| Objective: In this study,endoplasmic reticulum stress model rats with normal liver fibrosis and induced liver fibrosis were selected as research objects.Resveratrol was used as a tool to investigate the effect of endoplasmic reticulum stress on the expression and activity of UGTs metabolic enzyme in liver fibrosis disease model.In vitro incubation experiments,selective inhibitors of UGT1A1-specific bilirubin,UGT1A7 and UGT1A9 honokiol and nifluronic acid,specific inhibitors of UGT1A9,were added to investigate the UGTs subtype that played a major role in vitro incubation experiments of resveratrol mediated by liver microsomes and cell lysase in rats.The molecular mechanism of endoplasmic reticulum stress on resveratrol II phase metabolism was studied by constructing endoplasmic reticulum stress model cells and blocking endoplasmic reticulum stress pathway model cells.The results of this study will provide theoretical basis and experimental basis for related studies on endoplasmic reticulum stress affecting drug metabolism,and provide scientific guidance for the safe,rational and effective application of resveratrol in clinical practice.Method: In this study,rat models of endoplasmic reticulum stress induced by liver fibrosis,endoplasmic reticulum stress HepG2 cell model and endoplasmic reticulum stress pathway blocking HepG2 cell model were firstly established.Resveratrol and its metabolites were quantitatively analyzed by uplc-pda method.An in vitro incubation model of glucose aldehyde acidification reaction was established,and the conversion factor between the metabolites of resveratrol and the mother drug was obtained by hydrolysis method.Western blot was used to measure and compare the normal and model endoplasmic reticulum stress cell models of rats in the normal group and the model group.The protein expressions of UGT1A1,UGT1A7 and UGT1A9 in normal and endoplasmic reticulum stress cell models of rats in normal and model groups were measured and compared by Western blot.Application of normal and model group in the rat liver microsomal,endoplasmic reticulum stress model and normal cells,endoplasmic reticulum stress three pathways PERK,IRE1,ATF6 pathway inhibitor,the blocking of endoplasmic reticulum stress pathway induced by GSK2606414,STF083010,AEBSF.HepG2 cell lysis solution model of resveratrol in vitro metabolic reactions,enzyme were determined its reaction rate,using GraphPad Prism 5 software for data processing,analysis of rat liver tissue and human liver cells endoplasmic reticulum stress in the model and characteristics of the impact of resveratrol phase II metabolism;In vitro incubation experiments,the selective inhibitors honokiol of UGT1A1-specific bilirubin,UGT1A9 specific nifluronic acid,UGT1A7 and UGT1A9 were added to analyze and determine the UGTs subtype that plays a major role in the two-phase metabolism of rat liver tissue and human liver cell model.Results: Rat models of endoplasmic reticulum stress induced by liver fibrosis were successfully prepared,including endoplasmic reticulum stress HepG2 cell model and endoplasmic reticulum stress pathway blocking HepG2 cell model.The extracellular metabolism test of liver microsomes and cell lysis fluid confirmed that resveratrol was biotransformed into a glucose aldehyde acidification metabolite(M1)in rat liver microsomes,and resveratrol was biotransformed into two glucose aldehyde acidification metabolites(M1,M2).Western blot results showed that rat liver endoplasmic reticulum stress was activated in the model group.Compared with the normal group,GRP78 protein expression level in the model group was significantly increased(p<0.05),PERK phosphorylation level,ATF4 expression level,and eIF2 alpha phosphorylated protein expression level were significantly increased(p<0.001).Endoplasmic reticulum stress induced by liver fibrosis had an effect on the expression of UGTs metabolic enzyme in rats,and the expression of UGT1A1,UGT1A7 protein of phase II drug metabolic enzyme was significantly increased(p<0.01).In the model group,endoplasmic reticulum stress pathway was activated and GRP78 protein expression was significantly increased(p<0.001).At the same time,PERK phosphorylation level,IRE1,XBP1 protein expression increased significantly(p<0.001),ATF6 protein expression increased slightly(p>0.05),nuclear protein expression increased significantly(p<0.001).Nrf2 protein expression in nucleus was significantly increased(p<0.001).Endoplasmic reticulum stress can affect the expression of UGTs protein,resulting in a significant decrease in the expression of UGT1A1 and UGT1A9,phase II drug metabolism enzymes(p<0.05).The results showed that the metabolic rate of resveratrol in rat liver microsomes in the normal group was higher than that in the model group,but there was no significant difference.In rat liver microsomal mediates glucose aldehyde acid in vitro incubation experiment,to join the bilirubin UGT1A1 specific inhibitors,rat liver microsomal resveratrol glucose metabolism aldehyde acidification rate reduced significantly(p<0.001),join the UGT1A7,UGT1A9 selective inhibitors honokiol,resveratrol metabolic rate slightly lower,but no significant difference.In vitro incubation experiments mediated by cell lysates in the normal group,the metabolic rate of gluconal acidification was significantly higher than that in the endoplasmic reticulum stress model group(p<0.001).When bilirubin was added,the metabolic rate of resveratrol in cell lysates was slightly reduced with no significant difference(p>0.05),while when niflonic acid,a specific inhibitor of UGT1A9,was added,the metabolic rate of glucose aldehyde acidification of resveratrol was significantly reduced(p<0.001).After the three endoplasmic reticulum stress pathways were inhibited respectively,the metabolic rate of resveratrol glucose aldehyde acidification showed an upward trend,but no statistical significance.Conclusion: Three activation pathways of endoplasmic reticulum stress all have effects on resveratrol II drug metabolism.In the endoplasmic reticulum stress model rats induced by liver fibrosis,endoplasmic reticulum stress in the liver can cause changes in the expression of phase II drug metabolism enzymes UGT1A1 and UGT1A7 proteins,and UGT1A1 plays an important role in the glucose aldehyde acidification metabolism of resveratrol.In the endoplasmic reticulum stress model HepG2 cells,UGT1A1 and UGT1A9 had significant changes in protein expression and activity under endoplasmic reticulum stress,and had important effects on the glucose aldehyde acidification metabolism of resveratrol.The protein expression changes of UGT1A1,UGT1A7 and UGT1A9 may be regulated by nuclear receptor Nrf2,which will be further confirmed by subsequent experiments. |
PDF Full Text Request