Abstract：Liver is one of the most important organ for aquatic animals, which plays a pivotal role in regulating various physiological processes, such as metabolism, conversion, detoxification, secretion and storage. The diversity of function makes the liver more vulnerable to the destruction of the exogenous substances. Quercetin (QC), a kind of flavonoids, derived from many plants, has diverse pharmacological effects including immune regulation, antioxidant, antitumor, hypoglycemic effects, reduction of blood fat, and so on. Although numerous studies about the healthy aspects of QC on humans and mammals have been reported, there is a lack report about its healthy effects on aquatic animals. This study was to explore the protective effect of quercetin on CCl4-induced liver injury in common carp (Cyprinus carpio). In this study, 1,1-dipheny l-2-picrylhydrazyl radical (DPPH) was applied to evaluate the radical scavenging capacity of QC. The hepatocytes were treated with different concentrations of QC before (pre-treatment), after (post-treatment) and both before and after (pre- and post-treatment) incubation with 8 mmol/L CCl4. Then the activities of glutamatepyruvate transaminase (GPT), aspartate transaminase (GOT), lactate dehydrogenase (LDH), and superoxide dismutase (SOD), as well as the release amount of malondialdehyde (MDA) in the supernatant were measured and the hepatocytes viability were assayed by MTT. The mRNA expression levels of cytochrome enzymes P450 1A (CYP1A), 3A (CYP3A), inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were detected by Real-time quantitative PCR (qRT-PCR). Also, the relative content of nuclear transcription factors-κB (NF-κB c-Rel and p65) were detected by Western blot. The results showed that QC can scavenge the radicals effectively even at low concentration. QC markedly inhibited the level of GOT, GPT and LDH, indicating that QC had a positive effect on the protection of cytoplasm enzyme from leaking. QC significantly decreased MDA and elevated SOD, suggesting that QC could alleviate the lipid peroxidation and improve the antioxidant capacity.QC positively inhibited the expression levels of CYP1A and CYP3A. Thus, we deduced that QC could intervene the CYP450 enzymes to reduce the metabolism injury. Furthermore, QC significantly inhibited the activities of c-Rel and p65, the mechanism might be that QC suppressed free radicals to alleviate the inflammatory response. Administration of QC in the pre-treatment group showed the best effect on the inhibition of CCl4-induced liver injury, followed by the pre- and post-treatment group, post-treatment group of somewhat less. Each group with QC at the concentration of 0.1 and 0.2 mg/mL showed signifficant effect on the protection of damaged cells. The findings presented that QC can protect the hepatocytes from the chemical damage and positively inhibited the mRNA expression of CYP1A and CYP3A, and immune inflammatory response.
鲍启德，杨兰兰，王利，等. 2008. 异甘草酸镁对CCl4诱导小鼠急性肝损伤的保护作用[J]. 世界华人消化杂志, 9:1004-1007（Bao Q D, Yang L L, Wang L, et al. 2003. Protective effects of magnesium isoglycyrrhizinate against carbon tetrachloride-induced acute liver injury in mice[J]. World Chinese Journal of Digestology, 9:1004-1007） 贾睿，曹丽萍，丁炜东，等. 2011. 鲫鱼肝细胞分离与原代培养方法的优化[J].华北农报, 26( 12) :206-212 (Jia R, Cao L P, Ding W D, et al. 2011.Optimization of Isolation and Primary culture of hepatocytes in Crucian carp (Carassius auratus). Acta Agriculturae Boreali-Sinica. 26(12) :206-212) 何娅娣，艾 明，阳学风. 2005. 槲皮素对大鼠肝损伤保护作用的实验研究[J]. 杭州师范学院学报(自然科学版), 4(3):165-167 ( He Y D, Ai M, Yang X F. 2005. A preliminary study on protective effects of Quercetin on experin ental liver injury in rats[J]. Journal of Hangzhou Normal University(Natural Science),4(3):165-167) 胡泽华，王琳琳，唐清，等. 2010. 内毒素血症肝损伤幼年大鼠肝脏NF-κB的变化[J].中国当代儿科杂志,2010,12(10):804-8.( Hu Z H, Wang L L, Tang Q. 2010. NF-κB levels in the liver of yong rats with endotoxemic liver injury[J]. Chinese Journal of Contemporary Pediatrics,12(10):804-808) 刘爽，姚平，李珂,等. 2007. 槲皮素对大鼠原代肝细胞酒精性氧化损伤的防护作用[J].营养学报, 29(3):288-291.(Liu S, Yao P, Li K. 2007. Protection of quercetin against ethanol-induced oxidative rat primary hepatocytes[J]. Acta Nutrimenta Sinica，29(3):288-291) 谭赛男，陆彩玲，崔熠, 等. 2008. 槲皮素对结肠癌细胞生长的影响[J]. 细胞生物学志， 30:105-108 (Tan S N, Lu C L, Cui Y, et al. 2008. Effect of quercetin on growth of colon cancer cell line RKO[J]. 30:105-108) 王超，李侃，张燕翔，等. 槲皮素固体分散体对四氯化碳致小鼠急性肝损伤的保护作用[J]. 时珍国医国药，2012,23（9）：2206-2207(Wang C, Li K, Zhang Y X et al. 2012. Protective effects of the solid dispersion of quercetin on acute liver injury induced by carbon tetrachloride in mice[J]. Lishizhen medicine and material medica research, 23（9）：2206-2207) 杨句容，魏来，赵春景. 2010. 槲皮素对实验性大鼠急性肝损伤的保护作用[J]. 