Effect of Pure Total Flavonoids from Citrus on Hepatic SIRT1/PGC-1α Pathway in Mice with NASH
【Abstract】Objective: To observe the effect of pure total flavonoids from Citrus (PTFC) on the hepatic fatty degen-eration，inflammation，oxidative stress and SIRT1/PGC-1α expressions in mice with non-alcohol steatohepatitis (NASH), and discuss the action mechanism of PTFC on NASH. Method: Mice were given high-fat diet for 16 weeks to induce the NASH model. Since the seventh week after the model establishment，the mice were intervened with 100, 50 and 25 mg·kg -1·d -1 PTFC for 10 weeks. The pathologic changes in hepatic tissues were observed with HE staining. The contents of TG，CHOL in hepatic tissue，as well as the levels of AST，ALT in serum were detected by using the biochemical process. The expression of SIRT1，PGC-1α and MnSOD mRNA in hepatic tissues were detected with Real-time PCR assay. SIRT1，PGC-1α protein and 8-OHdG expressions were determined with the immunohistochemical method. The SOD level in hepatic tissues was tested by the xanthine oxidase method. The MDA content in hepatic tissues was examined by the thiobarbituric acid method. Result: The contents of TG，CHOL，NAFLD activity scores and ALT level in serum in hepatic tissues of mice in the model induced by fat-rich diet were remarkably higher than that of the control group (P < 0.01). The SIRT1，PGC-1α，MnSOD mRNA and protein expression in hepatic tissues were significantly lower than that of the control group (P < 0.01). The expres-sion of 8-OHdG and the content of MDA in hepatic tissues were significantly higher than that of the control group (P < 0.01). After the intervention with different doses of PTFC，the NAFLD activity scores，the content of TG and the level of AST in serum were notably lower than that of the control group (P < 0.01，P < 0.05); whereas the SIRT1，PGC-1α，MnSOD mRNA and protein expression were remarkably higher than that of the control group (P < 0.01，P < 0.05)，with the significant decrease in the expression of 8-OHdG and the content of MDA (P < 0.01). Conclusion: Oxidative stress/lipid peroxidation enhancement in NASH mice induced by high-fat diet may be relat-ed to the changes in SIRT1/PGC-1α signal transduction pathway. PTFC could enhance the anti-oxidant capacity in liver，relieve the damage of reactive oxygen during the fatty acid metabolic process，and prevent NASH from the occurrence and development by regulating the SIRT1/PGC-1α signal pathway.
【Keywords】 Non-alcoholic Steatohepatitis; Oxidative Stress; SIRT1; PGC-1α; Pure Total Flavonoids from Citrus;
 Day C P, James O F W. Steatohepatitis: a tale of two hits [J].Gastroenterology, 1998, 114(4): 842.
 Hwang J W, Yao H, Caito S, et al. Redox regulation of SIRT1 in in-flammation and cellular senescence [J]. Free Radic Biol Med, 2013, 61C: 95.
 Chen Y R, Fang S R, Fu Y C, et al. Calorie restriction on insulin re-sistance and expression of SIRT1 and SIRT 4 in rats [J]. Biochem Cell Biol, 2010, 88(4): 715.
 Purushotham A, Schug T T, Xu Q, et al. Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation [J]. Cell Metab, 2009, 9(4): 327.
 Banks A S, Kon N, Knight C, et al. SIRT1 gain of function increases energy efficiency and revents diabetes in mice [J]. Cell Metab, 2008, 8(4): 333.
 Yang X Q, Zhang H D & Li L. The antioxidants and antilipemic effects of flavonoids extracted from pomelo (Citrus grandis L. osbeck) peel [J]. Acta Nutrimenta Sinica, 2004, 26(5): 378–380 (In Chinese).
 Fatty Liver and Alcoholic Liver Disease Study Group of Chinese Liver Disease Association. Diagnostic criteria of nonalcoholic fatty liver dis-ease (the 2010 revised edition) [J]. Chin J Hepatol, 2003, 11(2): 71 (In Chinese).
 Xu L M, Liu P, Lu Gang, et al. Alterations of collagen type Ⅰ, Ⅳ and laminin in perisinusoids of fibrotic rat liver [J]. Chinese Journal of Di-gestion, 1995, 15(3): 146-148 (In Chinese).
 Zhao X M, Ye X Q & Xi Y F. Study on the extraction of flavonoids and antioxidation in huyou peels [J]. Journal of Fruit Science, 2003, 20(4): 261-265 (In Chinese).
 Jiang J P. Effect mechanism of Citrus grandis (L.) Osbeck peels on antioxidant flavonoid components gathering and blood fat reducing [D]. Nanchang, Jiangxi University of Traditional Chinese Medicine, 2011 (In Chinese).
 Chen L L, Deng X Q & Li N X. Effects of calorie restriction on SIRT1 expression in liver of nonalcoholic fatty liver disease experiment with rats [J]. Natl Med J China, 2007, 87(20): 1434-1436 (In Chinese).
 Li M, Chin J F, Mossman B T, et al. Down-regulation of manganese superoxide through phosphorylation of FOXO3a by AKt inexplanted vascular smooth muscle cells from old rats [J]. J Biol Chem, 2006, 28(1): 40429.
 Hou X, Xu S, Maitland-Toolan K A, et al. SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase [J]. J Biol Chem, 2008, 283(29): 20015.
 Wu Z, Puigserver P, Andersson U, et al. Mechanisms controlling mi-tochondrial biogenesis and respiration through the thermogenic coacti-vator PGC-1 [J]. Cell, 1999, 98(1): 115.
 F Picard, M Kurtev, N Chung, et al. SIRT1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma [J]. Nature, 2004, 429(6993): 771.
 D J Lomb, G Laurent, M C Haigis. Sirtuins regulate key aspects of lipid metabolism [J]. Biochim Biophys Acta, 2010, 1804(8): 1652.
 Lin J, Wu P H, Tarr P T, et al. Defects in adaptive energy metabolism with CNS-linked hyperactivity in PGC-1alpha null mice [J]. Diabetes,2006, 55(1): 120.
 Lu Y, Lu Y Q & Li H X. Relationship between the levels of serum 8-OHdG and macrovascular disease in type 2 diabetic patients [J]. Su-zhou University Journal of Medical Science, 2007, 27(5): 736-738 (In Chinese).
 Jiang H D, Fang W J, Fu X C, et al. Relationships between structure and activity for flavonoids to lipid peroxidation in liver microsomer from rat [J]. Chemical Journal of Chinese Universities, 2003, 24(12): 2221-2223 (In Chinese).
 Zhao X M, Zhu D Y, Ye X Q, et al. The study progress of the Citrus flavonoids [J]. Natural Product Research and Development, 2002, 14(1): 89-92 (In Chinese).
 Jagetia G C, Reddy T K. Modulation of radiation-induced alteration in the antioxidant status of mice by naringin [J]. Life Sci, 2005, 77(7): 780.