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Hepatoprotective effects of Yulangsan polysaccharide against isoniazid and rifampicin-induced liver injury in mice
Journal of Ethnopharmacology 152 (2014) 201–206
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Research Paper
Hepatoprotective effects of Yulangsan polysaccharide against isoniazid and rifampicin-induced liver injury in mice Yizhu Dong 1,2, Jianchun Huang 1, Xing Lin, Shijun Zhang, Yang Jiao, Tao Liang, Zhaoni Chen, Renbin Huang n Department of Pharmacology, Guangxi Medical University, Nanning 530021, China
art ic l e i nf o
a b s t r a c t
Article history: Received 2 September 2013 Received in revised form 31 December 2013 Accepted 1 January 2014 Available online 8 January 2014
Ethnopharmacological relevance: Yulangsan polysaccharide (YLSPS) is often used in popular folk medicine in the Guangxi Zhuang Autonomous Region of China as a chief ingredient of Millettia pulchra, which is used as an hepatic protection, anti-aging and memory improving agent. In this study, the hepatoprotective effects of YLSPS against isoniazid (INH) or rifampicin and isoniazid (RFPþINH)-induced liver injury were investigated in mice. Materials and methods: The liver injury was induced by intragastric administration of INH or RFP þINH daily for 10 days. During the experiment, the model group received INH or RFPþ INH only, and the normal control group received an equal volume of saline. Treatment groups received not only INH or RFPþ INH but also the corresponding drugs, DDB (200 mg/kg/day) or YLSPS (100, 200, and 400 mg/kg/day) 2 h after the administration of INH or RFPþ INH. Results: Analysis experiments showed that YLSPS significantly alleviated liver injury as indicated by the decreased levels of ALT and AST and the increased levels of SOD, GSH and GSH-Px. Moreover, YLSPS could effectively reduce the pathological tissue damage. The research on the mechanisms underlying the hepatoprotective effect showed that YLSPS was able to reduce lipid peroxidation and activate the antioxidative defense system. Conclusion: Our results show that YLSPS is effective in attenuating hepatic injury in the INH or RFPþ INHinduced mouse model, and could be developed as a new drug for treatment of liver injury. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Yulangsan polysaccharide (YLSPS) Hepatoprotective effects Rifampin Isoniazid
1. Introduction Tuberculosis is a fatal communicable disease that is easily spread among people. The WHO has declared that tuberculosis is a global emergency, with 9 million new cases and almost 2 million deaths per year worldwide (WHO, 2008). Multi-drug resistant strains of Mycobacterium tuberculosis have emerged. The administration of isoniazid (INH) or rifampicin and isoniazid (RFPþINH) is the most common medication prescribed against tuberculosis. These treatment protocols produce many metabolic and morphological aberrations in the liver because the liver is the main detoxifying site for these antitubercular drugs. Treatment with INH or RFPþINH can also induce hepatitis through a multiple step mechanism. This induction is characterized by a fall in serum albumin concentration and a rise in serum globulin concentration (Saad et al., 2010). These effects are related to the severity and duration of the disease. Peroxidation of endogenous lipids has n
Corresponding author. Tel.: þ 86 771 5339805. E-mail address: [email protected] (R. Huang). 1 These authors contributed equally to this work. 2 Present address: Beihai People0 s Hospital, Beihai 536000, China.
0378-8741/$ - see front matter & 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2014.01.001
been shown to be a major factor in the cytotoxic action of INH or RFPþINH. Antitubercular-drug-mediated oxidative damage is generally attributed to the formation of highly reactive oxygen species, which act as stimulators of lipid peroxidation and a source of destruction and damage to cell membranes (Georgieva et al., 2004; Santhosh et al., 2007). Alterations of various cellular defense mechanisms consisting of enzymatic and non-enzymatic components have been reported in INH- or RFPþ INH-induced hepatoxicity (Tasduq et al., 2005). Marine polysaccharides have been shown to have a number of medicinal applications. Yulangsan (YLS) is the root of Millettia Pulchra (Benth.) Kurz var. Laxior (Dunn) Z. Wei (Lin et al., 2014). The extract of YLS has been demonstrated to be an effective antioxidant and is used in the treatment of neurological and cardiovascular diseases (Huang et al., 2003; Huang et al., 2008). Yulangsan polysaccharide (YLSPS) is the major effective ingredient in the extract of YLS (Lin et al., 2014), which is often used as an hepatic protection agent in folk medicine (Guangxi FDA, 2008; Dai, 2009). YLS is capable of both inhibiting peroxidation in vitro and suppressing the production of an excess of free radicals in vivo (Jiao et al., 2004). Furthermore, YLSPS alleviated the acute hepatic injury in mice and CCl4 induced liver fibrosis in rats (Duan et al., 2007, 2008; Fu et al., 2009). Based on these reports,
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it would be of great interest to see if YLSPS has beneficial effects on antitubercular drug-induced hepatoxicity and to explore its underlying mechanism. To test our hypothesis, a classic INH or RFPþINH-induced liver injury model was chosen to study the hepatoprotective effects of YLSPS in mice. The effect of YLSPS on liver injury was compared with that of dimethyl diphenyl bicarboxylate (DDB). DDB is a antihepatitis drug used in the therapy of chronic persistent hepatitis, chronic active hepatitis, drug-induced injury and other diseases (Hassan et al., 2010). DDB has been shown to directly protect hepatocyte DNA from oxidative damage, and it is capable of inhibiting tumor necrosis factor (TNF)-alpha mRNA expression in liver tissue. These effects of DDB have resulted in the prevention of liver damage (Gao et al., 2005). Park et al. (2005) have also demonstrated that DDB protects the liver from chemical-induced injury potentiated by glutathione (GSH) deficiency; DDB has the additional advantage of lowering plasma lipids. The markers of liver oxidative stress and anti-oxidative defense systems have also been examined for the investigation of the hepatoprotective mechanisms of YLSPS.
