Effects Of Glycogen Synthase Kinase 3? On Hepatic Injury In Insulin Resistant Mice Induced By Bisphenol A

Objective To explore the effects of bisphenol A(BPA)on hepatic injury induced by insulin resistance(IR)in mice and the activity of glycogen synthase kinase 3?(GSK3?)-related protein by animal experiments.GSK3? inhibitor was used to down-regulate GSK3? activity to investigate the regulation of GSK3? of liver inflammatory injury in BPA-induced IR mice,and to provide experimental evidence for the prevention and treatment of BPA exposure hazards in the environment.Methods 72 four-week-old SPF male C57BL/6J wild-type mice were housed in a SPF-level environment and fed freely.The mice were randomly divided into NCD group,NCD+GSK3? inhibitor group,HFD group,HFD+GSK3? inhibitor group,HFD+1000 nM BPA group,HFD+1000 nM BPA+GSK3? inhibitor group.NCD stands for normal diet and HFD group stands for high fat diet,the mice were fed for 8 weeks and 16 weeks.BPA was dissolved in DMSO(final concentration not exceeding 1%),and formulated into a concentration of 1000 nmol/L,which was ingested by drinking water.GSK3? inhibitors were chlorinated by intraperitoneal injection of 21.5 mg/kg GSK3? inhibitor LiCl at 6th-8th weeks and 14th-16 th weeks.At 8th and 16 th weeks,detecting FG and FI,and assessing the IR index;performing ITT and GTT;Six mice in each group were anesthetized with cervical dislocation and blood was taken from the eyeball.The levels of AST and ALT in serum were detected,calculating liver coefficient.Some livers were fixed in 10% formaldehyde,and the rest were placed in liquid nitrogen for quick freezing and stored at-80 °C.Histopathological changes of liver tissue were observed by HE.IHC was used to detect the expressions of IL-1?,TNF-?and IL-6 in liver.WB was used to detect expressions of GSK3?,p-GSK3?(Ser9)protein and insulin pathway-related proteins.Results1 The general conditions of mice.During the experiment,each group of mice was generally in good condition.At different time points,there was no significant difference in daily average water consumption and food consumption between the groups.2 The effect of GSK3? inhibitor on BPA-induced changes in body weight and liver coefficient in mice.2.1 BPA induces increased body weight and liver coefficient in mice.Compared with the NCD group,the body weight of the HFD group increased at different time points,and the difference was statistically significant from the 10 th week(P<0.05).Compared with the HFD group,the weight of mice in the HFD+1000 nmol/L BPA group increased significantly from the 14 th week,and the difference was statistically significant(P<0.05).Compared with the NCD group,the liver coefficient of the HFD group was significantly increased at 8th week and 16 th week,and the difference was statistically significant(P<0.05).Compared with the HFD group,the liver coefficient of the HFD+1000 nmol/L BPA exposed group increased significantly at 16 th week,and the difference was statistically significant(P<0.05).2.2 Effect of GSK3? inhibitor on BPA-induced increase in body weight and liver coefficient in mice.Compared with the HFD group,the body weight of the HFD+GSK3? inhibitor groupdecreased,and the difference was statistically significant from the 15 th week(P<0.05).Compared with the HFD+1000 nmol/L BPA group,the body weight of the HFD+1000nmol/L BPA+GSK3? inhibitor group decreased significantly at the 7th,8th,and 15 th and 16 th weeks,and the difference was statistically significant(P <0.05).Compared with HFD+1000nmol/L BPA group,the liver coefficient of HFD+1000nmol/L BPA+GSK3? inhibitor group decreased significantly at 16 th week,the difference was statistically significant(P<0.05).3 The effect of GSK3? inhibitor on the IR of mice induced by BPA.3.1 Effects of BPA exposure on FG and FI and HOMA-IR in mice.Compared with the NCD group,FG was significantly increased in the HFD group at 8th and 16 th weeks,and the difference was statistically significant(P<0.05).Compared with the HFD group,the FG of the HFD+1000 nmol/L BPA group was significantly increased at 16 th week,and the difference was statistically significant(P<0.05).Compared with the HFD group,the FI level of the HFD+1000 