| Objective: To establish an in vitro model of non-alcoholic fatty liver by using animal serum pharmacological methods to induce steatosis of HepG2 cells by free fatty acids,to observe the effect of Jiawei Yinshao powder-containing serum on this cell model and the effect of autophagy;further explore this Fang treats non-alcoholic fatty liver mechanism.Method:HepG2 cells were cultured for 24 h by different final concentrations of free fatty acids(oleic acid and palmitic acid molar ratio of 2 to 1),and the best HepG2 cell steatosis was established by CCK-8,oil red O staining and photoelectric colorimetry.The induced concentration of the model;the CCK-8 method was used to determine the optimal concentration of rat-containing serum in each group;after the establishment of an in vitro model of non-alcoholic fatty liver by inducing HepG2 cells through the optimal concentration of FFA,the experiment was divided into 4 groups: blank control group,Model group,traditional Chinese medicine group and Yishanfu group,photoelectric colorimetry was used to detect the content of TG,AST and ALT in cells;Real-time PCR was used to detect the expression levels of Beclin1 and LC3-II mRNA;Western blot was used to determine the protein of Beclin1 and LC3-II expression.Result:1.CCK-8 method showed that HepG2 cells had different final concentrations of free fatty acids(oleic acid and palmitic acid molar ratio of 2 to 1)after 24 hours of induction,when the final concentration exceeded 0.5mmol/L,HepG2 cell viability was obvious Of inhibition.2.Oil red O staining method showed that: compared with the blank control group,the formation of intracellular lipid droplets in the model group was significantly increased;meanwhile,the photoelectric colorimetric method showed that compared with the blank control group,the intracellular TG content and cell supernatant in the model group The content of AST and ALT in the liquid increased significantly(P<0.05).3.Compared with fetal bovine blank serum group,5%,10% and 15% rat blank serum,modified Yinshao powder-containing serum and Yishanfu medicine-containing serum were cultured for 48 h.The concentration of drug serum has the smallest effect on cell viability.4.Photoelectric colorimetry showed that compared with the blank control group,the intracellular TG content and the AST and ALT content in the cell supernatant of the model group increased significantly(P<0.05);compared with the model group,the traditional Chinese medicine group and Yi Shan The content of TG in cells and the content of AST and ALT in cell supernatant were significantly reduced(P<0.05).5.Real-time fluorescence quantitative PCR results: HepG2 cells were induced by0.5mmol/L oleic acid and palmitic acid mixture for 24 h and then cultured with 5%drug-containing serum for 3h: there was no significant difference in the expression levels of LC3-II and Beclin1 mRNA between the groups;After 6 hours of culture: Compared with the blank control group,the expression of LC3-II and Beclin1 mRNA in HepG2 cells of the model group decreased(P<0.05).Compared with the model group,the expression of LC3-II and Beclin1 mRNA in the Chinese medicine group and the Yishanfu group were not.Significant difference;when cultured for 12h: Compared with the blank control group,theexpression levels of Beclin1 and LC3-II mRNA in HepG2 cells of the model group were significantly reduced(P<0.05).Compared with the model group,Beclin1 and LC3 in the Chinese medicine group and Yishanfu group-II mRNA expression level was significantly increased(P<0.05);at 12 h,the traditional Chinese medicine group up-regulated Beclin1 and LC3-II mRNA expression had the best effect.Incubation for 24h: There was no significant difference in Beclin1 and LC3-II mRNA expression levels between the groups.6.WesternBlot method results showed that: HepG2 cells were induced by 0.5mmol/L oleic acid and palmitic acid mixture for 24 h and then cultured with 5% drug-containing serum for 3h: there was no significant difference in the expression of LC3-II and Beclin1 between the groups;culture At 6h: Compared with the blank control group,the expression of LC3-II and Beclin1 protein in HepG2 cells in the model group was reduced(P<0.05).Compared with the model group,there was no significant difference in the expression of LC3-II and Beclin1 protein between the traditional Chinese medicine group and the Yishanfu group;Culture 12h:Compared with the blank control group,the Beclin1 and LC3-II protein expression in HepG2 cells in the model group was significantly reduced(P<0.05).Compared with the model group,the Beclin1 and LC3-II protein expression in the Chinese medicine group and the Yishanfu group Significantly increased(P<0.05);at 12 h,the Chinese medicine group had the best effect of up-regulating the expression of Beclin1 and LC3-II.24 h incubation: There was no significant difference in Beclin1 and LC3-II protein expression between the groups.in conclusion:1.Jiawei Yinshao powder-containing serum has the effect of reducing FFA-induced excessive accumulation of lipids in HepG2 cells and the degree of liver damage,thereby improving the disorder of liver cell lipid metabolism.2.Jiawei Yinshao powder-containing serum significantly up-regulated the expression of Beclin1,LC3-II mRNA and Beclin1,LC3-II protein in HepG2 cells.3.In the process of FFA-induced steatosis of HepG2 cells,the expression of autophagy-related genes in the cells decreased.Jiawei Yinshao powder-containing serum can significantly up-regulate the expression of Beclin1,LC3-II mRNA and Beclin1,LC3-II.Protein expression,thereby activating type III PI3K/Beclin1 signaling pathway,the mechanism of Jiawei Yinshao powder-containing serum regulating NAFLD in vitro may be related to the increase of intracellular autophagy.4.This experiment is based on the type III PI3K/Beclin1 signaling pathway to explore the mechanism of Jiawei Yinshao powder in the treatment of NAFLD.As far as the current research progress is concerned,the pathogenesis of NAFLD is more complicated,and its occurrence is often accompanied by oxidative stress and inflammation.Whether Shao San can reduce its oxidative stress and inflammatory response through autophagy to protect the liver is worthy of further experimental research. |
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