- Email: [email protected]
Ambroxol Alleviates Hepatic Ischemia Reperfusion Injury by Antioxidant and Antiapoptotic Pathways
Ambroxol Alleviates Hepatic Ischemia Reperfusion Injury by Antioxidant and Antiapoptotic Pathways K. Jiang, X. Wang, X. Mao, H. Lao, J. Zhang, G. Wang, Y. Cao, I. Tong, and F. Zhang ABSTRACT Background. Hepatic ischemia/reperfusion (HI/R) injury is a common pathologic process caused by many clinical settings, such as liver resection, liver transplantation, hypovolemic shock, and trauma. The use of ambroxol, which acts as a mucolytic agent, provides antioxidant and anti-inflammatory effects. Methods. A rat model of HI/R was induced by clamping the hepatic artery, the hepatoportal vein, and the bile duct with a vascular clamp for 30 minutes followed by reperfusion for 6 hours under anesthesia. The sham group underwent laparotomy without hepatic ischemia. The ambroxol group was injected into the tail vein in the ambroxol group 5 minutes before HI/R at one dose of 20 mg/kg, 80 mg/kg, or 140 mg/kg. The control group underwent the same procedure as the ambroxol group but with administration of physiological saline. Liver injury was evaluated by biochemical and histopathological examinations. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed in serum samples. Superoxide dismutase (SOD), catalase (CAT), malondiadehyde (MDA), and glutathione (GSH) were spectrophotometrically measured. Furthermore, caspase-3, Bcl-2 and Bax expression as well as the level of c-Jun N-terminal kinases (JNK) we estimated activation. Results. Wistar rats that received 20, 80 mg or 140 mg of ambroxol displayed reduced HI/R injury compared with controls. Use of ambroxol reduced the histologic injury and significantly decreased serum ALT and AST levels. In addition, ambroxol enhanced the activity of hepatic tissue SOD and CAT, increasing GSH but decreasing MDA tissue contents. In the ambroxol group, Bcl-2 expression was increased and Bax and caspase-3 decreased compared with the controls. Furthermore, ambroxol reduced levels of phosphorylated JNK (P < .05). Conclusion. These results indicated that ambroxol attenuated rat HI/R through upregulation of intracellular antioxidant and anti-apoptotic signaling pathways.
I
SCHEMIA/REPERFUSION injury (I/R) occurs by the interrupted blood flow for a short period in the tissues and consequently aggravate the tissue injury after a period of ischemia. The liver often suffers I/R injury after some surgical procedures, such as liver transplantation and elective hepatic resection. The injury, which may also occur during hypovolemic shock or after severe trauma, may lead to liver failure, which results in a high morbidity and mortality. Therefore, hepatic I/R (HI/R) has always been an obstacle to the development of liver surgery. In previous studies, several mechanisms have been suggested to contribute to the pathophysiology of HI/R injury, including reactive oxygen species (ROS). ROS can result in chromosomal malformation, nucleic acid base changes, or DNA rupture which can induce hepatocellular apoptosis or necrosis.1 Chandra et al showed
From the Department of General Surgery, the Fifth Affiliated Hospital of Harbin Medical University, Daqing, Heilongjiang Province. (K.J., H.L., J.Z., I.T., F.Z.), the Department of General Surgery, Daqing Oilfield General Hospital, Daqing, Heilongjiang Province (X.W.), the Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, Changsha (X.M.), the Department of Pathology, the Fifth Affiliated Hospital of Harbin Medical University, Daqing, Heilongjiang Province (G.W.), and Daqing Campus Harbin Medical University, Daqing, Heilongjiang Province (Y.C.), China. Supported by the Nature Science Foundation of Heilongjiang Province (D200927). Address reprint requests to Prof. F Zhang, First Department of General Surgery, the Fifth Affiliated Hospital of Harbin Medical University, Building Street, Daqing, Heilongjiang Province, China. E-mail: [email protected]
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.04.007
Transplantation Proceedings, 45, 2439e2445 (2013)
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that increased tissue H2O2 can cause apoptosis by upregulating the Fas-FasL system. Furthermore, mitochondrial membranes are potentially damaged by H2O2, which can contribute to the release of pro-apoptotic substances therein. In addition, injured mitochondria promoted the shift of transcriptional factors into the nucleus, such as p53, and nuclear factor-kappa B (NF-kB). In addition, ROS enhances the expression of pro-apoptotic genes and impairs survivalrelated genes.2 In contrast, natural antioxidants alleviate I/ R injury. Changes in superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) reductase attenuate I/R injures in animal. ROS have been shown to exert a central role contributing to HI/R injury.3e5 Apoptosis is an important mechanism in HI/R injury. Kohli et al confirmed that 50% to 70% of liver sinusoidal endothelial cells and 40% to 60% of hepatocytes underwent apoptosis.6 Ambroxol hydrochloride (trans-4-[2-amino-3, 5-dibrombeombenzylamino]-cyclohexanol HCL), where chemical structure is shown in Fig 1, has been prescribed as a mucolytic for viscid or excessive mucus. Recent studies have shown anti-oxidative and anti-inflammatory properties of ambroxol, in vitro and in vivo.7 Drews et al has observed that ambroxol is beneficial for organ preservation in a transplantation model.8 However, Hausen et al did not show that pretreatment with ambroxol alleviated reperfusion injury in experimental lung transplantation.9 The present study examined the dose-related anti-oxidative and anti-inflammatory actions of ambroxol. Therefore, the present study was conducted to assess the hepatic I/R injury by histopathological and biochemical examinations. MATERIALS AND METHODS Animals Male Wistar rats weighing 240 40 g were obtained from our Animal Research Center for use consistent with internationally accepted principles. Measures were taken to minimize suffering and reduce the number of animals.
Induction of HI/R and Drug Administration HI/R was induced according to the method described previously.10e12 Under the chloral hydrate (200 mg/kg) and ether anesthesia, rats underwent a median laparotomy. The hepatoportal vein, hepatic arterial, and hepatic duct were separated to be occluded with an atraumatic vascular clamp for 30 minutes, followed by 6 hours reperfusion. During the reperfusion period the body temperature of the animals was kept constant with a heating blanket.
Fig 1. The chemical structure of ambroxol.
