| Objective Bisphenol A(BPA),as the representative substance of(Environmental Endocrine Disruptors,EEDs),can be widely found in daily necessities.It was detected in mineral water bottle,medical instrument and food packaging coating;Studies have shown that BPA is associated with reproductive damage,fetal deformities,and tumorgenesis.Especially in recent years,with the increasing incidence of obesity and diabetes,the effects of BPA on glycolipid metabolism have attracted attention,but the mechanism remains to be clarified.Under physiological conditions,insulin can regulate blood sugar through PI3K/AKT/FOXO pathway after binding with its receptor.When interfered by foreign chemicals,insulin cannot play its normal physiological function and loses its role in regulating blood sugar,resulting in glucose metabolism disorder.PHLDA3,a P53 target gene discovered in recent years,regulates the occurrence of endocrine tumors by inhibiting the activity of AKT,which is a key factor in the physiological role of insulin.Therefore,does BPA affect the physiological role of insulin through the p53-Phlda3-Akt pathway,leading to abnormal glucose metabolism? In addition,BPA is a type of EEDs,and many research results show that different doses of BPA have bidirectional effects,that is,the toxic effects of low dose and high dose are inconsistent under the same experimental conditions.Therefore,due to the diversity of dose-response relationships,the US FDA set the AEL for BPA at 5 mg/kg,while the EU set it at 50 μg/kg,a 100-fold difference.So whether there is a two-way effect on sugar metabolism is also our concern.In this study,rat models and cell models of BPA exposure at different doses were established to observe the effects of BPA on glucose metabolism through IR/PI3K/AKT signaling pathway,and to investigate whether PHLDA3 interfered,providing toxicological basis for further investigation of the relationship between BPA and glucose metabolism disorder.MethodAnimal experiments: A total of 30 8-week-old SD rats were randomly divided into 3groups according to body weight after 1 week of adaptive feeding: control group,BPA 50μg/kg group and BPA 5 mg/kg group,with 10 rats in each group(half male and half female).The toxin was injected intraperitoneally with a volume of 2 ml/kg once a day for a total of 21 days.The control group was given same volume of solvent corn oil.Waiting the end of poisoning,chloral hydrate anesthesia,abdominal aorta blood collection and serum separation for use,we can weight rat organs and body weight and frozen it at-80°C for follow-up experiments.The m RNA expression horizontal and protein horizontal of key factors in Ins R-PI3K-AKT signaling pathway were down by RT-PCR and Western blot.Cell experiments: Hep G2 cell line was used to set up control group(Con),0.01,0.1,1,10,50,100 μmol/L BPA groups.IRS1 inhibitor(10 μmol/L)and P53 inhibitor(30μmol/L)were used to block the insulin signaling pathway and P53/PHLDA3 signaling pathway,respectively.24 h later,CCK8 was used to detect the cell viability of each group,and RT-PCR and Western blot were used to detect the related factor IRS1.The m RNA expression levels and protein levels of PI3K、AKT、FOXO1、G6PC、PEPCK、P53 and PHLDA3 were changed.Result1.Effects of BPA on growth and related organ coefficients in ratsAfter 3 weeks of BPA exposure,compared with the control group,the coat of rats in all groups was smooth,the movement was free,and the body weight of rats showed an increasing trend,and there was no significant difference between the groups.Compared with the control group,the liver,kidney and pancreas organ mass and organ coefficient showed a downward trend,and the liver mass and organ coefficient in 50 μg/kg BPA group were significantly lower than those in the control group(P<0.05),while the 5mg/kg BPA group and other organs had no significant changes.2.The effects of BPA on the liver blood glucose level and the expression of related genes in ratsCompared with the control group,the blood glucose level in the 50 μg/kg BPA group had no significant change,and the blood glucose level in the 5 mg/kg BPA group was significantly increased at the 3rd week(P<0.05).Meanwhile,the m RNA and protein expressions of PEPCK and G6 PC genes related to glucose metabolism showed a bidirectional effect in liver,and the m RNA and protein expressions of G6 PC decreased significantly in low-dose group,while significantly increased in high-dose PEPCK and G6 PC group(P<0.05),with statistical significance(P<0.05).3.The effect of BPA on Ins R/PI3K/AKT signaling pathway in rat liverCompared with the control group,the m RNA expression levels of IRS1 and AKT and the protein expression level of PI3 K in 50 μg/kg BPA group were significantly increased(P<0.05).In 5 mg/kg BPA group,m RNA levels of IRS1 and PI3 K were significantly down-regulated,protein expression of IRS1 and phosphorylation of AKT were significantly down-regulated(P<0.05),and the expression level of FOXO1 protein was decreased,with statistical significance(P<0.05).Effects of different doses of BPA on P53/PHLDA3 signaling pathway4.The effect of BPA on Hep G2 cell