Protective Effect And Mechanism Of Asiatic Acid From Potentilla Chinensis On Lipid Metabolism In Nonalcoholic Fatty Liver Cells

Objective:LO2 and HepG2 cells were stimulated by oleic acid（OA）to induce the nonalcoholic fatty liver disease（NAFLD）cell model.The aim of this study was to investigate the protective effect and mechanism of asiatic acid from potentilla chinensis（AAPC）on lipid metabolism in LO2 and HepG2 cells by detecting oxidative stress,inflammation,and the AMPK/LXRα/SREBP-1c/ACC1/FAS pathway.Methods:1.Establishment of nonalcoholic fatty liver cell modelLO2 cells and HepG2 cells were cultured with different concentrations of oleic acid（OA）for 24 h.Then,the most suitable concentration of OA that could induce the stable cell model of non-alcoholic fatty liver was screened by performing oil red O staining and detecting the cell viability,triglyceride（TG）and lactate dehydrogenase（LDH）.2.The effect of AAPC on steatotic cellsThe 50%inhibitive concentration(IC₅₀)of AAPC on LO2 cells and HepG2cells was detected by MTT assay to assess the cytotoxity of AAPC.Cells were treated with 0.5 mmol/L of OA and various concentrations of AAPC.The effect of AAPC on cell vitality was examined by MTT assay to screen the proper concentration of AAPC for the further experiments.Then,the cells were divided into 5 groups:normal control group,model group,low-,medium-and high-dosage of AAPC-treated groups.AO/EB and Annexin V-FITC/PI detection kits were used to detect cell apoptosis.Intracellular lipid droplets in different groups were detected by Oil Red O staining.The biochemical parameters,including triglyceride（TG）,free fatty acids（FFAs）,high densitylipoprotein（HDL）,lowdensity lipoprotein（LDL）,lactate dehydrogenase（LDH）,aspartate aminotransferase（AST）and alanine aminotransferase（ALT）,were detected by commercial kits.Oxidative stress was assessed by detecting the level of malondialdehyde（MDA）and superoxide dismutase（SOD）using the commercial kits,respectively.The mRNA expressions of SREBP-1c,FAS,LXRα,TNF-αand IL-6 were detected by RT-PCR.The protein expression of SREBP-1c was assessed by immunocytochemistry.Moreover,the protein expressions of FAS and ACC1were detected by immumofluorescence assay.In addition,the expressions of SREBP-1c,FAS,LXRα,ACC1,BAX,BCL2,NF-κBp65,AMPK and phospho-AMPK were analyzed by Western blotting.Results:1.Establishment of nonalcoholic fatty liver cell modelsMTT results showed that OA significantly inhibited the proliferation of LO2 and HepG2 cells in a concentration-dependent manner.Oil red O staining showed that the contents of intracellular lipid droplets was significanty increased in the model group.Moreover,the content of TG and LDH in the model group was significantly increased compared with the normal control group in a dose-dependent manner.Taken together,the above results indicated that NAFLD cells model were successfully induced by oleic acid at a concentration of 0.5 mmol/L.2.The effect of AAPC on steatotic cells（1）The effect of AAPC on cells proliferationMTT results showed that AAPC significantly inhibited the proliferation of LO2 and HepG2 cells in a dose-dependent manner（P<0.01）,and the IC₅₀ were98.182 and 71.070μmol/L,respectively.Meanwhile,AAPC significantly inhibited steatotic cells proliferation in a dose-dependent manner（P<0.01）.Compared to the normal control group,the low-,medium-and high-dosages of AAPC had little effect on the apoptotic rate and morphplogy.（2）The effect of AAPC on lipid accumlation in steatotic cellsThe result of Oil Red O staining showed that the number of the lipid droplets was significantly increased in the model group compared with the normal control group.But the production of lipid droplets was notably suppressed by AAPC.Moreover,the levels of TG,FFAs and LDL were increased significantly in the model group（P<0.01）,whereas HDL were significantly decreased（P<0.01）.However preteatment with low-,medium-and high-dosage of AAPC for 24 h could reverse the abnormal levels of the biochemical indicators induced by OA（P<0.05 or P<0.01）.The results suggest that AAPC significantly alleviates lipid accumulation,which may contribute to the prevention and treatment of nonalcoholic fatty liver disease.（3）The effect of AAPC on hepatic injury-related enzymes in steatotic cellsCompared with the normal control group,the contents of AST,ALT and LDH were increased significantly in model group（P<0.01）.However pretreatment with AAPC for 24 h markedly decreased these enzymes’levels in a concentration-dependent manner（P<0.01）.The Results suggest that AAPC can reduce the contents of AST,ALT and LDH in steatotic cells,alleviating liver cell damage.（4）The effect of AAPC on oxidative stress in steatotic cellsCompared with the normal control group,MDA content was increased significantly in model group（P<0.01）,while SOD activity was significantly decreased（P<0.01）.However pretreatment with low-,medium-and high-dosage of AAPC for 24 h could reverse the abnormal levels of both the enzymes（P<0.01）.The results of RT-PCR showed that OA obviously increased the mRNA expression of TNF-αand IL-6mRNA（P<0.05 or P<0.01）.Treatment with AAPC significantly decreased the mRNA expressions of TNF-αand IL-6（P<0.05 or P<0.01）.Western blot results revealed that OA markedly increased the expression of NF-κBp65 protein（P<0.01）,while pretreatment with AAPC significantly the protein expression of NF-κBp65（P<0.01）.These results indicate that AAPC reduces oxidative stress and inflammation reaction via inhibiting the NF-κB pathway,which is beneficial to improve lipid metabolism.（5）The effect of AAPC on the AMPK/LXRα/SREBP-1c/ACC1/FAS signaling pathway in steatotic cellsPT-PCR analysis showed that the mRNA expressions of SREBP-1c,FAS and LXRαin model group were significantly increased compared with the normal control group（P<0.01）.In contrast,AAPC significantly down-regulated these genes mRNA expressions（P<0.05 or P<0.01）.The immunocytochemical staining results showed that OA induced a significant increase in the percentage of SREBP-1c positive staining cells;however,treatment with AAPC resulted in a contrary trend.Western blot and immunofluoresence staining analysis showed that OA significantly enhanced the protein expressions of SREBP-1c,FAS,LXRαand ACC1 and suppressed the expression of AMPK and p-AMPK（P<0.01）.OA did not affect the protein expressions of BAX and BCL2.Treatment with AAPC markedly decreased the protein expressions of SREBP-1c,FAS,LXRαand ACC1,and significantly increased AMPK and p-AMPK protein levels（P<0.05or P<0.01）;AAPC had little effect on the protein expression of BAX and BCL2.The results indicate that AAPC inhibits lipid metabolism maybe through inhibition of AMPK/LXRα/SREBP-1c/ACC1/FAS signaling pathway.Conclusions:Asiatic acid from potentilla chinensis significantly alleviates lipid accumulation and therefore ameliorates the abnormal lipid metabolism in oleic acid-induced nonalcoholic fatty liver cells,and its mechanism may be related to theinhibitionofoxidativestress,inflammatoryreaction,and theAMPK/LXRα/SREBP-1c/ACC1/FAS signaling pathway.