Thirty rats were randomly assigned to five groups (six rats each): sham, control, and ambroxol hydrochloride (Sigma-Aldrich, Shanghai, China) treatments (20 mg/kg, 80 mg/kg, or 140 mg/kg). The sham group merely underwent a median laparotomy without occlusion of the hepatoportal vein, hepatic artery and hepatic duct. The control and ambroxol groups underwent HI/R before injections of the same volume of physiological saline or ambroxol though the tail veil. At the end of the reperfusion period, rats were sacrificed to collect blood and liver samples. Blood samples were centrifuged at 3000 g for 5 minutes to collect serum for determinations of alanine transaminase (ALT) and asparate transaminase (AST) levels. The hepatic tissue samples were stored at 80 C before measurements of tissue malondialdehyde (MDA), GSH, CAT, and SOD levels, together with estimation of caspase-3, Bcl-2, and Bax activities. Separate hematoxylin-eosin (HE) stained tissue samples underwent scoring under light microscopy.
Histologic Examination Liver specimens were fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 12 hours, followed by 2 days in 30% sucrose buffer. Serial coronal sections (6-mm thick) obtained using a microtome were stained with HE for light microscopy. The liver histopathological assessment was performed in blinded fashion.
Biochemical Analysis Automated analyzer autobiochemical analyzer (Toshiba, Tokyo, Japan) was used as described previously10e13 to measure AST and ALT (U/L).
The Activities of SOD, CAT, GSH, and MDA The enzymatic activities of SOD, and CAT, GSH, and MDA were measured according to the manufacturer’s instructions using commercial assay kits (Nanjing Jian Cheng Bioengineering Institute, China) SOD activity, (U/mg protein) in hepatic tissue homogenate was estimated by evaluating the rate of inhibition of nucleotide oxidation. CAT (U/mg protein) was assayed by quantifying the flaxen complex configurated by ammonium molybdate and the reminder peroxide at 405 nm. The GSH content mg GSH per gram protein was assayed by quantifying the rate of oxidation of reduced to oxidized glutathione by H2O2. The MDA concentration was determined by measuring thiobarbituricacid reactive substances (nmol MDA per milligram protein) at 532 nm.
Western Blot Hepatic samples were homogenized in an ice-cold lysis buffer, 10 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 10% glycerol, 1% Nonidet P-40, 5 mmol/ ethylene diamine tetraacetic acid (EDTA), and protease inhibitor cocktail. The resulting homogenate was centrifuged at 13,200 g for 20 minutes at 4 C. The supernate total protein content was estimated using a bicinchonininc acid (BCA) protein assay kit with bovine serum albumin as the standard (Beyotime Institute of Biotechnology, China). Equal 50 mg protein amounts were loaded into 10% sodium dodecyl sulfate (SDS)e polyacrylamide gels proteins were transferred onto nitrocellulose membranes (Millipore, MA), which have been blocked in 5% nonfat dry milk in Tris-buffered saline with Tween-20 (TBST) for 1 hour. Thereafter the manufactures were incubated at 4 C overnight with affinity-purified anti-caspase-3 (1:300; Cell Signaling, USA), anti-Bcl-2 (1:200; Santa Cruz, CA, USA), anti-Bax (1:200; Santa Cruz, CA, USA), anti-JNK (1:500; Santa Cruz, CA, USA) or antib-actin (1:2000; Santa Cruz, CA, USA). After three TBST rinses at
AMBROXOL 10-minute intervals, the membranes were incubated for 2 hours with peroxidase-labeled goat anti-rabbit (immunoglobulin 1:5000; Santa Cruz). Immunodetection was implemented with enhanced chemiluminesecence (ECL; Applygen) followed by x-ray film exposured. b-actin was the internal reference for quantification. Immunoblots scanned by the densitometer were subjected to grey analysis with Quantity One software (BioRad).
Statistical Analysis Comparisons between groups were analyzed with one-way analysis of variance and then Dunnett’s test using SPSS 13.0 software. P < .05 was considered to be statistically significant.
RESULTS Histopathological Examination
Figures 2A and 2F show the sham group to display normal appearances. In contrast, Figs 2B and 2G show color fading of hepatocytes cytoplasm with nuclear condensation. These changes were notably reduced among the group treated with ambroxol (20 mg/kg; Figs 2C and 2G). Furthermore, the 80 mg/kg and 140 mg/kg ambroxol cohorts showed significant improvement as in Figs 2I, 2E, and 2J. Serum ALT and AST
Compared with the sham group, the control group levels of serum ALT, as liver damage markers, were significantly increased (Fig 3A) from 34.33 8.66 to 2055.17 222.60 U/L (P < .05, n ¼ 6). The ambroxol group (20 mg/kg, 80 mg/kg, 140 mg/kg) content was markedly decreased from 2055.17 222.60 to 1385.83 212.20 U/L (P < .05, n ¼ 6); 905.50 121.26 (P < .05, n ¼ 6), and 845.67 107.97 U/L (P < .05, n ¼ 6), respectively, compared with the control group. Similar to serum ALT, level control group AST was notably greater than the sham group (Fig 3B) from 90.83 23.23 to 2477.33 238.23 U/L (P < .05, n ¼ 6). The ambroxol group (20 mg/kg, 80 mg/kg, 140 mg/kg) showed dramatically decreased expression from 2055.17 222.60 to 1727.00 243.85 U/L (P < .05, n ¼ 6); 1057.67 197.25 (P < .05, n ¼ 6), and 982.33 122.81 U/L (P < .05, n ¼ 6), respectively, compared with the control group. The Activities of Antioxidant Enzymes (SOD, CAT) and the Content of MDA and GSH in Hepatic Tissue
Figure 4A shows the activity of SOD as one of the most important antioxidant enzymes, to be significantly reduced among the controls from 314.91 35.81 to 204.41 34.53 U/ mg protein (P < .05, n ¼ 6) compared with the sham group. After administration with ambroxol (80 mg/kg, or 140 mg/kg), the SOD activity was significantly enhanced from 204.41 34.53 to 265.87 34.37 (P < .05, n ¼ 6), and 269.26 33.11 U/ mg protein (P < .05, n ¼ 6), although there was no significant difference when treated with 20 mg/kg of ambroxol. Similar to SOD, the CAT activity in the control group decreased from 34.02 4.38 (P < .05, n¼6) to 22.37 3.14 U/mg protein (P < .05, n ¼ 6) compared with the sham cohort. After 80 mg or 140 mg, ambroxol the CAT activity was notably enhanced from 22.37 3.14 to 28.79 3.25 (P < .05, n ¼ 6) or 27.33
