Abstract
Nonalcoholic steatohepatitis (NASH) is a liver disease defined as the dynamic condition of hepatocellular injury during the progress of nonalcoholic fatty liver disease (NAFLD). Total flavonoids from the dry and immature fruits of Citrus Paradisi cv.Changshanhuyou (accepted species name: Citrus × aurantium L) (Qu Zhi Qiao, QZQ) are purified and named TFCH. This study was purposed to investigate and analyze the effect of TFCH on NASH model through Nuclear factor erythroid 2-related factor 2 (Nrf2)- antioxidant response elements pathway in vivo and in vitro. In vivo study was performed using male C57BL/6 mice fed with high fat diet 16 weeks for NASH model. After 7-week modeling, mice in TFCH-treated group were daily treated with intragastric administration of TFCH at 25 mg/kg, 50 mg/kg, 200 mg/kg, respectively, for successive 8 weeks. Histopathological and immunohistochemical analyses were conducted for evaluating severity of NASH-mice model and the effect of TFCH treatment. In vitro experiment was performed by using human LX-2 cells and cultured with Free fatty acid (FFA) (Oleic acid: palmitic: l: 0.5 mmol/L) for 24 h and then treated with TFCH at different concentrations (0, 25, 50, 100, 200 mg/ml) for 6 h,12 h, and 24 h. Anti-apoptosis effect of TFCH on LX-2 cells cultured with FFA was revealed by the CCK-8 assay. Lipid parameters and oxidative stress markers were measured in vivo and in vitro, results showed that TFCH dose-dependently and greatly increased the antioxidant ability and reduced the oxidative damage in NASH model. The protein expression of Nrf2 and the downstream target genes in mice liver and human LX-2 cells were tested by Western blot analysis to investigate the possible molecular mechanisms of TFCH. Our results indicated that TFCH up-regulated protein expression of these genes and have the significant influence in activating the Nrf2-ARE signaling pathway. This study shows Nrf2-ARE signaling pathway may provide novel therapeutic opportunities for NASH therapy in the future.
Highlights
Nonalcoholic fatty liver disease (NAFLD) is the common liver disease wreaking havoc on people's lives
Low density lipoprotein cholesterol (LDL-C), High density lipoprotein cholesterol (HDL-C), Total cholesterol (TC) detection kit, Triglyceride (TG) detection kit, aspartate aminotransferase. detection kit, alanine aminotransferase detection kit, malondialdehyde (MDA) detection kit, Total Superoxide dismutase (SOD) detection kit, Free fatty acid (FFA) detection kit, and Glutathione Peroxidase (GSH-Px) detection kit were provided by Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). 8-iso-PGF2a ELISA kit was obtained from MEIMIAIN (Wuhan, USA)
Primary antibodies for nuclear factor-erythroid 2 related factor 2 (Nrf2), hemeoxygenase1 (HO1), NAD(P)H: quinone oxidoreductase 1 (NQO1), r-glutamylcysteine synthetase (r-GCS), and Glutathione-S-Transferases (GST) were obtained from Affinity Biosciences (Shanghai, China)
Summary
Nonalcoholic fatty liver disease (NAFLD) is the common liver disease wreaking havoc on people's lives. Progress of NAFLD traces from simple steatosis to nonalcoholic steatohepatitis (NASH), and pericellular fibrosis in NASH can slowly lead to cirrhosis and liver carcinoma. NASH patients developing fibrosis progression account for approximately 40%. Inflammation and oxidative stress are key factors in the pathogenesis of NASH, which can be applied as intervention targets for NASH treatment (Marra and Svegliati-Baroni, 2018). Elevated eactive oxygen species (ROS) generation causes damage in hepatocytes through systemic oxidative stress, triggering inflammation and fiborsis and leading to NASH (Spahis et al, 2017). Many studies demonstrated that Nrf2-ARE signaling pathway plays a key role in response to oxidative stress during NASH progress (Fisher et al, 2008; Lee et al, 2014). Oxidative stress plays a crucial role in pathogenesis of NASH and underscore solutions
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