Explore The Mechanisms Of QSHY Regulating Nonalcoholic Fatty Liver Triglyceride And Cholesterol Metabolism Via LXR Pathway

Aim:Based on the results of network pharmacology prediction,the mechanisms of QSHY decoction regulating triglyceride and cholesterol metabolism in non-alcoholic fatty liver was explored via the LXR pathway.Methods:1.Based on the network pharmacology method,the studied effective chemical components of QSHY decoction were searched in the existing database,the corresponding target proteins of the effective chemical components were searched,the target genes were further obtained.We also searched the disease background genes of NAFLD.Duplicated genes were removed separately,then the intersection of the two was taken,finally the interaction network of the two gene groups was obtained.The GO and KEGG pathways of these genes were analyzed in the DAVID and IPA data platforms,at last we got the predicted pathways of QSHY decoction treating NAFLD.2.1)Establish a model of hepatocyte TG deposition in vitro?TG deposition in Hep G2cells induced by LXR agonist GW3965?,observe the effect of QSHY decoction on TG deposition and the expression of LXR and SREBP1c gene and protein?RT-PCR,western blotting?.2.2)Establish a NAFLD mouse model induced by high-fat diet,high-sugar drinking water?HFHSD?,and 32 C57BL/6J mice were divided into 4 groups:normal,model,QSHY decoction and PUFA according to random principle.After 14 weeks of modeling,starting from the 15^th week,the medication group was administered with QSHY decoction or PUFA,and the normal and model groups were given the same amount of drinking blank water.The intragastrical administration was conducted for 6 weeks.The pathological changes of liver tissue?HE,oil red O staining?,serum ALT,liver triglyceride?TG?content,and m RNA and protein expression levels of LXR and SREBP1c in liver tissues were observed?RT-PCR,western blotting?.3.1)Establish a model of hepatocyte CHO deposition in vitro?cholesterol modeling?,and observe the effects of QSHY on CHO deposition,the m RNA and protein expression levels?RT-PCR,western blotting?of SREBF2,ABCG5,ABCG8.3.2)The mouse NAFLD model is the same as above?2.2?.Observing the mouse serum,liver and fecal free cholesterol?FC?,total cholesterol?TC?level,frozen liver tissue section total free cholesterol?FILIPIN staining?,the m RNA and protein expression liver SREBF2,CYP7A1,ABCG5,ABCG8 and small intestine LXR,NPC1L1,ABCG5?RT-PCR,western blotting?.Result:1.Based on the network pharmacology method,the top 10 signaling pathways of treating NAFLD by QSHY are predicted to be acute phase response signaling,induction of apoptosis by HIV1,neuroinflammation signaling pathway,hepatic fibrosis/hepatic stellate cell activation,LXR/RXR activation,FXR/RXR activation,Myc mediated apoptosis signaling,apoptosis signaling,endoplasmic reticulum stress pathway,IL-6signaling.2.In vitro,QSHY significantly reduced the TG deposition of Hep G2 cells induced by LXR agonist GW3965,inhibited the m RNA expression of LXR target gene SREBP-1c,and QSHY significantly inhibited LXR and SREBP-1c protein expression.In vivo,QSHY significantly reduced reduced liver TG accumulation in experimental NAFLD models,and inhibited the m RNA and protein expression of liver LXR and SREBP-1c.At the same time,QSHY decreased serum ALT levels and decreased liver inflammatory factors,such as the m RNA levels of TNF,IL6.3.In vitro,we found that QSHY significantly inhibited the increase of intracellular free cholesterol induced by cholesterol and inhibited the expression of cholesterol synthesis proteins SREBF2.In vivo,it showed that QSHY significantly inhibited the expression of SREBF2 and CYP7A1 in the liver,decreased the biosynthesis of CHO and the conversion of liver CHO to bile acid?TBA?,increased the liver protein levels of ABCG5 and ABCG8,increased the excretion of liver CHO,decreased the deposition of liver CHO.And in small intestine,QSHY decoction decreased the m RNA levels of NPC1L1,increased the protein levels of ABCG5,increased the fecal CHO content,increased the excretion of fecal CHO.Conclusion:1.QSHY may inhibit the expression of DNL genes and proteins by inhibiting the expression of upstream regulator LXR in the liver,thereby inhibiting hepatic lipogenesis and reducing hepatic steatosis.2.QSHY increases CHO excretion and reduces CHO accumulation in mice by reducing cholesterol biothynthesis,regulating downstream targets of the LXR pathway,such as promoting CHO excretion protein expression,inhibiting CHO absorption protein expression.