2. Materials and methods 2.1. Chemicals YLSPS was prepared by a method described previously (Lin et al., 2014). The root of the Millettia Pulchra (Benth.) Kurz var. Laxior (Dunn) Z. Wei was dried, powdered, and extracted three times with boiling water. The polysaccharide in the filtrate was precipitated fractionally with alcohol. The protein in the product was removed by the Sevag method and further purified using DEAE ion exchange cellulose (DEAE-52). The components of the saccharide were determined by GC and TLC. The results showed that YLSPS was composed of D-glucose and D-arabinose in a molar ratio of 90.79% and 9.21%, with an average molecular weight of 14,301 Da. Rifampicin (RFP) was purchased from Guangdong Southern China Pharmaceutical Group Co., Ltd (Guangdong, China); isoniazid (INH) was purchased from Shantou Jinshi General Pharmaceutical Factory (Shantou, China); dimethyl diphenyl bicarboxylate (DDB) was purchased from Guangzhou Xingqun Pharmaceutical Co., Ltd (Guangzhou, China); alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-Px), Coomassie (Bradford) protein assay kits were acquired from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China); all other chemicals were of analytical grade.
intragastrically; (ii) the INH-treated model group, animals received INH (100 mg/kg/day) intragastrically for 10 days; (iii) the DDBtreated group (positive control group), animals received DDB (200 mg/kg/day) intragastrically 2 h after administration of INH (100 mg/kg/day) for 10 days; (iv) the low-, medium- and highdosage YLSPS-treated groups, animals received YLSPS (100, 200 or 400 mg/kg) intragastrically 2 h after administration of INH (100 mg/kg/day) for 10 days. The drugs were dissolved in distilled water and diluted with physiologic saline for the animal tests. RFP þINH-induced liver injury model: After an acclimatization period of 1 week, the animals were divided into six groups (five male mice and five female mice/group) and treated for 10 days as follows: (i) the normal control group, animals received saline intragastrically; (ii) the RFPþ INH-treated model group, animals received RFP (100 mg/kg/day)þ INH (100 mg/kg/day) intragastrically for 10 days; (iii) the DDB-treated group (positive control group), animals received DDB (200 mg/kg/day) intragastrically 2 h after administration of RFP (100 mg/kg/day)þINH (100 mg/kg/day) for 10 d; (iv) the low-, medium- and high-dosage YLSPS-treated groups, animals received YLSPS (100, 200 or 400 mg/kg) intragastrically 2 h after administration of RFP (100 mg/kg/day)þINH (100 mg/kg/day) for 10 d. At the final stage of the experiment, the animals were fasted overnight and sacrificed on the next day by cervical dislocation immediately after withdrawal of blood from the retrobulbar vessels. Afterwards, the whole blood was centrifuged and serum samples were collected. Liver samples were dissected out and washed immediately with ice-cold saline to remove as much blood as possible. One fraction of the liver samples was immediately stored at 80 1C for future analysis; another fraction was excised and fixed in a 10% formalin solution for histopathologic analysis.
2.2. Animals Kunming mice of both sexes, weighing 207 2 g, SPF, were provided by the Experimental Animal Center of Guangxi Medical University (Guangxi, China). The studies were conducted according to protocols approved by our institutional ethical committee. All mice were housed under controlled conditions with temperature of 25 72 1C, relative humidity of 607 10%, room air changes 12–18 times/h, and a 12-h light/dark cycle. Food and water were available ad libitum. 2.3. Treatment INH-induced liver injury model: after an acclimatization period of 1 week, the animals were divided into six groups (five male mice and five female mice/group) and treated for 10 days as follows: (i) the normal control group, animals received saline
Fig. 1. (A) Effect of YLSPS on liver index in INH-induced hepatic injury mice. Results are presented as the mean7 S.E. (%) (n¼ 10). ap o0.05 compared with normal control group, bpo 0.05 compared with INH model group. (B) Effect of YLSPS on liver index in RFP þINH-induced hepatic injury mice. Results are presented as the mean7 S.E. (%) (n¼ 10). apo 0.05 compared with normal control group, bpo 0.05 compared with RFP þINH model group.
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2.4. Calculation of the liver index The liver index was calculated according to the formula: (mouse liver weight/mouse weight) 100%. 2.5. Analysis of oxidative stress markers Serum levels of ALT and AST were measured using commercially available kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer0 s instructions. 2.6. Estimation of antioxidant enzymes Liver tissues were washed with normal saline to remove any blood and blood clots, and they were then homogenized on ice in Tris–HCl (5 mmol/L containing 2 mmol/L EDTA, pH 7.4). Homogenates were centrifuged at 1000 g for 15 min at 4 1C. Aliquoted samples of the supernatants were used immediately for the assays of SOD, GSH and GSH-Px. All these enzymes were determined using commercially available kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer0 s instructions and our previous study. 2.7. Assay of lipid peroxidation products Lipid peroxidation was assessed by the measurement of the levels of MDA, which were determined following the instructions in the assay kit (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China). The MDA content was expressed as nmol per milligram protein.
Fig. 2. (A) Effects of YLSPS on serum ALT and AST. Results are presented as the mean 7 S.E. (%) (n ¼10). ap o 0.05 compared with normal control group, bp o 0.05 compared with INH model group. (B) Effects of YLSPS on serum ALT, AST. Results are presented as the mean 7 S.E. (%) (n ¼10). ap o 0.05 compared with normal control group, bp o 0.05 compared with RFPþ INH model group.