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1.98 U/mg protein (P < .05, n¼6), respectively, compared with the controls although 20 mg of ambroxol had no effect (Fig 4B). Furthermore, Fig 4C shows the GSH quantity in the control group dramatically decreased from 2.99 0.17 to 1.87 0.15 mg/g protein (P < .01, n ¼ 6) compared with the sham group. In contrast, the content of GSH was increased after administration of ambroxol (20 mg/kg, 80 mg/kg, 140 mg/kg), to 2.01 0.16; 2.38 0.11 (P < .05, n ¼ 6), and 2.26 0.13 mg/ g protein (P < .05, n ¼ 6). Additionally, the MDA content (Fig 4D), marker of lipid peroxidation, was significantly increased in control group hepatic tissue from 4.64 0.90 to 7.59 0.92 nmol/mg protein (P < .05, n ¼ 6), compared with the sham cohert. After administration of ambroxol (20 mg/kg, 80 mg/kg, or 140 mg/kg), the MDA content decreased to 6.87 0.65, 5.49 0.64 (P < .05, n ¼ 6) or 5.48 0.54 nmol/mg protein (P < .05, n ¼ 6), respectively. The B-cell Lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-3 Activity
Western blot analysis of Bcl-2, Bax, and caspase-3 proteins in hepatic tissue was performed. Figure. 5 (BeD) showed the control cohort, Bcl-2 to be remarkably reduced (1.47 0.04 to 0.80 0.04; P < .05, n ¼ 6) compared with the sham group. The 140-mg/kg ambroxol group revealed a remarkable increases to 1.09 0.04 (P < .05, n ¼ 6). The control displayed significantly increased protein expression of Bax from 0.88 0.06 to 1.16 0.05 (P < .05, n ¼ 6) compared with the sham group. The ambroxol 80mg /kg and 140 mg/kg groups were dramatically decreased (0.97 0.07; P < .05; n ¼ 6 and 0.94 0.03; P < 0.05; n ¼ 6), respectively compared with controls. Western blots of caspase-3 antibody showed the anticipated bands of 17 kDa. Compared with the sham control. Control Caspase-3 level in hepatic tissue was abundantly increased from 0.91 0.05 to 1.22 0.07 (P < .05, n ¼ 6). However, the caspase-3 protein levels after ambroxol 80 and 140 mg/kg were remarkably decreased to 0.92 0.07 (P < .05, n ¼ 6) and 0.87 0.07 (P < .05, n ¼ 6). The Phosphorylated Level of c-Jun N-terminal Kinases (JNK)
Figure 6 shows that the activated JNK phospho-JNK level in the control group was notably enhanced from 0.80 0.04 to 1.27 0.08 (P < .05, n ¼ 6) compared with the sham group. However, JNK activation in the group treated with 140 mg/kg ambroxol was significantly reduced to 0.78 0.05 (P < .05, n ¼ 6). DISCUSSION
Ambroxol, a common mucoactive drug with secretolytic actions, is frequently used to treat respiratory diseases. Ambroxol is not only an expectorant but also has other effects, including antioxidative13e15 and anti-inflammatory activities.16 Some studies have shown ambroxol to mitigate I/R effects.8 But there is no prior evidence of protection from hepatic warm I/R injury as shown herein by remarkable actions on serum transaminases (ALT and AST) and histology. HE staining showed 80 mg/kg or 140 mg/kg
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Fig 2. Effects of ambroxol on the hepatic tissue 6 hours after hepatic reperfusion with hematoxylin-eosin staining. The representative micrographs of hepatic tissue from (A) and (F), sham-operated; (B) and (G), control-treated; (C) and (H), ambroxol (20 mg/kg)etreated; (D) and (I), ambroxol (80 mg/kg)etreated; (E) and (J), ambroxol (140 mg/kg)etreated groups were established. (Original magnifications [AeE]; 400 [FeJ]). The pykcnosis in the control and amboxol (140 mg/kg) groups was marked. (Scale bar: 10 mm.)
ambroxol to significantly alleviate hepatic damage compared with controls. Many factors contribute to I/R injury with ROS accumulation, massive granulocyte infiltration, and hepatocyte apoptosis. ROS play significant roles during HI/R injury.17,18 Enhanced hepatic antioxidant ability can reduce
damage induced by I/R. Transgenic mice overexpressing SOD and CAT display significant improved HI/R injury compared with normal hosts.19 Administration intravenous GSH after HI/R protects hepatocytes and improves animal survival.20 The MDA level, a criterion to evaluate the severity of reperfusion injury, is increased during I/R.
AMBROXOL
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Fig 3. Effects of ambroxol on the level of serum alanine transaminase (ALT) and aspartate transaminase (AST) of rat that underwent hepatic ischemia reperfusion 6 hours after ischemia (mean SD, n ¼ 6) as the (A) and (B) show, respectively. ## P < .05 versus. sham-operated group, *P < .05, **P < .05, and ***P < .05 versus control-treated group. (Abbreviations: sham, sham-operated; control, controltreated; A20, ambroxol [20 mg/kg]etreated; A80, ambroxol [80 mg/kg]etreated; A140, ambroxol [140 mg/kg]etreated groups.)
Under physiological conditions, ROS are quickly detoxified by endogenous enzymes and low-molecular weight antioxidantsdSOD, CAT and GSH. In our study, the SOD and CAT, activities after applying ambroxol, were dramatically higher compared with controls, but the MDA content significantly lower. The present results indicated that the actions of ambroxol to alleviate HI/R injury might be associated with its antioxidant activity.
Hepatocyte apoptosis also plays an important part in the damage induced by I/R. Multiple factors activate apoptotic cascade proteins. The caspase family is progressively activated by apoptotic stimuli. Caspase-3, an executioner of apoptosis, plays a dominant role.21 Much evidence supports the role of up regulated caspase-3 after hepatic ischemia. In addition, Bcl-2 family plays important roles in hepatocyte apoptosis, particularly Bcl-2
Fig 4. Effects of ambroxol on the activities of antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT]) and the concentration of malondiadehyde (MDA) and glutathione (GSH) on hepatic tissue 6 hours after reperfusion (mean SD, n ¼ 6). (A)e(D) show the effects of ambroxol on MDA, GSH, SOD, and CAT activities in hepatic tissue, respectively. ##P < .05 versus sham-operated group, **P < .05, ***P < .05 versus control-treated group. (Abbreviations: sham, sham-operated; control, control-treated; A20, ambroxol [20 mg/kg]etreated; A80, ambroxol [80 mg/kg]etreated; A140, ambroxol [140 mg/kg]etreated groups.)
