Based On Lipid Metabolism And Inflammation To Explore The Mechanism Of Gegen Qinlian Decoction In Improving Nonalcoholic Steatohepatitis

Objective: In order to observe the effect of Gegen Qinlian Decoction on lipid metabolism and inflammation aspects of non-alcoholic steatohepatitis-related,and explore its mechanism of action.Method:In vivo experiments: The NASH model was established by continuous feeding with high fat diet for 24 weeks.High dose(GGQLD-H,11.2 g/kg·d),medium dose(GGQLD-M,3.73 g/kg·d)and low dose(GGQLD-L,1.24 g/kg·d)of Gegen Qinlian Decoction and pioglitazone(5.25 mg/kg·d)were treated for 7 weeks.Serum TG,CHO,LDL-C,TBA,LPS and NEFA were measured by colorimetry.Steatosis,inflammation and balloon like change of liver tissue were observed by HE staining to evaluate the effect of Gegen Qinlian Decoction.In vitro experiments.(1)The in vitro model of NASH was established by treating Hep G2 cells with free fatty acid(palmitic acid : Oleic acid=1:2)for 24 hours,and intervention with different concentrations of Gegen Qinlian Decoction-containing serum.Oil red O staining and detection of intracellular TG content were used to observe degree of cellular steatosis.ELISA was used to determine the level of IL-6 in the culture supernatant.q RT-PCR and WB to detect the relative expression of TLR4 gene and protein.(2)Transcriptomics technology was used to analyze the effect of RAW264.7 cell inflammation model after the intervention of Gegenqinlian Decoction-containing serum,and the bioinformatics analysis of GO classification and KEGG pathway enrichment of the differential genes was carried out.Then thepotential target was obtained by combining the literature.Finally,some of the differential genes were verified by q RT-PCR.(3)Proteomics technology was used to analyze the effect of RAW264.7 cell inflammation model after the intervention of Gegen Qinlian Decoction-containing serum.The bioinformatics analysis of the differential proteins was performed using DAVID and String databases,and then combined with the literature to obtain possible biomarkers.Result:1.In vivo experiments,(1)Rat body mass and pathological morphology: The body weight of the model group was significantly increased every week compared with the normal group.Its pathological sections also showed obvious steatosis,inflammatory cell infiltration,and hepatocyte ballooning.NAS score was greater than the normal group(P<0.01).Compared with the model group,Gegen Qinlian Decoction body weight of each group has decreased,but there is no significant difference,and the liver pathological state of drug intervention has different degrees improvement,in which the NAS score of Gegen Qinlian Decoction high-and low-dose groups decreased significantly(P<0.05,P<0.01).(2)Serum biochemical indicators: The serum levels of TG,CHO,LDL-C,NEFA,LPS,and TBA in the model group were significantly higher than those in the normal group(P<0.01,P<0.05).Gegen Qinlian Decoction high-dose serum TG,CHO,LDL-C,NEFA,LPS,TBA,middle-dose serum LPS,TBA,low-dose serum TG,CHO,LPS,TBA and pioglitazone serum TG,CHO,NEFA,LPS,TBA were significantly lower than the model group(P<0.01,P<0.05).2.(1)Oil Red O staining and TG: after treatment with FFA,the intracellular red lipid droplet particles increased,and the TG level was also significantly higher than that of the control group(P<0.01).Gegen Qinlian Decoction intervention group intracellular red lipid droplet and TG level were reduced compared with the FFA group(P<0.01).The results of ELISA showed that the IL-6 level of the model group was significantly higher than that of the control group(P<0.05).The IL-6 levels of Gegen Qinlian Decoction and pioglitazone groups were significantly lower than that of the model group(P<0.05).The results of q RT-PCR and WB showed that the expression levels of TLR4 m RNA and protein in the model group were significantly higher than those inthe control group(P<0.01,P<0.05).Compared with the model group,the TLR4 m RNA and protein levels of Gegen Qinlian Decoction high and medium dose groups were significantly down-regulated(P<0.01,P<0.05),and the expression of TLR4 protein in Gegen Qinlian Decoction low-dose group and pioglitazone group was significantly down-regulated(P<0.05,P<0.01).(2)Transcriptomics analysis identified 293 differentially expressed genes,of which 274 were up-regulated and 19 were down-regulated.After bioinformatics analysis and related literature review,the possible targets of Gegen Qinlian Decoction for improving NASH were obtained,which were Mat1 a,Hnf4α,Cbs,and Pparα genes,respectively.The results of q RT-PCR verification showed that compared with the control group,the model Hnf4αm RNA,Cbs m RNA and Pparα m RNA were all down-regulated(P<0.01),compared with the model group,Gegen Qinlian Decoction increased the expression levels of Hnf4α m RNA,Cbs m RNA and Pparα m RNA in varying degrees.The results of the transcriptome study are consistent.(3)Proteomics analysis obtained 1424 differentially expressed proteins,of which 755 were up-regulated and 669 were down-regulated.Through bioinformatics analysis and literature review,the differentially expressed proteins related to NASH are Ldlr,Nos3,Lpin3,and Mapk14.Network analysis of the above proteins showed that only Ldlr,Mapk14,and Nos3 proteins participated in the interaction.Conclusion:1.Gegen Qinlian Decoction can improve steatosis,inflammation reaction and other aspects of NASH,TLR4 signal pathway may be a way to improve inflammation.2.The Mat1 a,Hnf4α,Cbs,Pparα genes and Ldlr,Nos3,Mapk14 proteins obtained from the omics research may be the potential targets for Gegen Qinlian Decoction to treat NASH.