viabilityCompared with the control group,the cell viability was increased in 0.01,1,10 μM BPA groups for 24 h(P<0.05),and the cell viability was significantly decreased in 100μmol/L BPA and 500 μM BPA groups(P<0.05).Compared with the control group,0.01μM and 0.1μM BPA groups increased cell viability(P<0.05),and 100 μM and 500 μM BPA significantly decreased cell viability(P<0.05).5.The effects of BPA on glucose metabolizing enzymes in Hep G2 cellsCompared with the control group,the protein expressions of PEPCK and G6 PC in the 100 μM BPA group were significantly up-regulated,with statistical significance(P <0.05).The m RNA expression level of G6 PC in 1 μM and 10 μM BPA groups was significantly down-regulated,and the difference was statistically significant(P < 0.05).6.The effect of BPA on Ins R/PI3K/AKT signaling pathway in Hep G2 cellsCompared with the control group,AKT m RNA expression was up-regulated in(1,10,100 μM)BPA group,IRS1 and PI3 K protein expression was up-regulated in 1 μM BPA group,and PI3 K protein expression in 10 μM BPA group,the differences were statistically significant(P < 0.05).Compared with the control group,the phosphorylated water of AKT in the 100 μM BPA group was decreased,and the expression of PI3 K and FOXO1 protein was up-regulated,with statistical significance(P < 0.05).7.Effects of IGF-IR inhibitor GSK1904529 A on Ins R/PI3K/AKT signaling pathway of Hep G2 cellsCompared with the control group,the protein levels of IRS1,PI3 K and AKT in GSK(10 μM)group were significantly down-regulated,and the protein expression levels of PI3 K in GSK + BPA combined group(0.1,10,100 μM)were down-regulated in different degrees,the difference was statistically significant(P < 0.05).The protein levels of IRS1 and AKT in GSK + BPA combined group(0.1 μM)were significantly decreased,while the expression of FOXO1 protein in GSK + BPA combined group(100 μM)was significantly up-regulated,with statistical significance(P < 0.05).Compared with GSK(10 μM),IRS1 and PI3 K protein expression levels in GSK + BPA combined group(1 μM)were significantly up-regulated,and IRS1 protein expression in GSK + BPA combined group(100 μM)was significantly up-regulated,with statistical significance(P < 0.05).8.Effects of BPA on P53 / PHLDA3 signaling pathway in Hep G2 cellsCompared with the control group,the expression of PHLDA3 protein in 1 μM BPA was up-regulated,the expression of P53 protein in 10 μM BPA was significantly increased(P < 0.05),the expression of P53 and PHLDA3 protein in 100 μM BPA was significantly increased(P < 0.05),and the expression trend of P53 m RNA was consistent.9.Effects of P53 inhibitor PFT-α on PHLDA3 expression in Hep G2 cellsBy specifically inhibiting the post-transcriptional activity of P53,P53 inhibitor PFT-α down-regulated the protein expression level of PHLDA3 and up-regulated the phosphorylation level of AKT.The results showed that compared with the control group,the protein expression of PHLDA3 in PFT-α group was significantly down-regulated(P< 0.05).The expression level of PHLDA3 protein in PFT-α + BPA combined groups(10,100 μM)was down-regulated(P< 0.05).The expression of P53 protein in the PFT-α +BPA combined group was significantly up-regulated at 0.10 μM,and down-regulated at100 μM in the PFT-α + BPA combined group(P< 0.05).10.Effect of BPA blocking Ins R/PI3K/AKT signaling pathway on P53/PHLDA3 in Hep G2 cellsIGF-Ins R inhibitor GSK1904529 A inhibited the Ins R/PI3K/AKT signaling pathway by specifically inhibiting the activity of insulin receptors,and up-regulated the protein expression levels of P53 and PHLDA3(P < 0.05).The results showed that compared with the control group,the protein expressions of P53 and PHLDA3 in GSK + BPA combined groups(0.1,1,10 μM)were significantly up-regulated,and the differences were statistically significant(P < 0.05).Compared with GSK(10 μM),the protein expression level of PHLDA3 in GSK + BPA combined groups(10,100 μM)was significantly up-regulated(P < 0.05).11.The effect of blocking the P53/PHLDA3 signaling pathway BPA on AKT expression in Hep G2 cellsCompared with the control group,the phosphorylation of AKT at 30 μM was significantly up-regulated.The phosphorylation of AKT in the PFT-α + BPA combined groups(0.1 and 100 μM)was significantly up-regulated,and the difference was statistically significant(P < 0.05).Compared with 30 μM PFT-α,the phosphorylation of AKT in the PFT-α + BPA combined group(0.1μM,1μM)was significantly decreased(P< 0.05).Conclusion1.BPA exposure in vivo increases blood glucose by interfering with the expression of G6 PC and PEPCK,which can induce liver glucose metabolism disorder.2.The effect of BPA on Ins R/PI3K/AKT signaling pathway showed a non-monotonic dose effect,that is,the m RNA and protein expressions of key factors were up-regulated at low dose,but down-regulated at high dose.3.In vitro experiments have demonstrated that BPA interferes with cellular glucose metabolism mainly through Ins R/PI3K/AKT signaling pathway,and also affects insulin signaling pathway by regulating AKT expression through P53/PHLDA3 pathway. |
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