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Fig 5. Effects of ambroxol on the protein expressions of Bcl-2, Bax, and Caspase-3 in hepatic tissue (mean SD, n ¼ 6); (A) shows the representative image of immunoblots. (B) (C), and (D) were the quantitative analysis of the protein levels of Bcl-2 Bax and Caspase-3 from six hepatic tissue in each group. The data were normalized to the loading control b-actin. Abbreviations: sham, sham-operated; control, controltreated; A20, ambroxol [20 mg/kg]etreated; A80, ambroxol [100 mg/kg]etreated; A140, ambroxol [140 mg/kg]etreated groups. ## P < .05 versus. sham-operated group. *P < .05, **P < .05 versus control-treated group.
and Bax. The Bcl-2, a key regulator of apoptosis, is beneficial for cell survival by inhibiting caspase activation. However, Bax exerts the reverse role to regulate apoptosis. The Bcl-2/Bax ratio determines the fate cells after an apoptotic stimulus. If the Bcl-2 level is higher than that of Bax, caspase activation is suppressed; when Bax is higher caspase is activated and the cell undergoes apoptosis.22e24 Our study showed that the caspase-3 activity in the group treated with ambroxol was remarkably decreased compared with the controls with, higher
Fig 6. Effects of ambroxol on the protein expressions of JNK and P-JNK in hepatic tissue (mean SD, n ¼ 6); (A) showed the representative image of immunoblots. (B) was the quantitative analysis of the protein levels of JNK and P-JNK from six hepatic tissues in each group. The data were normalized to the loading control b-actin. (Abbreviations: sham, sham-operated; control, control-treated; A20, ambroxol [20 mg/kg]etreated; A80, ambroxol [100 mg/kg]etreated; A140, ambroxol [140 mg/ kg]etreated groups.) ##P < .05 versus sham-operated group, **P < .05 versus. control-treated group.
Bcl-2/Bax ratios. Ambroxol alleviated HI/R injury by suppressing apoptosis. ROS promote JNK activation.25e27 JNK is highly phosphorylated during warm HI/R injury and cold ischemia/ warm injury.28 JNK phosphorylation promotes cell apoptosis.29,30 It is well known that administration of JNK inhibitor alleviates I/R injury.31 Treatment with ambroxol reduced JNK activation by inhibiting c-jun phosphorylation. In conclusion, amborxol reduced HI/R by enhancing antioxidant and anti-apoptotic activities.
AMBROXOL
REFERENCES 1. Eguchi M, Miyazaki T, Masatsuji-Kato E, et al. Cytoprotection against ischemia-induced DNA cleavages and cell injuries in the rat liver by pro-vitamin C via hydrolytic conversion into ascorbate. Mol Cell Biochem. 2003;252:17. 2. Chandra J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress. Free Radic Biol Med. 2000;29:323. 3. Jaeschke H. Reactive oxygen and ischemia/reperfusion injury of the liver. Chem Biol Interact. 1991;79:115. 4. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med. 1985;312:159. 5. Sasaki H, Matsuno T, Nakagawa K, et al. Superoxide induces hepatocyte apoptosis during the early phase of reperfusion after murine liver ischemia. Transplant Proc. 1998;30:2958. 6. Kohli V, Selzner M, Madden JF, et al. Endothelial cell and hepatocyte deaths occur by apoptosis after ischemia-reperfusion injury in the rat liver. Transplantation. 1999;67:1099e1105. 7. Stetinova V, Herout V, Kvetina J. In vitro and in vivo antioxidant activity of ambroxol. Clin Exp Med. 2004;4:152. 8. Drews G, Tannapfel A, Gnauk HG, et al. Protective effects of ambroxol in hypothermic liver preservation a transplant study. J Invest Surg. 2000;13:197. 9. Hausen B, Bahra M, Mueller P, et al. Donor pretreatment with ambroxol or dexamethasone fails to ameliorate reperfusion injury in experimental lung transplantation. Transpl Int. 1998;11:186. 10. Zhang F, Wang X, Tong L, et al. Matrine attenuates endotoxin-induced acute liver injury after hepatic ischemia/reperfusion in rats. Surg Today. 2011;41:1075. 11. Zhang F, Mao Y, Qiao H, et al. Protective effects of taurine against endotoxin-induced acute liver injury after hepatic ischemia reperfusion. Amino Acids. 2010;38:237. 12. Jiang H, Meng F, Li W, et al. Splenectomy ameliorates acute multiple organ damage induced by liver warm ischemia reperfusion in rats. Surgery. 2007;141:32. 13. Gillissen A, Bartling A, Schoen S, et al. Antioxidant function of ambroxol in mononuclear and polymorphonuclear cells in vitro. Lung. 1997;175:235. 14. Gillissen A, Scharling B, Jaworska M, et al. Oxidant scavenger function of ambroxol in vitro: a comparison with N-acetylcysteine. Res Exp Med (Berl). 1997;196:389. 15. Nowak D, Antczak A, Krol M, et al. Antioxidant properties of Ambroxol. Free Radic Biol Med. 1994;16:517.
2445 16. Beeh KM, Beier J, Esperester A, et al. Antiinflammatory properties of ambroxol. Eur J Med Res. 2008;13:557. 17. Kang KJ. Mechanism of hepatic ischemia/reperfusion injury and protection against reperfusion injury. Transplant Proc. 2002;34: 2659. 18. Teoh NC, Farrell GC. Hepatic ischemia reperfusion injury: pathogenic mechanisms and basis for hepatoprotection. J Gastroenterol Hepatol. 2003;18:891. 19. He SQ, Zhang YH, Venugopal SK, et al. Delivery of antioxidative enzyme genes protects against ischemia/reperfusioninduced liver injury in mice. Liver Transpl. 2006;12:1869. 20. Schauer RJ, Gerbes AL, Vonier D, et al. Glutathione protects the rat liver against reperfusion injury after prolonged warm ischemia. Ann Surg. 2004;239:220. 21. Perry DK, Smyth MJ, Stennicke HR, et al. Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis. J Biol Chem. 1997;272:18530. 22. Yao Y, Huang C, Li ZF, et al. Exogenous phosphatidylethanolamine induces apoptosis of human hepatoma HepG2 cells via the bcl-2/Bax pathway. World J Gastroenterol. 2009;15:1751. 23. Jin S, Dai CL. Attenuation of reperfusion-induced hepatocyte apoptosis is associated with reversed bcl-2/bax ratio in hemihepatic artery-preserved portal occlusion. J Surg Res. 2012;174:298. 24. Mackey TJ, Borkowski A, Amin P, et al. Bcl-2/Bax ratio as a predictive marker for therapeutic response to radiotherapy in patients with prostate cancer. Urology. 1998;52:1085. 25. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell. 2000;103:239. 26. Li C, Jackson RM. Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol. 2002;282: C227. 27. Torres M. Mitogen-activated protein kinase pathways in redox signaling. Front Biosci. 2003;8:369. 28. Bendinelli P, Piccoletti R, Maroni P, et al. The MAP kinase cascades are activated during post-ischemic liver reperfusion. FEBS Lett. 1996;398:193. 29. Aoki H, Kang PM, Hampe J, et al. Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes. J Biol Chem. 2002;277:10244. 30. Deng X, Xiao L, Lang W, et al. Novel role for JNK as a stress-activated Bcl2 kinase. J Biol Chem. 2001;276:23681. 31. Uehara T, Bennett B, Sakata ST, et al. JNK mediates hepatic ischemia reperfusion injury. J Hepatol. 2005;42:850.