Fig. 3. Effects of YLSPS on liver SOD, GSH-Px, MDA and GSH. Results are presented as the mean7 S.E. (%) (n¼ 10). ap o0.05 compared with normal control group, b p o 0.05 compared with INH model group.
Fig. 4. Effects of YLSPS on liver SOD, GSH-Px, MDA and GSH. Results are presented as the mean7 S.E. (%) (n¼ 10). ap o0.05 compared with normal control group, b p o 0.05 compared with RFP þINH model group.
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2.8. Pathological examination A portion of the liver tissue was instantly fixed in 10% phosphate buffered formalin, processed by routine histology procedures, and then embedded in paraffin. Tissue sections (5 μm thick) were stained with hematoxylin–eosin (HE) and subsequently examined using a light microscope for histopathological examination.
control group, indicating that INH or RFP þ INH-induced hypertrophy of liver tissue. Conversely, the liver index in the high and the medium dosage LYSPS-treated groups and the DDB-treated groups was significantly reduced compared with that in the model group (P o0.05), and there was no significant difference compared with the normal control group. These results showed that the hepatic intumescence induced by INH or RFPþ INH had been decreased after treatment with YLSPS (Fig. 1A and B).
2.9. Statistical analysis 3.2. Effects of YLSPS on serum ALT and AST Statistical analysis was performed using SPSS 13.0 for Windows. One-way analysis of variance (ANOVA) was used to compare the means among different groups, and Tukey0 s test was used in the post hoc multiple comparisons. Minimum level of statistical significance was fixed at 0.05.
3. Results
Evaluation of the extent of liver injury in the mice was based on an analysis of serum ALT and AST activities. Compared with the normal control group, the activities of the two enzymes in the model groups treated with INH or RFPþINH were significantly increased. Conversely, animals treated with all dosages of YLSPS and DDB exhibited a significant decrease in the activities of ALT and AST compared with those in the model group (Po0.05) (Fig. 2A and B).
3.1. Effects of YLSPS on liver index in mice
3.3. Effects of YLSPS on antioxidant enzyme and lipid peroxidation
The liver index showed a significant increase in the model groups treated with INH or RFPþINH compared to the normal
Liver injury provoked a significant reduction in liver SOD and GSH-Px activities, produced a significant depletion of liver GSH
Fig. 5. Histologic results of tissues stained with HE under light microscopy in INH-induced liver injury mice (100 ). (A) Normal control; (B) INH-treated model; (C) 200 mg/kg DDBþINH; (D) 100 mg/kg YLSPSþ INH; (E) 200 mg/kg YLSPSþ INH; (F) 400 mg/kg YLSPSþ INH.
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Fig. 6. Histologic results of tissues stained with HE under light microscopy in RFPþ INH-induced liver injury mice (100 ). (A) Normal control; (B) RFPþ INH-treated model; (C) 200 mg/kg DDBþ RFPþ INH; (D) 100 mg/kg YLSPSþ RFPþ INH; (E) 200 mg/kg YLSPS þRFP þINH; (F) 400 mg/kg YLSPSþ RFPþ INH.
content, and led to a remarkable promotion of liver MDA content in the model group treated with INH compared with the normal control group. However, the results showed that liver SOD activities were increased by treatment with different dosage of YLSPS, and GSH-Px activities were increased by treatment with high-, and medium-dosages of YLSPS (Fig. 3A); the GSH concentration was clearly increased and the liver MDA production level was markedly decreased after treatment with different dosages of YLSPS (Fig. 3B). Liver injury provoked a significant reduction of liver SOD and GSH-Px activities, produced a significant depletion of liver GSH content, and resulted in a remarkable promotion of liver MDA content in the model group treated with RFP þ INH compared with normal control group. The results showed that liver SOD activities were clearly increased by treatment with high-dosage YLSPS; the GSH-Px activities were notably increased by treatment with highand medium-dosage of YLSPS (Fig. 4A) and the GSH concentrations were obviously increased by treatment with high- and medium-dosages of YLSPS. The liver MDA production level was markedly decreased after treatment with a high-dosage of YLSPS (Fig. 4B). 3.4. Histopathological examination Histological changes were assessed using hematoxylin–eosin (HE)-stained liver tissue sections from each treatment group. The
normal control group had normal lobular architecture with central veins and radiating hepatic cords (Figs. 5A and 6A). Typical pathological characteristics including predominantly macrovesicular steatosis, mononuclear inflammatory infiltrates in the portal triad region and marked sinusoidal dilatations in the model groups treated with INH or RFPþINH confirmed the successful establishment of liver injury (Figs. 5B and 6B). In contrast, the groups treated with YLSPS and DDB did not show pathological damages (Figs. 5D and F and 6D and F).
