Study On Intervention Effect And Mechanism Of Electrophilic Compounds In Xiaoheiyao Against Hepatocyte Steatosis

Nonalcoholic fatty liver disease(NAFLD)is a primary manifestation of metabolic syndrome in the liver,whose important manifestation is liver steatosis,the occurrence of NAFLD is closely related to lipid metabolism disorder,mitochondrial dysfunction and oxidative stress.Nrf2-Keap1 signaling pathway that can regulate redox status to improve the lipid metabolism and mitochondrial function is considered to be an important way to intervene the NAFLD.Electrophilic compounds with electron-deficiency functional group can activate Nrf2-Keap1 signaling pathway by modifying sulfydryl group of cysteine in Keap1,which are developing and potential functional factors for the intervention of NAFLD.Xiaoheiyao is the root and stem of Inula nervosa Wall.belonging to the genus Inula of composite family,which is a kind of new resource food with potential electrophilic compounds,the identification of the electrophilic compounds in Xiaoheiyao is not exactly clear,the effect on steatosis intervention of the electrophilic compounds in Xiaoheiyao are still undefined.In our study,we identified the electrophilic compounds in Xiaoheiyao based on the targeted knocking-out method by GSH functionalized affinity magnetic particles(GSH-MNPs)and molecular networking based on Global Natural Product Social Molecular Networking(GNPS),the intervention effect and its possible mechanism of electrophilic compounds in Xiaoheiyao on steatosis are investigated based on free fatty acid(FFA)(oleic acid:palmitic acid,2:1)induced HepG2 cells model.Firstly,the effects on lipid accumulation and ROS production of the ethanol extract of Xiaoheiyao without and with electrophilic compounds knocked-out by GSH-MNPs were evaluated,the results showed that at the concentration of 0.5?8.0?g/m L,the ethanol extract of Xiaoheiyao without and with electrophilic compounds knocked-out had the activities of reducing the lipid accumulation and ROS production,the activities of the ethanol extract with electrophilic compounds knocked-out reduced compared to the ethanol extract without electrophilic compounds knocked-out,the reduction of activities were significant at the concentration of 8.0?g/m L(P<0.05).The chemical constituents of the ethanol extract without and with electrophilic compounds knocked-out were analyzed by high performance liquid chromatography tandem mass spectrometry,GNPS molecular networking and multivariate statistical analysis,the results indicated that there were chemical composition differences between the ethanol extract without and with electrophilic compounds knocked-out,38compounds varied significantly(P<0.05,Fold Change>2)were further discovered,the chemical structure of 3 electrophilic compounds were identified as 13-OxoODE,9(10)-EpOME,10-Isobutyryloxy-8,9-epoxythymol isobutyrate(XHY-1),which were found in Xiaoheiyao for the first time.Next,the electrophilic compounds in the petroleum ether,ethyl acetate and water fraction of Xiaoheiyao were manifested by FITC quantitatively,the results showed that the distribution of electrophilic compounds was ethyl acetate fraction>petroleum ether fraction>water fraction.The effects on steatosis intervention of different fractions of Xiaoheiyao with and without electrophilic compounds knocked out were further evaluated,the results indicated that at the concentration of 0.5?2.0?g/m L,the fractions without and with electrophilic compounds knocked-out had the activities of reducing the lipid accumulation and ROS production,the activities of the fractions with electrophilic compounds knocked-out reduced compared to the fractions without electrophilic compounds knocked-out,which were the most significant in ethyl acetate fraction.The effects on lipid metabolism,redox status and mitochondrial function of ethyl acetate fraction of Xiaoheiyao with and without electrophilic compounds knocked out were assessed,the results showed that at the concentration of 0.5?2.0?g/mL,the ethyl acetate fraction without and with electrophilic compounds knocked-out had the activities of improving the lipid metabolism,redox status and mitochondrial function,the activities of the ethyl acetate fraction without electrophilic compounds knocked-out were better compared to the ethyl acetate fraction with electrophilic compounds knocked-out,the reduction of activities were significant at the concentration of 2.0?g/mL(P<0.05),the improvement on lipid metabolism of components knocked out were mainly reflected in the upregulated expressions of PPAR?,the downregulated expressions of SREBP1c,FAS and the reduction of TG content;the improvement on redox status were mainly reflected in the upregulated expressions of Nrf2,GCLC,NQO1,Gpx1 and the increase of antioxidants level;the improvement on mitochondrial function were mainly reflected in the upregulated expressions of IDH3,ND1,ATP5A1 and the increase of the levels of acetyl-CoA,NADH/NAD~+,ATP.Finally,XHY-1 was tracked and isolated from the ethyl acetate fraction by column chromatography and charactered by ~1H-NMR.The effects on redox status,lipid metabolism,mitochondrial function of XHY-1 in FFA-induced HepG2 cells were investigated.The results indicated that at the concentration of 1?10?M,XHY-1 could upregulate the intracellular expressions of Nrf2 and its downstream antioxidant genes and improve the antioxidant capacity,XHY-1 could upregulate the intracellular expressions of PPAR?,CPT1A,downregulate the expressions of SREBP1c,FAS,ACC1 and decrease the level of lipid accumulation,XHY-1could upregulate the expressions of OGDH,ND1 and ATP5A1 and increase the levels of acetyl-CoA,NADH/NAD~+,mitochondrial membrane potential and ATP,the activities performed best at the concentration of 10?M.The results of covalent docking based on MOEdock showed that XHY-1 binded with the sulfydryl group of Cys151 on Keap1 to activate the Nrf2 signaling pathway.In conclusion,Xiaoheiyao had the effect of interventing steatosis,the electrophilic compounds were determined as active components in Xiaoheiyao,the ethyl acetate fraction was determined as the most electrophilic compounds-enriched fraction of Xiaoheiyao.XHY-1,the electrophilic compounds in Xiaoheiyao,might improve intracellular redox status caused by XHY-1 binding with sulfydryl of cysteine in Keap1 to improve lipid metabolism and mitochondrial function to further interfere steatosis in liver cells,which has the application prospect of NAFLD intervention.