nmol/L BPA group was significantly increased at 8th and 16 th weeks,and it was more obvious at 16 th week than at 8th week,and the difference was statistically significant(P<0.05).Compared with NCD group,HOMA-IR was significantly increased in HFD group at8 th and 16 th weeks,with statistical difference(P<0.05).Compared with the HFD group,HOMA-IR in the HFD+1000 nmol/L BPA group was significantly increased at 16 th week,and the difference was statistically significant(P<0.05).3.2 Effects of GSK3? inhibitors on changes of FG,FI and HOMA-IR in mice induced by BPA.Compared with the HFD group,the FG of the HFD+GSK3? inhibitor group wassignificantly decreased at 8th and 16 th weeks,and the difference was statistically significant(P<0.05).Compared with the HFD+1000 nmol/L BPA group,the FG of the HFD+1000 nmol/L BPA+GSK3? inhibitor group was significantly decreased at 16 th week,and the difference was statistically significant(P<0.05).Compared with the HFD group,the FI level of the HFD+GSK3? inhibitor group was significantly decreased at 16 th week.Compared with the HFD+1000 nmol/L BPA group,the FI level of the HFD+1000 nmol/L BPA+GSK3? inhibitor group was significantly lower at 16 th week,and the difference was statistically significant(P<0.05).Compared with the HFD group,HOMA-IR was significantly decreased in the HFD+GSK3? inhibitor group at 8th and 16 th weeks,and significantly more significant at 16 th week than at 8th week(P<0.05).Compared with HFD+1000 nmol/L BPA group,HOMA-IR of HFD+1000 nmol/L BPA+GSK3? inhibitor group was significantly decreased at 16 th week,and the difference was statistically significant(P<0.05).3.3 Effect of BPA exposure on GTT and ITT in mice.The area under curve(AUC)of the GTT showed that compared with the NCD group,the AUC of GTT in the HFD group was significantly higher at 8th and 16 th weeks.Compared with the HFD group,the AUC of GTT in the HFD+1000 nmol/L BPA group was significantly higher at 8th and 16 th weeks,and significantly higher at 16 th week than at 8th week(P<0.05).The AUC of the ITT showed that compared with the NCD group,the AUC of the ITT in the HFD group was significantly higher at 8th and 16 th weeks,and the difference was statistically significant(P<0.05).Compared with the HFD group,the AUC of ITT in the HFD+1000 nmol/L BPA group was significantly higher at 8th and 16 th weeks,and the difference was statistically significant(P<0.05).3.4 Effect of GSK3? inhibitor on the changes of GTT and ITT in mice induced by BPA.After intraperitoneal injection of glucose solution,the area under curvethe(AUC)of GTT showed that compared with HFD+1000nmol/L BPA mice,the AUC of GTT in the HFD+1000nmol/L BPA+GSK3? inhibitor group was significantly decreased at 8th and16 th weeks,and the difference was statistically significant(P<0.05).After intraperitoneal injection of insulin into the mice,compared with HFD+1000nmol/L BPA mice,the AUC of ITT in the HFD+1000nmol/L BPA+GSK3?inhibitor group was significantly decreased at 8th and 16 th weeks,the difference was statistically significant(P<0.05).4 Effect of GSK3? inhibitor on liver inflammatory injury in IR mice induced by BPA.4.1 Effect of BPA exposure on liver injury in mice.Liver function results showed that compared with the HFD group,serum levels of AST and ALT in the HFD+1000nmol/L BPA group were significantly higher at 8th and 16 th weeks,and the difference was statistically significant(P<0.05).The histopathological results of the liver showed that compared with the NCD group,liver tissue histopathological changes were aggravated in the HFD group at 8 and 16 weeks.Compared with the HFD group,the liver tissue lesions of the HFD+1000nmol/L BPA group were more severe at 8th and 16 th weeks.4.2 Effect of GSK3? inhibitor on liver damage in mice exposed to BPA.Liver function results showed that compared with the HFD group,the serum levels of AST and ALT in the HFD+GSK3? inhibitor group decreased significantly at 16 th week.Compared with HFD+1000nmol/L BPA group,the levels of serum AST and ALT in the HFD+1000nmol/L BPA+GSK3? inhibitor group were significantly lower at 8th and16 th weeks,and the difference was statistically significant(P<0.05).Liver histopathology