (1 Kg/mL) for 8 min, then washed with phosphate-buffered saline and examined by a fluorescence microscope (Nikon Eclipse TE2000-U; NIKON, Japan) with 200 magnification.
TUNEL Assay Paraffin sections were dewaxed as described above, and the assay of apoptosis in hepatic tissues was performed by TUNEL assay according to the kit. Briefly, the fluorescein (green)-labeled deoxyuridine triphosphate solution was added on the sections and incubated at 37-C for 1 hr, then washed with phosphate-buffered saline, and the images were photographed using fluorescence microscopy (Nikon Eclipse TE2000-U) with 200 magnification. After photographing, the slides were stained with converter-POD, then counter stained with DAB solution and hematoxylin. The images were obtained using microscope (Leica DM4000B) with 200 magnification.
Quantitative Real-Time Polymerase Chain Reaction Assay Total RNA samples from the left lateral lobe of livers were extracted by using RNAiso Plus reagent following the manufacturer’s protocol. Reverse transcription polymerase chain reaction (PCR) was performed using PrimeScript RT reagent kit following the manufacturer’s instructions with a TC-512 PCR system (TECHNE, UK). The levels of mRNA expression were quantified by real-time PCR with SYBR PremixEx TaqII (Tli RNaseH Plus) and ABI 7500 Real Time PCR System (Applied Biosystems, Grand Island, NY). The sequences of the primers for rats are shown in (Table S1, SDC, http://links.lww.com/TP/B10). A no-template control was analyzed in parallel for each gene, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was selected as the housekeeping gene in our study. Finally, the unknown template was calculated through the standard curve for quantitative analysis.
Transplantation
with 5% dried skim milk in triethanolamine buffered saline solution + Tween (TTBS) at room temperature, the membrane was individually incubated for overnight at 4-C with primary antibodies (Table S2, SDC, http://links.lww.com/TP/B10). Then the membrane was incubated at room temperature for 2 hr with horseradish peroxidase-conjugated antibodies at a 1:2,000 dilution (Beyotime Institute of Biotechnology, China). Protein expression was detected by an enhanced chemiluminescence method and imaged using Bio-Spectrum Gel Imaging System (UVP, Upland, CA). To eliminate the variations of protein expression, the data were adjusted to GAPDH expression (integral optical density value of target protein vs. integral optical density of GAPDH).
Immunohistochemical Assay Deparaffinized sections were incubated in 3% hydrogen peroxide (H2O2) for 30 min and normal goat serum to block nonspecific protein binding for 30 min. The sections were then incubated overnight at 4-C with rabbit anti-PARP and anti-HMGB-1 antibodies at a 1:100 dilution, followed by incubation in biotin-labeled goat anti-rabbit immunoglobulin G and horseradish peroxidase-conjugated streptavidin for 15 min, respectively. The slides were incubated with DAB solution for 10 min at room temperature, counterstained by hematoxylin and mounted with a permanent mounting medium. Image was taken by a light microscope (Leica DM4000B) with 200 magnification.
Immunofluorescent Assay The sections were incubated in a moist box for overnight at 4-C with the rabbit anti-caspase-9 antibody (1:100, dilution) after the blocking of nonspecific protein binding using normal goat serum for 30 min, then incubated with tetramethyl rhodamine isothiocynate conjugated goat antirabbit immunoglobulin G for 3 hr. The photographs were captured by a fluorescent microscopy (Olympus BX63, Japan) with 200 magnification.
Statistical Analysis All values for each group were given as mean and standard deviation. The data were analyzed by one-way analysis of variance coupled with leastsignificant difference in Post Hoc Multiple Comparisons using the SPSS Statistics 13.0 (IBM, New York). Differences were considered significant when P is less than 0.05 or 0.01.
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8.
Western Blot Assay Total protein was extracted from the livers of different groups using appropriate cold lysis buffer containing 1-mM phenylmethylsulfonyl fluoride based on the manufacturer’s instructions. Sample was loaded onto the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (10%Y15%), separated electrophoretically and transferred onto a PVDF membrane (Millipore, Billerica, MA). After blocking nonspecific binding sites for 3 hr
& Volume 98, Number 6, September 27, 2014
9. 10.
Arab HA, Sasani F, Rafiee MH, et al. Histological and biochemical alterations in early-stage lobar ischemia-reperfusion in rat liver. World J Gastroenterol 2009; 15: 1951. Kapan M, Gumus M, Onder A, et al. The effects of ellagic acid on the liver and remote organs’ oxidative stress and structure after hepatic ischemia reperfusion injury caused by pringle maneuver in rats. Bratisl Lek Listy 2012; 113: 274. Kim SJ, Park JG, Lee SM. Protective effect of heme oxygenase-1 induction against hepatic injury in alcoholic steatotic liver exposed to cold ischemia/reperfusion. Life Sci 2012; 90: 169. Conti A, Scala S, D’Agostino P, et al. Wide gene expression profiling of ischemia reperfusion injury in human liver transplantation. Liver Transpl 2007; 13: 99. Matsuda A, Jacob A, Wu R, et al. Milk fat globulee EGF factor VIII ameliorates liver injury after hepatic ischemia-reperfusion. J Surg Res 2013; 180: 37. Caban A, Oczkowicz G, Abdel-Samad O, et al. Influence of Kupffer cells on the early phase of liver reperfusion. Transplant Proc 2002; 34: 694. Hines IN, Grisham MB. Divergent roles of superoxide and nitric oxide in liver ischemia and reperfusion injury. J Clin Biochem Nutr 2011; 48: 50. Mukhopadhyay P, Rajesh M, Horva´th B, et al. Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death. Free Radic Biol Med 2011; 50: 1368. Iwanaga K, Hasegawa T, Hultquist DE, et al. Riboflavin-mediated reduction of oxidant injury, rejection, and vasculopathy after cardiac allotransplantation. Transplantation 2007; 83: 747. Rah DK, Han DW, Baek HS, et al. Protection of rabbit kidney from ischemia/reperfusion injury by green tea polyphenol pretreatment. Arch Pharm Res 2007; 30: 1447.