现代医药卫生, 26(11): 1601-1602 (Yang J R, Wei L, Zhao C J. 2010. Protective effects of quercetin on the acute liver injury in mice [J]. Modern Medicine & Health, 26(11): 1601-1602) 尤红，王宝恩. 2000. 核因子-κB 在肝脏疾病中研究进展[J].临床肝胆杂志, 2000, 16:11-12.(You H, Wang B E. 2000. The development of Nuclear factor - kappa B in liver disease[J]. Chinese journal of clinical hepatology,16:11-12 朱玉婷，邢同岳，毛海芬. 2013. 槲皮素对高糖诱导的原代小胶质细胞炎性反应的抑制作用[J]. 中国医院药学杂志, 33（21）:1787-1790（Zhu Y T, Xing T Y, Mao H F. 2013. Inhibitory effect of quercetin on high-glucose induced inflammatory reaction in primary microglia cells[J]. Chinese Journal of Hospital Pharmacy, 33（21）:1787-1790） Arnold H, Plutah J, Braunbeck T. 1996. Sublethal effects of prolonged exposure to disulfoton in rainbow trout ( Oncorhynchus mykiss) :cytological alterations in the liver by a potent acetylcholine esterase inhibitor[J]. EcotoxicologyandEnvironmental Safety, 34(1) :43-55 Bastien M C, Leblond F, Pichette V, et al. 2010. Differential alteration of cytochrome P450 isoenzymes in two experimental models of cirrhosis[J].Canadian Journal of Ethnopharmacology, 131（9）：290-299 Braunbeck R T，Segne R H. 2000. Isolation and cultivation of teleost hepatocytes[J]. The Hepatocyte Review, 45( 2) :49-72 Cabello F C. 2006. Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment[J]. Environ Microbiol, 8(7):1137–1144 Gutteridge J M, Halliwell B. 2000. Free radicals and antioxidants in the year. A historical look to the future.Ann N Y Acad Sci, 899:136-47 Harman D. 1995. Free radical theory of aging:Alzheimer’s disease pathogenesis[J]. Age, 18(3): 97-119. Hassan M, Arash A, Hadi C, et al. 2010. The protective effect of Liquorice plant extract on CCl4-induced hepatotoxicity in common in common carp(Cyprinus carpio)[J]. Veterinary research forum, 1(3):158-164. Hattorit T，Ito M，Suzuki Y. 1991. Studies on antinephritic effects of plant components in rats.Effects of saikosaponins original-type anti-GBM nephritis in rats and its mechanisms[J]. Nihon Yakurigaku Zasshi Folia Pharmacol Jpn, 97(1)：13-19 Ma M，Liu G H，Yu Z H. 2009. Effect of the Lycium barbarum polysaccharides administratin on blood lipid metabolism and oxidative stress of mice fed high-fat diet in vivo[J].Food Chemis- try，113：872–877 Masada T, Hua Y, Xi G,et al. 2003. Overexpression of interleukin-1 receptor antagonist reduced brain edema induced by intracerebal hemorrhage and thrombin[J]. Acta Neurochir Suppl, 86(5):463-467. Masuda Y. 2006. Learning toxicology from carbon tetrachloride-induced hepatotoxicity [J].Yakugaku Zasshi, 126(10):885-899. Meng X, Ding Q . 2004. The causes of fish ‘‘liver and gall syndrome’’ and methods for prevention and control. Reserv Fish, 24:65–67 Mensor L L, Menezes F S, Leitao G G, et al. 2001. Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free rdical method[J]. Phytother Res, 15:127-130 Myers M S，Landahl J T，Krahn M M, et al. 1991. Relationship between hepatic neoplasms andrelated lesions and exposure to toxic chemicals in marine fish from tire U S West Coast Environ[J].Health Persp, 90:7-15. Nemeroff C B, Devane C L, Pollack B G. 1996. Newer antidepressants and the cytochrome P450 system[J]. The American Journal of Psychiatry, 153(3):311-320 Palmes D, Spiegel H U. 2004. Animal models of liver regeneration [J]. Biomaterials,25(9):1601. Sattu K, Radhakrishnan V, Pandi A,et al. 2007. The effects of quercetin on antioxidant status and tumor markers inthe lung and serum of mice treated with benzo(a)pyrene[J].Biol Pharm Bull, 30(12):2268-2273. Taniguchi M, Takeuchi T, Nakatsuka R,et al. 2004. Molecular process in acute liver injury and regeneration induced by carbon tetrachloride[J]. Life Sci, 75(13):1539-1549. Wai M L, Julie M, Proudfoot S S，et al. 2008. Metabolic transformation has a profound effect on anti-inflammatory activity of flavonoids such as quercetin ：Lack of association between antioxidant and lipoxygenase inhibitory activity.Biochemical pharmacology, 7 (5):1045-1053 Wang J H，Dong J C，Zhao J X，et al． 2011. Effects of different molecule weight Astragalus polysacharin isolated from annual astragalus membra-neaceus on experssions of inflammatory cytokines in RAW264．7 cells［J］． Journal of Jilin University: Medicine Edition，6( 12) :28-38 Weber L W, Boll M, Stampfl A. 2003. Hepatotoxicity and mechanism of action of haloalkanes:carbon tetrachloride as a toxicological model.Crit. Rev. Toxicol ,33:105-136. Yin G J，Cao L P，XU P，et al. 2011. Hepatoprotective and antioxidant effects of Glycyrrhiza glabra extract against carbon tetrachloride( CCl4) -induced hepatocyte damage in common carp ( Cyprinus carpio)[J]. Fish Physiology and Biochemistry, 37(1) :209-216 Yin G J, Cao L P, Xu P, et al. 2011. Hepatoprotective and antioxidant effects of Hibiscus sabdariffa extract against carbon tetrachloride induced hepatocyte damage in Cyprinus carpio[J].In Vitro Cell Dev Biol Animal, 47:10-15.