showed that Compared with the HFD group,the pathological changes of liver tissue in the HFD+GSK3? inhibitor group were significantly relieved at8 th and 16 th weeks.Compared with the HFD+1000nmol/L BPA group,liver lesions in the HFD+1000nmol/L BPA+GSK3? inhibitor group were significantly improved at 8th and 16 th weeks.4.3 Effect of BPA exposure on inflammatory factors in liver tissue of mice.Compared with the NCD group,the expressions of TNF-?,IL-1? and IL-6 in the liver tissues of the HFD group were significantly increased at 8th and 16 th weeks.Compared with the HFD group,the expressions of TNF-?,IL-1? and IL-6 in the liver tissues of HFD+1000nmol/L BPA group were significantly increased at 8th and 16 th weeks,and the difference was statistically significant(P<0.05)..4.4 Effects of GSK3? inhibitors on inflammatory factors in liver tissue of mice exposed to BPA.Compared with the HFD group,the expression of IL-1? in the liver tissue of HFD+GSK3? inhibitor group was significantly decreased at 8th and 16 th weeks,and the expression of TNF-? and IL-6 was significantly decreased at 16 th week.Compared with HFD+1000nmol/L BPA group,the expression of TNF-?,IL-1? and IL-6 in the liver of HFD+1000nmol/L BPA+GSK3? inhibitor group decreased significantly at 8th and 16 th weeks,and 16 th week is more obvious than 8th week,the difference was statistically significant(P<0.05).5 Effects of GSK3? inhibitor on the expression of GSK3?,p-GSK3? and insulin signaling pathway proteins in liver of BPA-induced IR mice.5.1 The effect of BPA exposure on the expression of GSK3?,p-GSK3? and insulin signaling pathway proteins in liver of mice.Compared with the NCD group,the expression of p-GSK3? in the liver of the HFD group was significantly decreased at 8th and 16 th weeks.Compared with HFD group,the expression of p-GSK3? in liver of HFD+1000nmol/L BPA group was significantly decreased at 8th and 16 th weeks(P<0.05).Compared with NCD group,the expression levels of the expressions of p-IRS1,p-PI3 K and p-AKT in liverP of HFD group were significantly decreased at 8th and 16 th weeks.Compared with the HFD group,the expression level of p-IRS1,p-PI3 K,p-AKT proteins in the liver of HFD+1000 nmol/L BPA group were significantly lower,and the difference was statistically significant(P<0.05).5.2 Effects of GSK3? inhibitor on the expression of GSK3?,p-GSK3? and insulin signaling pathway proteins in liver of mice exposed to BPA.Compared with NCD group,the expression of p-GSK3? in liver of NCD+GSK3?inhibitor group was significantly decreased at 8th and 16 th weeks.Compared with the HFD group,the expression of p-GSK3? in liver of HFD+GSK3? inhibitor group was significantly higher than that in the HFD group at 8th and 16 th weeks.Compared with HFD+1000nmol/L BPA group,the expression of p-GSK3? in liver of HFD+1000nmol/L BPA+GSK3? inhibitor group was significantly increased at 8th and16 th weeks.(P<0.05).Compared with the HFD group,the expression of p-IRS1,p-PI3 K and p-AKT levels in the liver of HFD+GSK3? inhibitor group were significantly higher at 8th and 16 th weeks.The increase was more obvious in 16 th week than in 8th week,and the difference was statistically significant(P<0.05).Compared with HFD+1000 nmol/L BPA group,the expression of p-IRS1,p-PI3 K and p-AKT in liver of HFD+1000nmol/LBPA+GSK3? inhibitor group was significantly increased at 8th and 16 th weeks,and the differences were statistically significant(P<0.05).Conclusions(1)Low-dose BPA exposure can aggravate the occurrence of IR and liver damage in HFD mice.(2)Low-dose BPA exposure can cause increased inflammatory cell infiltration and up-regulation of inflammatory factors in liver tissue of IR mice,and can significantly reduce the expression of GSK3?-S9 protein and lead to GSK3? activation in liver tissue.(3)GSK3? inhibitor can up-regulate the level of GSK3?-S9 in the liver of IR mice induced by BPA exposure,which leads to the decrease of GSK3? activity,which can significantly alleviate BPA-induced IR and improve BPA-induced liver pathological damage in IR mice,leading to hepatic inflammation,resulting in decreased inflammatory cell infiltration of liver and decreased expression of inflammatory factors.