4. Discussion and conclusion During the treatment of tuberculosis, hepatotoxicity is the most common serious complication. The administration of INH or RFPþINH produces many metabolic and morphological aberrations in the liver because the liver is the main detoxifying site for these antitubercular drugs (Santhosh et al., 2007). Thus, the discovery of a novel and effective medicine to reverse the antitubercular drug-induced liver injury has become a major focus in this field. YLSPS is often used as a hepatic protection agent in folk medicine, and its hepatoprotective effects have been confirmed in our previous studies. Currently, we have focused our research on the comprehensive analysis of its effective preclinical use, and expect to develop a novel drug for the treatment of
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antitubercular drug-induced hepatoxicity with high efficacy and low toxicity. INH and RFP-induced hepatitis is due to biotransformation of these drugs into reactive metabolites that are capable of binding to cellular macromolecules (Georgieva et al., 2004). As an alternative to inducing cellular damage by covalent binding, there is evidence that these antitubercular drugs cause cellular damage through the induction of oxidative stress, a consequence of dysfunction of hepatic antioxidant defense system. The role of oxidative stress in the mechanism of INH and RFP-induced hepatitis has been reported by Attri et al. (2000). In this study, a model of INH or RFPþINH-induced liver injury was successfully developed through INH or RFPþ INH infusion for 10 days in mice, results showed that the level of lipid peroxidation in the serum and liver tissue of INH and RFPþINH-treated mice significantly increased as compared to that of normal control mice. Increases in the level of lipid peroxides in liver reflected the hepatocellular damage. The depletion of antioxidant defenses and/ or increase in free radical production deteriorates the prooxidant– antioxidant balance, leading to oxidative stress-induced cell death (Sodhi et al., 1997). The depletion of reduced GSH is known to result in enhanced lipid peroxidation, and excessive lipid peroxidation can cause increased GSH consumption (Onyema et al., 2006), as observed in the present study. Intragastric administration of YLSPS reversed the INH and RFPþINH-induced effects, as this treatment resulted in significantly elevated activities of SOD and GSH-Px and an increased concentration of GSH. The levels of MDA were markedly decreased, indicating that YLSPS might inhibit lipid peroxidation and effectively recruit the antioxidative defense system in liver injury. Amino transferases are an important class of enzymes that link carbohydrate and amino acid metabolism, thereby clearly establishing the relationship between the intermediates of the citric acid cycle and amino acids. ALT and AST are well known diagnostic indicators of liver disease. In cases of liver damage with hepatocellular lesions and parenchymal cell necrosis, these marker enzymes are released from the damaged tissues into the bloodstream. In the present study, the activities of these enzymes were significantly higher in the serum of the model groups of mice that were administered antitubercular drugs compared to control mice. Treatment with YLSPS and DDB effectively decreased the hepatic injury scores and serum biomarkers, indicating that YLSPS was effective in the inhibition of liver injury. Furthermore, the hepatic histological changes in the INH or RFPþINH-induced liver injury model were found to be similar to those induced by human antitubercular drugs. Analysis of the pathology revealed that INH or RFPþINH administration caused damage to the hepatic architecture and produced histological changes such as micro- and macro-vesicular fatty infiltration, Kupffer cell hyperplasia and sinusoidal dilatation in the liver. Similar results were observed in our previous studies. Indeed, YLS was capable of both inhibiting peroxidation in vitro and suppressing the production of excess free radicals in vivo (Jiao et al., 2004). Furthermore, YLSPS alleviated the acute hepatic injury in mice and CCl4 induced liver fibrosis in rats (Duan et al., 2007; Duan et al., 2008; Fu et al., 2009). These results provided valuable reference for the hepatoprotection of YLSPS against INH or RFPþINH-induced liver injury. In the present study a preliminary exploration of the potential mechanism underlying the hepatoprotective effects of YLSPS was performed and pointed towards a radical scavenging action and antioxidant activity. Other possible mechanisms through which YLSPS interferes with INH and RFPINH-induced toxicity will be investigated in further research.
In conclusion, the INH or RFPþINH-induced hepatic injury was normalized by YLSPS co-administration, indicating a possible cytoprotective role of YLSPS against drug-induced hepatitis. Thus YLSPS should be regarded as a new and promising agent with a high potential in the prevention and treatment of antitubercular drug-induced liver injury.
Acknowledgments The authors gratefully acknowledge financial support from the Science and Technology Research Development of Guangxi Province (No. 2012GXNSFBA053094), Scientific Research and Technology Development Research Projects of Guangxi Province (Nos. 0630002-2 and 10124008-6), Science and Technology Foundation Platform Construction Project of Guangxi Province (No. 12-97-20). References Attri, S., Rana, S., Vaiphei, K., Sodhi, C., Katyal, R., Goel, R., Nain, C., Singh, K., 2000. Isoniazid- and rifampicin-induced oxidative hepatic injury protection by N-acetylcysteine. Human Exp. Toxicol. 19, 517–522. Dai, B., 2009. Chinese Modern Yao Medicine. Guangxi Science and Technology Publishing House, Nanning, China, pp. 401–405 Duan, X., Jiao, Y., Zhang, S., Huang, R., 2007. Protective effects of Yulangsan polysaccharide (YLS) on primary cultured rat hepatocytes injury induced by H2O2. LiShiZhen Med. Mater. Med. Res. 18, 1592–1593. Duan, X., Jiao, Y., Zhang, S., Lu, X., Huang, R., 2008. Effects of Yulangsan polysaccharide on proliferation and collagen I production of hepatic stellate cells. China J. Mod. Med. 4, 423–425. Fu, S., Huang, J., Wang, N., Huang, R., 2009. Protective effect of Yulangsan polysaccharide on acute alcohol hepatic Injury in mice. China Pharm. 