Study On Mechanism Of Qushi Huayu Decoction On Hepatic De Novo Lipogenesis In Nonalcoholic Fatty Liver Disease Based On IRE1?-XBP1s Pathways

Objective:The effect and main material basis of Qushi Huayu Decoction on inositol requiring enzyme 1-alpha(IRE1?)-spliced X-box binding protein 1(XBP1s)pathway and hepatic de novo lipogenesis(DNL)will be observed in high-fructose diet induce NAFLD mice in vivo and tunicamycin-induced HepG2 in vitro and to explain the possible mechanism of Qushi Huayu Decoction treatment of nonalcoholic fatty liver disease(NAFLD).Methods:The whole experiment was undertaked by two batchs in vitro and in vivo.The first batch:Effect of Qushi Huayu Decoction on Liver DNL Mediated by IRE1?-XBP1s pathway in high fructose induced non-alcoholic fatty liver disease in mice1.The NAFLD model of mice induced by high-fructose diet,and the normal diet was used as control.Changes in hepatic insulin resistance(IR),liver tissue histopathology,liver tissue triglyceride(TG)and DNL content,liver input and output lipids,liver TG and DNL synthesis key enzyme expression,carbohydrate response element-binding protein(CHREBP)and Sterol Regulatory Element-Binding Protein(SREBP1)expression and the IRE1?-XBP1s pathway were observed on DayO,Day3,Week1,Week2,and Week4.2.The NAFLD model of mice induced by high-fructose diet for 2 weeks,and the normal diet was used as control.Through the design of low,medium and high dose groups of Qushi Huayu Decoction,the effects of Qushi Huayu Decoction on liver IR,liver tissue histopathology,liver TG and DNL content,liver input and output lipids,liver TG and DNL synthesis key enzyme expression,liver CHREBP and SREBP1 expression and IRE1?-XBP1s pathway were observed.The second batch:Effect of Qushi Huayu Decoction and main material basis on hepatic de novo lipogenesis and IRE1?-XBP1s pathway in HepG2 cell induced by tunicamycin.1.Different concentrations of tunicamycin were used to induce HepG2 cells in vitro,and blank control space was set up.The effects of different concentrations of tunicamycin on intracellular TG,NEFA and IRE1?-XBP1s pathway were observed.2.The HepG2 cell model was induced by tunicamycin in vitro,and the effects of Qushi Huayu Decoction drug serum on intracellular TG and DNL synthesis and IRE1?-XBP1s pathway were observed.3.UHPLC-Q-Orbitrap HRMS analysis was used to identify potential blood components in the serum of rats given Qushi Huayu Decoction.4.CCK8(Cell Counting Kit 8)was used to detect the drug toxicity of constituents of Qushi Huayu Decoction migrating to blood,geniposide,resveratrol,kaempferol,polydatin,genipin,luteolin and chlorogenic acid to HepG2 cells.HepG2 cell models were induced in vitro by tunicamycin in a safe dose range to observe their effect on the IRE1?-XBP1s pathway mediating DNL.Results:The first batch:Effect of Qushi Huayu Decoction on hepatic de novo lipogenesis and IRE1?-XBP1s pathway in high fructose induced non-alcoholic fatty liver disease in mice1.With the prolongation of modeling time,compared with the normal group,the insulin resistance and liver tissue pathological changes of liver tissue in the high fructose diet group were significantly aggravated,serum and liver TG and NEFA levels were significantly increased,and liver tissue TG synthesis key enzymes were significantly increased.Glycerol 3-phosphate acyltransferase1(GPAT1),acyl glycerol 3-phosphate acyl transferase(AGPAT),di acyl glycerol O-acyl transferase(DGAT2),phosphatidic acid phosphatase(PAP)and DNL key enzyme acetyl-CoA carboxylase(ACC),fatty acid synthase(FAS),stearoyl-CoA dehydrogenase 1(SCD1)mRNA were significantly increased,CHREBP and SREBP1 mRNA were significantly increased,and phosphorylated IRE1? and XBP1s proteins and XBP1s mRNA expression were significantly increased.2.Qushi Huayu Decoction could significantly improve the insulin resistance and liver tissue pathological changes in the NAFLD model of mice induced by high-fructose diet for 2 weeks,significantly reduce serum and liver TG and NEFA levels,and reduce liver tissue TG synthesis key enzymes GPAT1,AGPAT,DGAT2,PNPLA3 mRNA and protein expression level,decreased the expression of DNL synthesis key enzymes ACC1,ACC2,FAS,SCD1 mRNA in the liver tissue,down-regulated CHREBP and SREBP1 mRNA expression,but no significant inhibition of CHREBP and SREBP1 nuclear protein expression and SREBP1 transcriptional activity,down-regulated phosphorylated IRE1? and XBP1s total protein and XBP1s mRNA and nuclear protein expression.The second batch:Effect of Qushi Huayu Decoction and main material basis on hepatic de novo lipogenesis and IRE1?-XBP1s pathway in HepG2 cell induced by tunicamycin.1.The cells were cultured using fetal bovine serum.Compared with the control group,0.5 ?g/ml and 1 ?g/ml tunicamycin could significantly increase the content of TG and NEFA in the HepG2 cells after 24 hours,and up-regulate XBP1s,ACACB and SCD1 mRNA expression.2.The cells were cultured using rat serum.The drug serum of Qushi Huayu Decoction from rats had no inhibitory effect on TG in HepG2 cells induced by 1 ?g/ml tunicamycin in vitro.In addition,after culturing HepG2 cells with normal rat serum,after 24 hours of stimulation of HepG2 cells with 0.5 ?g/ml and 1 ?g/ml tunicamycin,there was no statistically significant difference in intracellular TG compared with the control group.These results may be related to interference factors in rat serum culture.3.By comparing with the extracts of Qushi Huayu Decoction and the standard reference,combined with the extracted ion flow diagram and mass spectrum,the seven maj or blood components in the serum samples of the rats after oral administration of Qushi Huayu Decoction were confirmed,including geniposide,resveratrol,kaempferol,polydatin,genipin,luteolin and chlorogenic acid.4.The cells were cultured using fetal bovine serum.Within the safe dose range of the drug,it was found that geniposide,chlorogenic acid and polydatin,the constituents of Qushi Huayu Decoction migrating to blood,could significantly reduce the expression of XBP1s mRNA in the HepG2 cell model induced by 1?g/ml tunica in vitro,and reduce intracellular TG,DNL content and ACACB,SCD1 mRNA expression,reduce phosphorylated IRE1?,XBP1s,ACACB and SCD1 protein expression.Conclusion:1.Qushi Huayu Decoction could regulate the IRE1?-XBP1s pathway by inhibiting DNL thereby preventing NAFLD.2.Geniposide,chlorogenic acid and polydatin are the main effect material basis of Qushi Huayu Decoction for the regulation of IRE1?-XBP1s pathway to inhibit liver DNL and prevent NAFLD.