I
SCHEMIA/REPERFUSION injury (I/R) occurs by the interrupted blood flow for a short period in the tissues and consequently aggravate the tissue injury after a period of ischemia. The liver often suffers I/R injury after some surgical procedures, such as liver transplantation and elective hepatic resection. The injury, which may also occur during hypovolemic shock or after severe trauma, may lead to liver failure, which results in a high morbidity and mortality. Therefore, hepatic I/R (HI/R) has always been an obstacle to the development of liver surgery. In previous studies, several mechanisms have been suggested to contribute to the pathophysiology of HI/R injury, including reactive oxygen species (ROS). ROS can result in chromosomal malformation, nucleic acid base changes, or DNA rupture which can induce hepatocellular apoptosis or necrosis.1 Chandra et al showed
From the Department of General Surgery, the Fifth Affiliated Hospital of Harbin Medical University, Daqing, Heilongjiang Province. (K.J., H.L., J.Z., I.T., F.Z.), the Department of General Surgery, Daqing Oilfield General Hospital, Daqing, Heilongjiang Province (X.W.), the Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, Changsha (X.M.), the Department of Pathology, the Fifth Affiliated Hospital of Harbin Medical University, Daqing, Heilongjiang Province (G.W.), and Daqing Campus Harbin Medical University, Daqing, Heilongjiang Province (Y.C.), China. Supported by the Nature Science Foundation of Heilongjiang Province (D200927). Address reprint requests to Prof. F Zhang, First Department of General Surgery, the Fifth Affiliated Hospital of Harbin Medical University, Building Street, Daqing, Heilongjiang Province, China. E-mail: [email protected]
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.04.007
Transplantation Proceedings, 45, 2439e2445 (2013)
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that increased tissue H2O2 can cause apoptosis by upregulating the Fas-FasL system. Furthermore, mitochondrial membranes are potentially damaged by H2O2, which can contribute to the release of pro-apoptotic substances therein. In addition, injured mitochondria promoted the shift of transcriptional factors into the nucleus, such as p53, and nuclear factor-kappa B (NF-kB). In addition, ROS enhances the expression of pro-apoptotic genes and impairs survivalrelated genes.2 In contrast, natural antioxidants alleviate I/ R injury. Changes in superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) reductase attenuate I/R injures in animal. ROS have been shown to exert a central role contributing to HI/R injury.3e5 Apoptosis is an important mechanism in HI/R injury. Kohli et al confirmed that 50% to 70% of liver sinusoidal endothelial cells and 40% to 60% of hepatocytes underwent apoptosis.6 Ambroxol hydrochloride (trans-4-[2-amino-3, 5-dibrombeombenzylamino]-cyclohexanol HCL), where chemical structure is shown in Fig 1, has been prescribed as a mucolytic for viscid or excessive mucus. Recent studies have shown anti-oxidative and anti-inflammatory properties of ambroxol, in vitro and in vivo.7 Drews et al has observed that ambroxol is beneficial for organ preservation in a transplantation model.8 However, Hausen et al did not show that pretreatment with ambroxol alleviated reperfusion injury in experimental lung transplantation.9 The present study examined the dose-related anti-oxidative and anti-inflammatory actions of ambroxol. Therefore, the present study was conducted to assess the hepatic I/R injury by histopathological and biochemical examinations. MATERIALS AND METHODS Animals Male Wistar rats weighing 240 40 g were obtained from our Animal Research Center for use consistent with internationally accepted principles. Measures were taken to minimize suffering and reduce the number of animals.
Induction of HI/R and Drug Administration HI/R was induced according to the method described previously.10e12 Under the chloral hydrate (200 mg/kg) and ether anesthesia, rats underwent a median laparotomy. The hepatoportal vein, hepatic arterial, and hepatic duct were separated to be occluded with an atraumatic vascular clamp for 30 minutes, followed by 6 hours reperfusion. During the reperfusion period the body temperature of the animals was kept constant with a heating blanket.
Fig 1. The chemical structure of ambroxol.
Thirty rats were randomly assigned to five groups (six rats each): sham, control, and ambroxol hydrochloride (Sigma-Aldrich, Shanghai, China) treatments (20 mg/kg, 80 mg/kg, or 140 mg/kg). The sham group merely underwent a median laparotomy without occlusion of the hepatoportal vein, hepatic artery and hepatic duct. The control and ambroxol groups underwent HI/R before injections of the same volume of physiological saline or ambroxol though the tail veil. At the end of the reperfusion period, rats were sacrificed to collect blood and liver samples. Blood samples were centrifuged at 3000 g for 5 minutes to collect serum for determinations of alanine transaminase (ALT) and asparate transaminase (AST) levels. The hepatic tissue samples were stored at 80 C before measurements of tissue malondialdehyde (MDA), GSH, CAT, and SOD levels, together with estimation of caspase-3, Bcl-2, and Bax activities. Separate hematoxylin-eosin (HE) stained tissue samples underwent scoring under light microscopy.
Histologic Examination Liver specimens were fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 12 hours, followed by 2 days in 30% sucrose buffer. Serial coronal sections (6-mm thick) obtained using a microtome were stained with HE for light microscopy. The liver histopathological assessment was performed in blinded fashion.
Biochemical Analysis Automated analyzer autobiochemical analyzer (Toshiba, Tokyo, Japan) was used as described previously10e13 to measure AST and ALT (U/L).