6, 406–408. Guangxi Food and Drug Administration, 2008. Standards of Guangxi Zhuang Medicine. Guangxi Science and Technology Publishing House, Nanning, China p. 72 Gao, M., Zhang, J., Liu, G., 2005. Effect of diphenyl dimethyl bicarboxylate on concanavalin A‐induced liver injury in mice. Liver Int. 25, 904–912. Georgieva, N., Gadjeva, V., Tolekova, A., 2004. New isonicotinoylhydrazones with SSA protect against oxidative-hepatic injury of isoniazid. Trakia J. Sci. 2, 37–43. Hassan, A.A., Tawakkol, W., El Barawy, A.A., 2010. The hepatoprotective effect of dimethyl 4, 4-dimethoxy 5, 6, 5, 6-dimethylene dioxy-biphenyl-dicarboxylate (DDB) on aflatoxin B1 induced liver injury. Life Sci. J. 7, 148–153. Huang, R., Jiao, Y., Jiang, W., 2003. Effect of Yulangsan extract on the cardiac hemodynamics and the coronary flow in rats. Chin. J. Hosp. Pharm. 6, 321–322. Huang, Y., Chen, J., Huang, R., Lin, X., Wang, N., 2008. Anti-inflammatory effect of Yulangsan extracts and the mechanisms Chinese. J. New Drugs 20, 1764–1767. Jiao, Y., Duan, X., Huang, R., 2004. Scavenging and inhibiting effect of Yulangsan extract on superoxide anion free radical and hydroxy free radical. J. Guangxi Med. Univ. 1, 22–23. Lin, X., Huang, Z., Chen, X., Rong, Y., Zhang, S., Jiao, Y., Huang, Q., Huang, R., 2014. Protective effect of Millettia pulchra polysaccharide on cognitive impairment induced by D-galactose in mice. Carbohydr. Polym. 101, 533–543. Onyema, O.O., Farombi, E.O., Emerole, G.O., Ukoha, A.I., Onyeze, G.O., 2006. Effect of vitamin E on monosodium glutamate induced hepatotoxicity and oxidative stress in rats. Indian J. Biochem. Biophys. 43, 20. Park, E.Y., Ki, S.H., Ko, M.S., Kim, C.W., Lee, M.H., Lee, Y.S., Kim, S.G., 2005. Garlic oil and DDB, comprised in a pharmaceutical composition for the treatment of patients with viral hepatitis, prevents acute liver injuries potentiated by glutathione deficiency in rats. Chem. Biol. Interact. 155, 82–96. Saad, E.I., El-Gowilly, S.M., Sherhaa, M.O., Bistawroos, A.E., 2010. Role of oxidative stress and nitric oxide in the protective effects of α-lipoic acid and aminoguanidine against isoniazid–rifampicin-induced hepatotoxicity in rats. Food Chem. Toxicol. 48, 1869–1875. Santhosh, S., Sini, T.K., Anandan, R., Mathew, P.T., 2007. Hepatoprotective activity of chitosan against isoniazid and rifampicin-induced toxicity in experimental rats. Eur. J. Pharmacol. 572, 69–73. Sodhi, C., Rana, S., Mehta, S., Vaiphei, K., Attari, S., Mehta, S., 1997. Study of oxidative-stress in isoniazid-rifampicin induced hepatic injury in young rats. Drug Chem. Toxicol. 20, 255–269. Tasduq, S.A., Peerzada, K., Koul, S., Bhat, R., Johri, R.K., 2005. Biochemical manifestations of anti-tuberculosis drugs induced hepatotoxicity and the effect of silymarin. Hepatol. Res. 31, 132–135. WHO Report, 2008. Global Tuberculosis Control: Surveillance, Planning, Financing. World Health Organization, Geneva (WHO/HTM/TB/2008.393)
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Research Paper
Hepatoprotective effects of Yulangsan polysaccharide against isoniazid and rifampicin-induced liver injury in mice Yizhu Dong 1,2, Jianchun Huang 1, Xing Lin, Shijun Zhang, Yang Jiao, Tao Liang, Zhaoni Chen, Renbin Huang n Department of Pharmacology, Guangxi Medical University, Nanning 530021, China
art ic l e i nf o
a b s t r a c t
Article history: Received 2 September 2013 Received in revised form 31 December 2013 Accepted 1 January 2014 Available online 8 January 2014
Ethnopharmacological relevance: Yulangsan polysaccharide (YLSPS) is often used in popular folk medicine in the Guangxi Zhuang Autonomous Region of China as a chief ingredient of Millettia pulchra, which is used as an hepatic protection, anti-aging and memory improving agent. In this study, the hepatoprotective effects of YLSPS against isoniazid (INH) or rifampicin and isoniazid (RFPþINH)-induced liver injury were investigated in mice. Materials and methods: The liver injury was induced by intragastric administration of INH or RFP þINH daily for 10 days. During the experiment, the model group received INH or RFPþ INH only, and the normal control group received an equal volume of saline. Treatment groups received not only INH or RFPþ INH but also the corresponding drugs, DDB (200 mg/kg/day) or YLSPS (100, 200, and 400 mg/kg/day) 2 h after the administration of INH or RFPþ INH. Results: Analysis experiments showed that YLSPS significantly alleviated liver injury as indicated by the decreased levels of ALT and AST and the increased levels of SOD, GSH and GSH-Px. Moreover, YLSPS could effectively reduce the pathological tissue damage. The research on the mechanisms underlying the hepatoprotective effect showed that YLSPS was able to reduce lipid peroxidation and activate the antioxidative defense system. Conclusion: Our results show that YLSPS is effective in attenuating hepatic injury in the INH or RFPþ INHinduced mouse model, and could be developed as a new drug for treatment of liver injury. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Yulangsan polysaccharide (YLSPS) Hepatoprotective effects Rifampin Isoniazid
1. Introduction Tuberculosis is a fatal communicable disease that is easily spread among people. The WHO has declared that tuberculosis is a global emergency, with 9 million new cases and almost 2 million deaths per year worldwide (WHO, 2008). Multi-drug resistant strains of Mycobacterium tuberculosis have emerged. The administration of isoniazid (INH) or rifampicin and isoniazid (RFPþINH) is the most common medication prescribed against tuberculosis. These treatment protocols produce many metabolic and morphological aberrations in the liver because the liver is the main detoxifying site for these antitubercular drugs. Treatment with INH or RFPþINH can also induce hepatitis through a multiple step mechanism. This induction is characterized by a fall in serum albumin concentration and a rise in serum globulin concentration (Saad et al., 2010). These effects are related to the severity and duration of the disease. Peroxidation of endogenous lipids has n
Corresponding author. Tel.: þ 86 771 5339805. E-mail address: [email protected] (R. Huang). 1 These authors contributed equally to this work. 2 Present address: Beihai People0 s Hospital, Beihai 536000, China.
