Huazhi Rougan Granules attenuates steatosis in cell model of nonalcoholic fatty liver disease by inducing autophagy.
10.19540/j.cnki.cjcmm.20221111.401
- Author:
Ya-Min SHI
1
;
Zhi-Hui FU
1
;
Chun-Sheng ZHU
1
;
Xiao-Ping LI
1
Author Information
1. the First Affiliated Hospital of Zhengzhou University Zhengzhou 450052, China.
- Publication Type:Journal Article
- Keywords:
Huazhi Rougan Granules;
SIRT1/AMPK signaling pathway;
autophagy;
nonalcoholic fatty liver disease;
steatosis
- MeSH:
Humans;
Non-alcoholic Fatty Liver Disease/metabolism*;
Sirtuin 1/metabolism*;
AMP-Activated Protein Kinases/metabolism*;
Fatty Acids, Nonesterified/metabolism*;
Autophagy;
Liver
- From:
China Journal of Chinese Materia Medica
2023;48(7):1770-1778
- CountryChina
- Language:Chinese
-
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.