The Activities of SOD, CAT, GSH, and MDA The enzymatic activities of SOD, and CAT, GSH, and MDA were measured according to the manufacturer’s instructions using commercial assay kits (Nanjing Jian Cheng Bioengineering Institute, China) SOD activity, (U/mg protein) in hepatic tissue homogenate was estimated by evaluating the rate of inhibition of nucleotide oxidation. CAT (U/mg protein) was assayed by quantifying the flaxen complex configurated by ammonium molybdate and the reminder peroxide at 405 nm. The GSH content mg GSH per gram protein was assayed by quantifying the rate of oxidation of reduced to oxidized glutathione by H2O2. The MDA concentration was determined by measuring thiobarbituricacid reactive substances (nmol MDA per milligram protein) at 532 nm.
Western Blot Hepatic samples were homogenized in an ice-cold lysis buffer, 10 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 10% glycerol, 1% Nonidet P-40, 5 mmol/ ethylene diamine tetraacetic acid (EDTA), and protease inhibitor cocktail. The resulting homogenate was centrifuged at 13,200 g for 20 minutes at 4 C. The supernate total protein content was estimated using a bicinchonininc acid (BCA) protein assay kit with bovine serum albumin as the standard (Beyotime Institute of Biotechnology, China). Equal 50 mg protein amounts were loaded into 10% sodium dodecyl sulfate (SDS)e polyacrylamide gels proteins were transferred onto nitrocellulose membranes (Millipore, MA), which have been blocked in 5% nonfat dry milk in Tris-buffered saline with Tween-20 (TBST) for 1 hour. Thereafter the manufactures were incubated at 4 C overnight with affinity-purified anti-caspase-3 (1:300; Cell Signaling, USA), anti-Bcl-2 (1:200; Santa Cruz, CA, USA), anti-Bax (1:200; Santa Cruz, CA, USA), anti-JNK (1:500; Santa Cruz, CA, USA) or antib-actin (1:2000; Santa Cruz, CA, USA). After three TBST rinses at
AMBROXOL 10-minute intervals, the membranes were incubated for 2 hours with peroxidase-labeled goat anti-rabbit (immunoglobulin 1:5000; Santa Cruz). Immunodetection was implemented with enhanced chemiluminesecence (ECL; Applygen) followed by x-ray film exposured. b-actin was the internal reference for quantification. Immunoblots scanned by the densitometer were subjected to grey analysis with Quantity One software (BioRad).
Statistical Analysis Comparisons between groups were analyzed with one-way analysis of variance and then Dunnett’s test using SPSS 13.0 software. P < .05 was considered to be statistically significant.
RESULTS Histopathological Examination
Figures 2A and 2F show the sham group to display normal appearances. In contrast, Figs 2B and 2G show color fading of hepatocytes cytoplasm with nuclear condensation. These changes were notably reduced among the group treated with ambroxol (20 mg/kg; Figs 2C and 2G). Furthermore, the 80 mg/kg and 140 mg/kg ambroxol cohorts showed significant improvement as in Figs 2I, 2E, and 2J. Serum ALT and AST
Compared with the sham group, the control group levels of serum ALT, as liver damage markers, were significantly increased (Fig 3A) from 34.33 8.66 to 2055.17 222.60 U/L (P < .05, n ¼ 6). The ambroxol group (20 mg/kg, 80 mg/kg, 140 mg/kg) content was markedly decreased from 2055.17 222.60 to 1385.83 212.20 U/L (P < .05, n ¼ 6); 905.50 121.26 (P < .05, n ¼ 6), and 845.67 107.97 U/L (P < .05, n ¼ 6), respectively, compared with the control group. Similar to serum ALT, level control group AST was notably greater than the sham group (Fig 3B) from 90.83 23.23 to 2477.33 238.23 U/L (P < .05, n ¼ 6). The ambroxol group (20 mg/kg, 80 mg/kg, 140 mg/kg) showed dramatically decreased expression from 2055.17 222.60 to 1727.00 243.85 U/L (P < .05, n ¼ 6); 1057.67 197.25 (P < .05, n ¼ 6), and 982.33 122.81 U/L (P < .05, n ¼ 6), respectively, compared with the control group. The Activities of Antioxidant Enzymes (SOD, CAT) and the Content of MDA and GSH in Hepatic Tissue
Figure 4A shows the activity of SOD as one of the most important antioxidant enzymes, to be significantly reduced among the controls from 314.91 35.81 to 204.41 34.53 U/ mg protein (P < .05, n ¼ 6) compared with the sham group. After administration with ambroxol (80 mg/kg, or 140 mg/kg), the SOD activity was significantly enhanced from 204.41 34.53 to 265.87 34.37 (P < .05, n ¼ 6), and 269.26 33.11 U/ mg protein (P < .05, n ¼ 6), although there was no significant difference when treated with 20 mg/kg of ambroxol. Similar to SOD, the CAT activity in the control group decreased from 34.02 4.38 (P < .05, n¼6) to 22.37 3.14 U/mg protein (P < .05, n ¼ 6) compared with the sham cohort. After 80 mg or 140 mg, ambroxol the CAT activity was notably enhanced from 22.37 3.14 to 28.79 3.25 (P < .05, n ¼ 6) or 27.33
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1.98 U/mg protein (P < .05, n¼6), respectively, compared with the controls although 20 mg of ambroxol had no effect (Fig 4B). Furthermore, Fig 4C shows the GSH quantity in the control group dramatically decreased from 2.99 0.17 to 1.87 0.15 mg/g protein (P < .01, n ¼ 6) compared with the sham group. In contrast, the content of GSH was increased after administration of ambroxol (20 mg/kg, 80 mg/kg, 140 mg/kg), to 2.01 0.16; 2.38 0.11 (P < .05, n ¼ 6), and 2.26 0.13 mg/ g protein (P < .05, n ¼ 6). Additionally, the MDA content (Fig 4D), marker of lipid peroxidation, was significantly increased in control group hepatic tissue from 4.64 0.90 to 7.59 0.92 nmol/mg protein (P < .05, n ¼ 6), compared with the sham cohert. After administration of ambroxol (20 mg/kg, 80 mg/kg, or 140 mg/kg), the MDA content decreased to 6.87 0.65, 5.49 0.64 (P < .05, n ¼ 6) or 5.48 0.54 nmol/mg protein (P < .05, n ¼ 6), respectively. The B-cell Lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-3 Activity
Western blot analysis of Bcl-2, Bax, and caspase-3 proteins in hepatic tissue was performed. Figure. 5 (BeD) showed the control cohort, Bcl-2 to be remarkably reduced (1.47 0.04 to 0.80 0.04; P < .05, n ¼ 6) compared with the sham group. The 140-mg/kg ambroxol group revealed a remarkable increases to 1.09 0.04 (P < .05, n ¼ 6). The control displayed significantly increased protein expression of Bax from 0.88 0.06 to 1.16 0.05 (P < .05, n ¼ 6) compared with the sham group. The ambroxol 80mg /kg and 140 mg/kg groups were dramatically decreased (0.97 0.07; P < .05; n ¼ 6 and 0.94 0.03; P < 0.05; n ¼ 6), respectively compared with controls. Western blots of caspase-3 antibody showed the anticipated bands of 17 kDa. Compared with the sham control. Control Caspase-3 level in hepatic tissue was abundantly increased from 0.91 0.05 to 1.22 0.07 (P < .05, n ¼ 6). However, the caspase-3 protein levels after ambroxol 80 and 140 mg/kg were remarkably decreased to 0.92 0.07 (P < .05, n ¼ 6) and 0.87 0.07 (P < .05, n ¼ 6). The Phosphorylated Level of c-Jun N-terminal Kinases (JNK)