0378-8741/$ - see front matter & 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2014.01.001
been shown to be a major factor in the cytotoxic action of INH or RFPþINH. Antitubercular-drug-mediated oxidative damage is generally attributed to the formation of highly reactive oxygen species, which act as stimulators of lipid peroxidation and a source of destruction and damage to cell membranes (Georgieva et al., 2004; Santhosh et al., 2007). Alterations of various cellular defense mechanisms consisting of enzymatic and non-enzymatic components have been reported in INH- or RFPþ INH-induced hepatoxicity (Tasduq et al., 2005). Marine polysaccharides have been shown to have a number of medicinal applications. Yulangsan (YLS) is the root of Millettia Pulchra (Benth.) Kurz var. Laxior (Dunn) Z. Wei (Lin et al., 2014). The extract of YLS has been demonstrated to be an effective antioxidant and is used in the treatment of neurological and cardiovascular diseases (Huang et al., 2003; Huang et al., 2008). Yulangsan polysaccharide (YLSPS) is the major effective ingredient in the extract of YLS (Lin et al., 2014), which is often used as an hepatic protection agent in folk medicine (Guangxi FDA, 2008; Dai, 2009). YLS is capable of both inhibiting peroxidation in vitro and suppressing the production of an excess of free radicals in vivo (Jiao et al., 2004). Furthermore, YLSPS alleviated the acute hepatic injury in mice and CCl4 induced liver fibrosis in rats (Duan et al., 2007, 2008; Fu et al., 2009). Based on these reports,
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it would be of great interest to see if YLSPS has beneficial effects on antitubercular drug-induced hepatoxicity and to explore its underlying mechanism. To test our hypothesis, a classic INH or RFPþINH-induced liver injury model was chosen to study the hepatoprotective effects of YLSPS in mice. The effect of YLSPS on liver injury was compared with that of dimethyl diphenyl bicarboxylate (DDB). DDB is a antihepatitis drug used in the therapy of chronic persistent hepatitis, chronic active hepatitis, drug-induced injury and other diseases (Hassan et al., 2010). DDB has been shown to directly protect hepatocyte DNA from oxidative damage, and it is capable of inhibiting tumor necrosis factor (TNF)-alpha mRNA expression in liver tissue. These effects of DDB have resulted in the prevention of liver damage (Gao et al., 2005). Park et al. (2005) have also demonstrated that DDB protects the liver from chemical-induced injury potentiated by glutathione (GSH) deficiency; DDB has the additional advantage of lowering plasma lipids. The markers of liver oxidative stress and anti-oxidative defense systems have also been examined for the investigation of the hepatoprotective mechanisms of YLSPS.
2. Materials and methods 2.1. Chemicals YLSPS was prepared by a method described previously (Lin et al., 2014). The root of the Millettia Pulchra (Benth.) Kurz var. Laxior (Dunn) Z. Wei was dried, powdered, and extracted three times with boiling water. The polysaccharide in the filtrate was precipitated fractionally with alcohol. The protein in the product was removed by the Sevag method and further purified using DEAE ion exchange cellulose (DEAE-52). The components of the saccharide were determined by GC and TLC. The results showed that YLSPS was composed of D-glucose and D-arabinose in a molar ratio of 90.79% and 9.21%, with an average molecular weight of 14,301 Da. Rifampicin (RFP) was purchased from Guangdong Southern China Pharmaceutical Group Co., Ltd (Guangdong, China); isoniazid (INH) was purchased from Shantou Jinshi General Pharmaceutical Factory (Shantou, China); dimethyl diphenyl bicarboxylate (DDB) was purchased from Guangzhou Xingqun Pharmaceutical Co., Ltd (Guangzhou, China); alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-Px), Coomassie (Bradford) protein assay kits were acquired from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China); all other chemicals were of analytical grade.
intragastrically; (ii) the INH-treated model group, animals received INH (100 mg/kg/day) intragastrically for 10 days; (iii) the DDBtreated group (positive control group), animals received DDB (200 mg/kg/day) intragastrically 2 h after administration of INH (100 mg/kg/day) for 10 days; (iv) the low-, medium- and highdosage YLSPS-treated groups, animals received YLSPS (100, 200 or 400 mg/kg) intragastrically 2 h after administration of INH (100 mg/kg/day) for 10 days. The drugs were dissolved in distilled water and diluted with physiologic saline for the animal tests. RFP þINH-induced liver injury model: After an acclimatization period of 1 week, the animals were divided into six groups (five male mice and five female mice/group) and treated for 10 days as follows: (i) the normal control group, animals received saline intragastrically; (ii) the RFPþ INH-treated model group, animals received RFP (100 mg/kg/day)þ INH (100 mg/kg/day) intragastrically for 10 days; (iii) the DDB-treated group (positive control group), animals received DDB (200 mg/kg/day) intragastrically 2 h after administration of RFP (100 mg/kg/day)þINH (100 mg/kg/day) for 10 d; (iv) the low-, medium- and high-dosage YLSPS-treated groups, animals received YLSPS (100, 200 or 400 mg/kg) intragastrically 2 h after administration of RFP (100 mg/kg/day)þINH (100 mg/kg/day) for 10 d. At the final stage of the experiment, the animals were fasted overnight and sacrificed on the next day by cervical dislocation immediately after withdrawal of blood from the retrobulbar vessels. Afterwards, the whole blood was centrifuged and serum samples were collected. Liver samples were dissected out and washed immediately with ice-cold saline to remove as much blood as possible. One fraction of the liver samples was immediately stored at 80 1C for future analysis; another fraction was excised and fixed in a 10% formalin solution for histopathologic analysis.
