ABSTRACT
To investigate the effect of Huazhi Rougan Granules(HZRG) on autophagy in a steatotic hepatocyte model of free fatty acid(FFA)-induced nonalcoholic fatty liver disease(NAFLD) and explore the possible mechanism. FFA solution prepared by mixing palmitic acid(PA) and oleic acid(OA) at the ratio of 1∶2 was used to induce hepatic steatosis in L02 cells after 24 h treatment, and an in vitro NAFLD cell model was established. After termination of incubation, cell counting kit-8(CCK-8) assay was performed to detect the cell viability; Oil red O staining was employed to detect the intracellular lipid accumulation; enzyme-linked immunosorbnent assay(ELISA) was performed to measure the level of triglyceride(TG); to monitor autophagy in L02 cells, transmission electron microscopy(TEM) was used to observe the autophagosomes; LysoBrite Red was used to detect the pH change in lysosome; transfection with mRFP-GFP-LC3 adenovirus was conducted to observe the autophagic flux; Western blot was performed to determine the expression of autophagy marker LC3B-Ⅰ/LC3B-Ⅱ, autophagy substrate p62 and silent information regulator 1(SIRT1)/adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK) signaling pathway. NAFLD cell model was successfully induced by FFA at 0.2 mmol·L~(-1) PA and 0.4 mmol·L~(-1) OA. HZRG reduced the TG level(P<0.05, P<0.01) and the lipid accumulation of FFA-induced L02 cells, while elevated the number of autophagosomes and autophagolysosomes to generate autophagic flux. It also affected the functions of lysosomes by regulating their pH. Additionally, HZRG up-regulated the expression of LC3B-Ⅱ/LC3B-Ⅰ, SIRT1, p-AMPK and phospho-protein kinase A(p-PKA)(P<0.05, P<0.01), while down-regulated the expression of p62(P<0.01). Furthermore, 3-methyladenine(3-MA) or chloroquine(CQ) treatment obviously inhibited the above effects of HZRG. HZRG prevented FFA-induced steatosis in L02 cells, and its mechanism might be related to promoting autophagy and regulating SIRT1/AMPK signaling pathway.
Subject(s)
Humans , Non-alcoholic Fatty Liver Disease/metabolism , Sirtuin 1/metabolism , AMP-Activated Protein Kinases/metabolism , Fatty Acids, Nonesterified/metabolism , Autophagy , LiverABSTRACT
AIM To observe the clinical efficacy of Huazhi Rougan Granules (Artemisiae scopariae Herba,Cassiae Semen,Rhei Radix et Rhizoma,etc.) in the treatment of damp-heat accumulation type non-alcoholic steatohepatitis (NASH) and its influence on insulin resistance.METHODS During January 2014-January 2016,a total of one hundred and twenty patients with damp-heat accumulation type NASH,given diet and exercise guidance,were randomly and equally divided into control group (polyene phosphatidylcholine) and treatment group (Huazhi Rougan Granules),administrated for twelve weeks.The levels of serum alanine aminotransferase (ALT),aspartate aminotranferase (AST),gamma-glutamyltransferase (GGT),total cholesterol (TC),triglyceride (TG),γ-glutamyl transpeptadase (GGT),fasting plasma glucose (FPG),fasting insulin (FINS),HOMA-IR index,adiponectin (ADPN) and leptin,body mass index (BMI) and uhrasound examination were detected before and after the treatment,respectively.These data were compared in two groups.RESULTS Compared with the control group,the serum ALT,AST,GGT,TC,TG,FPG,FINS and HOMA-IR of the patients in the treatment group decreased significantly,and liver fat were effectively reduced,while serum adiponectin increased markedly (P ＜ 0.05).After the treatment,remarkable reductions in the levels of BMI,leptin were observed in both groups (P ＜ 0.05).CONCLUSION Huazhi Rougan Granules combined with lifestyle modulation can effectively improve the syndrome of damp-heat accumulation type NASH by lowing insulin resistance.