Figure 6 shows that the activated JNK phospho-JNK level in the control group was notably enhanced from 0.80 0.04 to 1.27 0.08 (P < .05, n ¼ 6) compared with the sham group. However, JNK activation in the group treated with 140 mg/kg ambroxol was significantly reduced to 0.78 0.05 (P < .05, n ¼ 6). DISCUSSION
Ambroxol, a common mucoactive drug with secretolytic actions, is frequently used to treat respiratory diseases. Ambroxol is not only an expectorant but also has other effects, including antioxidative13e15 and anti-inflammatory activities.16 Some studies have shown ambroxol to mitigate I/R effects.8 But there is no prior evidence of protection from hepatic warm I/R injury as shown herein by remarkable actions on serum transaminases (ALT and AST) and histology. HE staining showed 80 mg/kg or 140 mg/kg
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Fig 2. Effects of ambroxol on the hepatic tissue 6 hours after hepatic reperfusion with hematoxylin-eosin staining. The representative micrographs of hepatic tissue from (A) and (F), sham-operated; (B) and (G), control-treated; (C) and (H), ambroxol (20 mg/kg)etreated; (D) and (I), ambroxol (80 mg/kg)etreated; (E) and (J), ambroxol (140 mg/kg)etreated groups were established. (Original magnifications [AeE]; 400 [FeJ]). The pykcnosis in the control and amboxol (140 mg/kg) groups was marked. (Scale bar: 10 mm.)
ambroxol to significantly alleviate hepatic damage compared with controls. Many factors contribute to I/R injury with ROS accumulation, massive granulocyte infiltration, and hepatocyte apoptosis. ROS play significant roles during HI/R injury.17,18 Enhanced hepatic antioxidant ability can reduce
damage induced by I/R. Transgenic mice overexpressing SOD and CAT display significant improved HI/R injury compared with normal hosts.19 Administration intravenous GSH after HI/R protects hepatocytes and improves animal survival.20 The MDA level, a criterion to evaluate the severity of reperfusion injury, is increased during I/R.
AMBROXOL
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Fig 3. Effects of ambroxol on the level of serum alanine transaminase (ALT) and aspartate transaminase (AST) of rat that underwent hepatic ischemia reperfusion 6 hours after ischemia (mean SD, n ¼ 6) as the (A) and (B) show, respectively. ## P < .05 versus. sham-operated group, *P < .05, **P < .05, and ***P < .05 versus control-treated group. (Abbreviations: sham, sham-operated; control, controltreated; A20, ambroxol [20 mg/kg]etreated; A80, ambroxol [80 mg/kg]etreated; A140, ambroxol [140 mg/kg]etreated groups.)
Under physiological conditions, ROS are quickly detoxified by endogenous enzymes and low-molecular weight antioxidantsdSOD, CAT and GSH. In our study, the SOD and CAT, activities after applying ambroxol, were dramatically higher compared with controls, but the MDA content significantly lower. The present results indicated that the actions of ambroxol to alleviate HI/R injury might be associated with its antioxidant activity.
Hepatocyte apoptosis also plays an important part in the damage induced by I/R. Multiple factors activate apoptotic cascade proteins. The caspase family is progressively activated by apoptotic stimuli. Caspase-3, an executioner of apoptosis, plays a dominant role.21 Much evidence supports the role of up regulated caspase-3 after hepatic ischemia. In addition, Bcl-2 family plays important roles in hepatocyte apoptosis, particularly Bcl-2
Fig 4. Effects of ambroxol on the activities of antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT]) and the concentration of malondiadehyde (MDA) and glutathione (GSH) on hepatic tissue 6 hours after reperfusion (mean SD, n ¼ 6). (A)e(D) show the effects of ambroxol on MDA, GSH, SOD, and CAT activities in hepatic tissue, respectively. ##P < .05 versus sham-operated group, **P < .05, ***P < .05 versus control-treated group. (Abbreviations: sham, sham-operated; control, control-treated; A20, ambroxol [20 mg/kg]etreated; A80, ambroxol [80 mg/kg]etreated; A140, ambroxol [140 mg/kg]etreated groups.)
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Fig 5. Effects of ambroxol on the protein expressions of Bcl-2, Bax, and Caspase-3 in hepatic tissue (mean SD, n ¼ 6); (A) shows the representative image of immunoblots. (B) (C), and (D) were the quantitative analysis of the protein levels of Bcl-2 Bax and Caspase-3 from six hepatic tissue in each group. The data were normalized to the loading control b-actin. Abbreviations: sham, sham-operated; control, controltreated; A20, ambroxol [20 mg/kg]etreated; A80, ambroxol [100 mg/kg]etreated; A140, ambroxol [140 mg/kg]etreated groups. ## P < .05 versus. sham-operated group. *P < .05, **P < .05 versus control-treated group.
and Bax. The Bcl-2, a key regulator of apoptosis, is beneficial for cell survival by inhibiting caspase activation. However, Bax exerts the reverse role to regulate apoptosis. The Bcl-2/Bax ratio determines the fate cells after an apoptotic stimulus. If the Bcl-2 level is higher than that of Bax, caspase activation is suppressed; when Bax is higher caspase is activated and the cell undergoes apoptosis.22e24 Our study showed that the caspase-3 activity in the group treated with ambroxol was remarkably decreased compared with the controls with, higher
Fig 6. Effects of ambroxol on the protein expressions of JNK and P-JNK in hepatic tissue (mean SD, n ¼ 6); (A) showed the representative image of immunoblots. (B) was the quantitative analysis of the protein levels of JNK and P-JNK from six hepatic tissues in each group. The data were normalized to the loading control b-actin. (Abbreviations: sham, sham-operated; control, control-treated; A20, ambroxol [20 mg/kg]etreated; A80, ambroxol [100 mg/kg]etreated; A140, ambroxol [140 mg/ kg]etreated groups.) ##P < .05 versus sham-operated group, **P < .05 versus. control-treated group.