2.2. Animals Kunming mice of both sexes, weighing 207 2 g, SPF, were provided by the Experimental Animal Center of Guangxi Medical University (Guangxi, China). The studies were conducted according to protocols approved by our institutional ethical committee. All mice were housed under controlled conditions with temperature of 25 72 1C, relative humidity of 607 10%, room air changes 12–18 times/h, and a 12-h light/dark cycle. Food and water were available ad libitum. 2.3. Treatment INH-induced liver injury model: after an acclimatization period of 1 week, the animals were divided into six groups (five male mice and five female mice/group) and treated for 10 days as follows: (i) the normal control group, animals received saline
Fig. 1. (A) Effect of YLSPS on liver index in INH-induced hepatic injury mice. Results are presented as the mean7 S.E. (%) (n¼ 10). ap o0.05 compared with normal control group, bpo 0.05 compared with INH model group. (B) Effect of YLSPS on liver index in RFP þINH-induced hepatic injury mice. Results are presented as the mean7 S.E. (%) (n¼ 10). apo 0.05 compared with normal control group, bpo 0.05 compared with RFP þINH model group.
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2.4. Calculation of the liver index The liver index was calculated according to the formula: (mouse liver weight/mouse weight) 100%. 2.5. Analysis of oxidative stress markers Serum levels of ALT and AST were measured using commercially available kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer0 s instructions. 2.6. Estimation of antioxidant enzymes Liver tissues were washed with normal saline to remove any blood and blood clots, and they were then homogenized on ice in Tris–HCl (5 mmol/L containing 2 mmol/L EDTA, pH 7.4). Homogenates were centrifuged at 1000 g for 15 min at 4 1C. Aliquoted samples of the supernatants were used immediately for the assays of SOD, GSH and GSH-Px. All these enzymes were determined using commercially available kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer0 s instructions and our previous study. 2.7. Assay of lipid peroxidation products Lipid peroxidation was assessed by the measurement of the levels of MDA, which were determined following the instructions in the assay kit (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China). The MDA content was expressed as nmol per milligram protein.
Fig. 2. (A) Effects of YLSPS on serum ALT and AST. Results are presented as the mean 7 S.E. (%) (n ¼10). ap o 0.05 compared with normal control group, bp o 0.05 compared with INH model group. (B) Effects of YLSPS on serum ALT, AST. Results are presented as the mean 7 S.E. (%) (n ¼10). ap o 0.05 compared with normal control group, bp o 0.05 compared with RFPþ INH model group.
Fig. 3. Effects of YLSPS on liver SOD, GSH-Px, MDA and GSH. Results are presented as the mean7 S.E. (%) (n¼ 10). ap o0.05 compared with normal control group, b p o 0.05 compared with INH model group.
Fig. 4. Effects of YLSPS on liver SOD, GSH-Px, MDA and GSH. Results are presented as the mean7 S.E. (%) (n¼ 10). ap o0.05 compared with normal control group, b p o 0.05 compared with RFP þINH model group.
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2.8. Pathological examination A portion of the liver tissue was instantly fixed in 10% phosphate buffered formalin, processed by routine histology procedures, and then embedded in paraffin. Tissue sections (5 μm thick) were stained with hematoxylin–eosin (HE) and subsequently examined using a light microscope for histopathological examination.
control group, indicating that INH or RFP þ INH-induced hypertrophy of liver tissue. Conversely, the liver index in the high and the medium dosage LYSPS-treated groups and the DDB-treated groups was significantly reduced compared with that in the model group (P o0.05), and there was no significant difference compared with the normal control group. These results showed that the hepatic intumescence induced by INH or RFPþ INH had been decreased after treatment with YLSPS (Fig. 1A and B).
2.9. Statistical analysis 3.2. Effects of YLSPS on serum ALT and AST Statistical analysis was performed using SPSS 13.0 for Windows. One-way analysis of variance (ANOVA) was used to compare the means among different groups, and Tukey0 s test was used in the post hoc multiple comparisons. Minimum level of statistical significance was fixed at 0.05.
3. Results
Evaluation of the extent of liver injury in the mice was based on an analysis of serum ALT and AST activities. Compared with the normal control group, the activities of the two enzymes in the model groups treated with INH or RFPþINH were significantly increased. Conversely, animals treated with all dosages of YLSPS and DDB exhibited a significant decrease in the activities of ALT and AST compared with those in the model group (Po0.05) (Fig. 2A and B).
3.1. Effects of YLSPS on liver index in mice
3.3. Effects of YLSPS on antioxidant enzyme and lipid peroxidation
The liver index showed a significant increase in the model groups treated with INH or RFPþINH compared to the normal
Liver injury provoked a significant reduction in liver SOD and GSH-Px activities, produced a significant depletion of liver GSH
Fig. 5. Histologic results of tissues stained with HE under light microscopy in INH-induced liver injury mice (100 ). (A) Normal control; (B) INH-treated model; (C) 200 mg/kg DDBþINH; (D) 100 mg/kg YLSPSþ INH; (E) 200 mg/kg YLSPSþ INH; (F) 400 mg/kg YLSPSþ INH.