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tao et al.
* 2014 Lippincott Williams & Wilkins
11. 12. 13. 14. 15. 16. 17.
18.
19. 20. 21. 22. 23.
Lv L, Jiang SS, Xu J, et al. Protective effect of ligustrazine against myocardial ischaemia reperfusion in rats: the role of endothelial nitric oxide synthase. Clin Exp Pharmacol Physiol 2012; 39: 20. Liu CZ, Zhou HY, Yan Q. Fingerprint analysis of Dioscorea nipponica by high-performance liquid chromatography with evaporative light scattering detection. Anal Chim Acta 2007; 582: 61. Li H, Huang W, Wen Y, et al. Anti-thrombotic activity and chemical characterization of steroidal saponins from Dioscorea zingiberensis C.H. Wright. Fitoterapia 2010; 81: 1147. Zhao X, Cong X, Zheng L, et al. Dioscin, a natural steroid saponin, shows remarkable protective effect against acetaminophen-induced liver damage in vitro and in vivo. Toxicol Lett 2012; 214: 69. Lu BN, Xu YS, Xu LN, et al. Mechanism investigation of dioscin against CCl4-induced acute liver damage in mice. Environ Toxicol Pharmacol 2012; 34: 127. Xu TT, Zheng LL, Xu LN, et al. Protective effects of dioscin against alcohol-induced liver injury. Arch Toxicol 2014; 88: 739. Mukhopadhyay P, Horva´th B, Zsengelle˙r Z, et al. Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemiaYreperfusion: therapeutic potential of mitochondrially targeted antioxidants. Free Radic Biol Med 2012; 53: 1123. Van Golen RF, Van Gulik TM, Heger M. Mechanistic overview of reactive species -induced degradation of the endothelial glycocalyx during hepatic ischemia/reperfusion injury. Free Radic Biol Med 2012; 52: 1382. Kim SJ, Moon YJ, Lee SM. Protective effects of baicalin against ischemia/reperfusion injury in rat liver. J Nat Prod 2010; 73: 2003. Fouad AA, Jresat I. Therapeutic potential of cannabidiol against ischemia/reperfusion liver injury in rats. Eur J Pharmacol 2011; 670: 216. Bahde R, Spiegel HU. Hepatic ischaemia-reperfusion injury from bench to bedside. Br J Surg 2010; 97: 1461. Bilzer M, Baron A, Schauer R, et al. Glutathione treatment protects the rat liver against injury after warm ischemia and Kupffer cell activation. Digestion 2002; 66: 49. Liu Q, Rehman H, Krishnasamy Y, et al. Role of inducible nitric oxide synthase in mitochondrial depolarization and graft injury after transplantation of fatty livers. Free Radic Biol Med 2012; 53: 250.
24. 25.
26. 27. 28.
29. 30. 31. 32. 33. 34. 35. 36.
611
Zhang S, Lu BN, Han X, et al. Protection of the flavonoid fraction from Rosa laevigata Michx fruit against carbon tetrachloride-induced acute liver injury in mice. Food Chem Toxicol 2013; 55: 60. Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophilinduced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions. Am J Physiol Gastrointest Liver Physiol 2006; 290: 1083. Liu SF, Newton R, Evans TW, et al. Differential regulation of cyclooxygenase-1 and cyclooxygenase-2 gene expression by lipopolysaccharide treatment in vivo in the rat. Clin Sci (Lond) 1996; 90: 301. Watanabe T, Kubota S, Nagaya M, et al. The role of HMGB-1 on the development of necrosis during hepatic ischemia and hepatic ischemia/reperfusion injury in mice. J Surg Res 2005; 124: 59. Taki-Eldin A, Zhou L, Xie HY, et al. Triiodothyronine attenuates hepatic ischemia/reperfusion injury in a partial hepatectomy model through inhibition of proinflammatory cytokines, transcription factors, and adhesion molecules. J Surg Res 2012; 178: 646. Kim J, Kim M, Song JH, et al. Endogenous A1 adenosine receptors protect against hepatic ischemia reperfusion injury in mice. Liver Transpl 2008; 14: 845. Yin XM, Ding WX. Death receptor activation-induced hepatocyte apoptosis and liver injury. Curr Mol Med 2003; 3: 491. Weng SY, Yang CY, Li CC, et al. Synergism between p53 and Mcl-1 in protecting from hepatic injury, fibrosis and cancer. J Hepatol 2011; 54: 685. Ikeda R, Che XF, Ushiyama M, et al. 2-Deoxy-d-ribose inhibits hypoxia-induced apoptosis by suppressing the phosphorylation of p38 MAPK. Biochem Biophys Res Commun 2006; 342: 280. Petrache I, Choi ME, Otterbein LE, et al. Mitogen-activated protein kinase pathway mediates hyperoxia induced apoptosis in cultured macrophage cells. Am J Physiol 1999; 277: 589. Uehara T, Bennett B, Sakata ST, et al. JNK mediates hepatic ischemia reperfusion injury. J Hepatol 2005; 42: 850. Wang Y, Singh R, Lefkowitch JH, et al. Tumor necrosis factor-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway. J Biol Chem 2006; 281: 15258. Zhang M, Ueki S, Kimura S, et al. Roles of dendritic cells in murine hepatic warm and liver transplantation-induced cold ischemia/reperfusion injury. Hepatology 2013; 57: 1585.
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