Bcl-2/Bax ratios. Ambroxol alleviated HI/R injury by suppressing apoptosis. ROS promote JNK activation.25e27 JNK is highly phosphorylated during warm HI/R injury and cold ischemia/ warm injury.28 JNK phosphorylation promotes cell apoptosis.29,30 It is well known that administration of JNK inhibitor alleviates I/R injury.31 Treatment with ambroxol reduced JNK activation by inhibiting c-jun phosphorylation. In conclusion, amborxol reduced HI/R by enhancing antioxidant and anti-apoptotic activities.
AMBROXOL
REFERENCES 1. Eguchi M, Miyazaki T, Masatsuji-Kato E, et al. Cytoprotection against ischemia-induced DNA cleavages and cell injuries in the rat liver by pro-vitamin C via hydrolytic conversion into ascorbate. Mol Cell Biochem. 2003;252:17. 2. Chandra J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress. Free Radic Biol Med. 2000;29:323. 3. Jaeschke H. Reactive oxygen and ischemia/reperfusion injury of the liver. Chem Biol Interact. 1991;79:115. 4. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med. 1985;312:159. 5. Sasaki H, Matsuno T, Nakagawa K, et al. Superoxide induces hepatocyte apoptosis during the early phase of reperfusion after murine liver ischemia. Transplant Proc. 1998;30:2958. 6. Kohli V, Selzner M, Madden JF, et al. Endothelial cell and hepatocyte deaths occur by apoptosis after ischemia-reperfusion injury in the rat liver. Transplantation. 1999;67:1099e1105. 7. Stetinova V, Herout V, Kvetina J. In vitro and in vivo antioxidant activity of ambroxol. Clin Exp Med. 2004;4:152. 8. Drews G, Tannapfel A, Gnauk HG, et al. Protective effects of ambroxol in hypothermic liver preservation a transplant study. J Invest Surg. 2000;13:197. 9. Hausen B, Bahra M, Mueller P, et al. Donor pretreatment with ambroxol or dexamethasone fails to ameliorate reperfusion injury in experimental lung transplantation. Transpl Int. 1998;11:186. 10. Zhang F, Wang X, Tong L, et al. Matrine attenuates endotoxin-induced acute liver injury after hepatic ischemia/reperfusion in rats. Surg Today. 2011;41:1075. 11. Zhang F, Mao Y, Qiao H, et al. Protective effects of taurine against endotoxin-induced acute liver injury after hepatic ischemia reperfusion. Amino Acids. 2010;38:237. 12. Jiang H, Meng F, Li W, et al. Splenectomy ameliorates acute multiple organ damage induced by liver warm ischemia reperfusion in rats. Surgery. 2007;141:32. 13. Gillissen A, Bartling A, Schoen S, et al. Antioxidant function of ambroxol in mononuclear and polymorphonuclear cells in vitro. Lung. 1997;175:235. 14. Gillissen A, Scharling B, Jaworska M, et al. Oxidant scavenger function of ambroxol in vitro: a comparison with N-acetylcysteine. Res Exp Med (Berl). 1997;196:389. 15. Nowak D, Antczak A, Krol M, et al. Antioxidant properties of Ambroxol. Free Radic Biol Med. 1994;16:517.
2445 16. Beeh KM, Beier J, Esperester A, et al. Antiinflammatory properties of ambroxol. Eur J Med Res. 2008;13:557. 17. Kang KJ. Mechanism of hepatic ischemia/reperfusion injury and protection against reperfusion injury. Transplant Proc. 2002;34: 2659. 18. Teoh NC, Farrell GC. Hepatic ischemia reperfusion injury: pathogenic mechanisms and basis for hepatoprotection. J Gastroenterol Hepatol. 2003;18:891. 19. He SQ, Zhang YH, Venugopal SK, et al. Delivery of antioxidative enzyme genes protects against ischemia/reperfusioninduced liver injury in mice. Liver Transpl. 2006;12:1869. 20. Schauer RJ, Gerbes AL, Vonier D, et al. Glutathione protects the rat liver against reperfusion injury after prolonged warm ischemia. Ann Surg. 2004;239:220. 21. Perry DK, Smyth MJ, Stennicke HR, et al. Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis. J Biol Chem. 1997;272:18530. 22. Yao Y, Huang C, Li ZF, et al. Exogenous phosphatidylethanolamine induces apoptosis of human hepatoma HepG2 cells via the bcl-2/Bax pathway. World J Gastroenterol. 2009;15:1751. 23. Jin S, Dai CL. Attenuation of reperfusion-induced hepatocyte apoptosis is associated with reversed bcl-2/bax ratio in hemihepatic artery-preserved portal occlusion. J Surg Res. 2012;174:298. 24. Mackey TJ, Borkowski A, Amin P, et al. Bcl-2/Bax ratio as a predictive marker for therapeutic response to radiotherapy in patients with prostate cancer. Urology. 1998;52:1085. 25. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell. 2000;103:239. 26. Li C, Jackson RM. Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol. 2002;282: C227. 27. Torres M. Mitogen-activated protein kinase pathways in redox signaling. Front Biosci. 2003;8:369. 28. Bendinelli P, Piccoletti R, Maroni P, et al. The MAP kinase cascades are activated during post-ischemic liver reperfusion. FEBS Lett. 1996;398:193. 29. Aoki H, Kang PM, Hampe J, et al. Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes. J Biol Chem. 2002;277:10244. 30. Deng X, Xiao L, Lang W, et al. Novel role for JNK as a stress-activated Bcl2 kinase. J Biol Chem. 2001;276:23681. 31. Uehara T, Bennett B, Sakata ST, et al. JNK mediates hepatic ischemia reperfusion injury. J Hepatol. 2005;42:850.