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Fig. 6. Histologic results of tissues stained with HE under light microscopy in RFPþ INH-induced liver injury mice (100 ). (A) Normal control; (B) RFPþ INH-treated model; (C) 200 mg/kg DDBþ RFPþ INH; (D) 100 mg/kg YLSPSþ RFPþ INH; (E) 200 mg/kg YLSPS þRFP þINH; (F) 400 mg/kg YLSPSþ RFPþ INH.
content, and led to a remarkable promotion of liver MDA content in the model group treated with INH compared with the normal control group. However, the results showed that liver SOD activities were increased by treatment with different dosage of YLSPS, and GSH-Px activities were increased by treatment with high-, and medium-dosages of YLSPS (Fig. 3A); the GSH concentration was clearly increased and the liver MDA production level was markedly decreased after treatment with different dosages of YLSPS (Fig. 3B). Liver injury provoked a significant reduction of liver SOD and GSH-Px activities, produced a significant depletion of liver GSH content, and resulted in a remarkable promotion of liver MDA content in the model group treated with RFP þ INH compared with normal control group. The results showed that liver SOD activities were clearly increased by treatment with high-dosage YLSPS; the GSH-Px activities were notably increased by treatment with highand medium-dosage of YLSPS (Fig. 4A) and the GSH concentrations were obviously increased by treatment with high- and medium-dosages of YLSPS. The liver MDA production level was markedly decreased after treatment with a high-dosage of YLSPS (Fig. 4B). 3.4. Histopathological examination Histological changes were assessed using hematoxylin–eosin (HE)-stained liver tissue sections from each treatment group. The
normal control group had normal lobular architecture with central veins and radiating hepatic cords (Figs. 5A and 6A). Typical pathological characteristics including predominantly macrovesicular steatosis, mononuclear inflammatory infiltrates in the portal triad region and marked sinusoidal dilatations in the model groups treated with INH or RFPþINH confirmed the successful establishment of liver injury (Figs. 5B and 6B). In contrast, the groups treated with YLSPS and DDB did not show pathological damages (Figs. 5D and F and 6D and F).
4. Discussion and conclusion During the treatment of tuberculosis, hepatotoxicity is the most common serious complication. The administration of INH or RFPþINH produces many metabolic and morphological aberrations in the liver because the liver is the main detoxifying site for these antitubercular drugs (Santhosh et al., 2007). Thus, the discovery of a novel and effective medicine to reverse the antitubercular drug-induced liver injury has become a major focus in this field. YLSPS is often used as a hepatic protection agent in folk medicine, and its hepatoprotective effects have been confirmed in our previous studies. Currently, we have focused our research on the comprehensive analysis of its effective preclinical use, and expect to develop a novel drug for the treatment of
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antitubercular drug-induced hepatoxicity with high efficacy and low toxicity. INH and RFP-induced hepatitis is due to biotransformation of these drugs into reactive metabolites that are capable of binding to cellular macromolecules (Georgieva et al., 2004). As an alternative to inducing cellular damage by covalent binding, there is evidence that these antitubercular drugs cause cellular damage through the induction of oxidative stress, a consequence of dysfunction of hepatic antioxidant defense system. The role of oxidative stress in the mechanism of INH and RFP-induced hepatitis has been reported by Attri et al. (2000). In this study, a model of INH or RFPþINH-induced liver injury was successfully developed through INH or RFPþ INH infusion for 10 days in mice, results showed that the level of lipid peroxidation in the serum and liver tissue of INH and RFPþINH-treated mice significantly increased as compared to that of normal control mice. Increases in the level of lipid peroxides in liver reflected the hepatocellular damage. The depletion of antioxidant defenses and/ or increase in free radical production deteriorates the prooxidant– antioxidant balance, leading to oxidative stress-induced cell death (Sodhi et al., 1997). The depletion of reduced GSH is known to result in enhanced lipid peroxidation, and excessive lipid peroxidation can cause increased GSH consumption (Onyema et al., 2006), as observed in the present study. Intragastric administration of YLSPS reversed the INH and RFPþINH-induced effects, as this treatment resulted in significantly elevated activities of SOD and GSH-Px and an increased concentration of GSH. The levels of MDA were markedly decreased, indicating that YLSPS might inhibit lipid peroxidation and effectively recruit the antioxidative defense system in liver injury. Amino transferases are an important class of enzymes that link carbohydrate and amino acid metabolism, thereby clearly establishing the relationship between the intermediates of the citric acid cycle and amino acids. ALT and AST are well known diagnostic indicators of liver disease. In cases of liver damage with hepatocellular lesions and parenchymal cell necrosis, these marker enzymes are released from the damaged tissues into the bloodstream. In the present study, the activities of these enzymes were significantly higher in the serum of the model groups of mice that were administered antitubercular drugs compared to control mice. Treatment with YLSPS and DDB effectively decreased the hepatic injury scores and serum biomarkers, indicating that YLSPS was effective in the inhibition of liver injury. Furthermore, the hepatic histological changes in the INH or RFPþINH-induced liver injury model were found to be similar to those induced by human antitubercular drugs. Analysis of the pathology revealed that INH or RFPþINH administration caused damage to the hepatic architecture and produced histological changes such as micro- and macro-vesicular fatty infiltration, Kupffer cell hyperplasia and sinusoidal dilatation in the liver. Similar results were observed in our previous studies. Indeed, YLS was capable of both inhibiting peroxidation in vitro and suppressing the production of excess free radicals in vivo (Jiao et al., 2004). Furthermore, YLSPS alleviated the acute hepatic injury in mice and CCl4 induced liver fibrosis in rats (Duan et al., 2007; Duan et al., 2008; Fu et al., 2009). These results provided valuable reference for the hepatoprotection of YLSPS against INH or RFPþINH-induced liver injury. In the present study a preliminary exploration of the potential mechanism underlying the hepatoprotective effects of YLSPS was performed and pointed towards a radical scavenging action and antioxidant activity. Other possible mechanisms through which YLSPS interferes with INH and RFPINH-induced toxicity will be investigated in further research.
In conclusion, the INH or RFPþINH-induced hepatic injury was normalized by YLSPS co-administration, indicating a possible cytoprotective role of YLSPS against drug-induced hepatitis. Thus YLSPS should be regarded as a new and promising agent with a high potential in the prevention and treatment of antitubercular drug-induced liver injury.
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