| Background The incidence of metabolic syndrome has increased year by year in the world,posing a great threat to human health in today’s world.Obesity-derived insulin resistance is the main feature of metabolic diseases.Bisphenol A(BPA)is a widely produced and used environmental endocrine disruptor,and its exposure is a risk factor that cannot be ignored in metabolic syndrome such as obesity.Many studies have shown that BPA exposure promotes the accumulation of ATMs and IL-1β secretion in adipose tissue of obesity induced by HFD,and aggravates obesity-derived insulin resistance through interference with insulin signaling pathways,but in BPA promoted obesity derived IR,the regulatory mechanisms by which immune cells secrete IL-1βhave not been elucidated.The NLRP3 inflammasome is an intracellular protein complex formed by the intracellular pattern recognition receptor NOD-like receptor family,which can be activated by a variety of pathogenic microorganisms and risk signals,associated with IL-1β activation and release,and involved a variety of major human diseases occur.However,BPA promotes obesity-derived IR,the regulation of BPA on NLRP3 inflammasome activation and secretion of IL-1β,and the mechanism of promoting HFD-induced insulin resistance in obese mice are not clear and need further study.Objective Based on the HFD-induced WT obesity mouse model,the effects of different doses of BPA exposure on the IR phenotype,insulin sensitivity to adipose tissue and NLRP3 inflammasome activation in obese mice induced by HFD were investigated in different exposure time.To investigate the effect of BPA on the activation of NLRP3 inflammasome in IR of mice with HFD.Methods Three-week-old SPF C57BL/6J wild-type male mice were randomly divided into NCD(normal diet)group,HFD(high-fat diet)group,and HFD+10 nmol/L BPA group,HFD+100 nmol/L BPA group and the HFD+1000 nmol/L BPA group,mice were exposed to different doses of BPA via drinking water.After 8 weeks and 16 weeks of drinking water exposure,measured body weight(BW),the insulin tolerance tests(ITT)and glucose tolerance tests(GTT).Fasting glucose(FG)and fasting insulin(FI)were measured by eyelids,using homeostasis model assessment(HOMA)calculate the insulin resistance index(HOMA-IR),enzyme-linked immunosorbent assay(ELISA)detection of IL-1β,IL-10,IL-6,TNF-α in serum.The mice were sacrificed and epididymal fat pad was weighed.Histopathologic(HP)was used to observe the adipose tissue inflammation and fat cell size,immunohistochemistry(IHC)was used to detect the expression of IL-1β and NLRP3 in adipose tissue,immunofluorescence(IF)was used to detect the expression of NLRP3 in ATMs,q RT-PCR analysis of NLRP3,ASC,caspase-1 and IL-1β m RNA expression levels,western Blot(WB)detection of NLPR3,pro-caspase-1 and caspase-1,pro-IL-1β and its cleavage product IL-1β protein expression,immunoblotting(IB)analysis of IRS-1 and p-IRS-1(Tyr612),AKT and p-AKT(Ser473),PI3Kp85 and p-PI3 K p85(Tyr458)in adipose tissue after intraperitoneal injection of insulin(1m U/g BW)for 5 min.Results1.Effect of BPA exposure on general condition and body weight of mice with HFD1.1 General condition Mice were in good condition during the experiment and no adverse reactions and death occurred.There was no significant difference in weekly mean water consumption and food consumption between the mice in different groups at the same time point.(P>0.05).1.2 Effect of BPA exposure on body weight of mice with HFD Compared with the NCD group,the average body weight of the mice in the HFD group increased in different exposure time.Compared with the HFD group,the average body weight of the BPA-exposed group increased in a dose-and time-dependent manner.Compared with the NCD group,the HFD+1000 nmol/L BPA group had a statistically significant difference from the 4th week of exposure(P<0.05).The HFD+10 nmol/L BPA group and the HFD+100 nmol/L BPA group had a statistically significant difference at the 9th week of exposure(P<0.05).The difference between the HFD group at the 10 th week of exposure was statistically significant(P<0.05).Compared with the HFD group,the HFD+1000 nmol/L BPA group had a statistically significant difference from the 11 th week of exposure(P<0.05).The HFD+1000 nmol/L BPA group reached the level of obesity at the 14 th week of exposure.1.3 Effect of BPA exposure on the fat pad coefficient in epididymis of mice with HFD Compared with the NCD group,the increase in the epididymal fat pad coefficient of the HFD group increased.Compared with the HFD group,the epididymal fat pad coefficient of the BPA-exposed group showed a dose-and time-dependent increase.Compared with the NCD group,the BPA-exposed group had statistically significant differences at 8 and 16 weeks of exposure(P<0.05).HFD group mice had statistically significant differences at 16 weeks(P<0.05).Compared with the HFD group,the HFD+100 nmol/L BPA group and the HFD+1000 nmol/L BPA group had statistically significant differences at 16 weeks(P<0.05).2.Effect of BPA exposure on IR phenotype in mice with HFD2.1 Effects of BPA exposure on FG and FI in mice with HFD Compared with the NCD group,both FG and FI were elevated in the HFD group and BPA-exposed group.Compared with the HFD group,there was no significant difference in FG between the BPA-exposed group mice at 8 and 16 weeks(P>0.05),and the FI in HFD+1000 nmol/L BPA group was statistically significant at 8 and 16weeks(P<0.05).Compared with NCD group,the difference of HOME-IR in BPA-exposed group mice at 8 and 16 weeks was statistically significant(P<0.05).Compared with the HFD group,the HOME-IR in BPA-exposed group was statistically significant 16 weeks(P<0.05).2.2 GTT and ITT After intraperitoneal injection of glucose,the blood glucose level of each group of mice was significantly increased.After 2 hours,the blood glucose concentration of the NCD group returned to normal.The blood glucose concentration of the HFD group and BPA-exposed group were still higher than the normal value.Compared with the NCD group,the area under the curve(AUC)of the BPA-exposed group was significantly increased,and the difference was statistically significant at 16 weeks(P<0.05).Compared with the HFD group,the AUC of the HFD+1000 nmol/L BPA group was significantly different at 16 weeks(P<0.05).After intraperitoneal injection of insulin,the blood glucose level of each group of mice was significantly decreased.After 2 hours,the NCD group returned to normal value,and the blood glucose concentration of the HFD group and BPA-exposed group still above normal.Compared with the NCD group,the AUC of the insulin resistance curve of the BPA-exposed group was significantly increased,and the difference was statistically significant at 16 weeks(P<0.05).Compared with the HFD group,the AUC of the HFD+1000 nmol/L BPA group was significantly different at 16 weeks(P<0.05).3.Effect of BPA exposure on insulin sensitivity in epididymis adipose tissue of mice with HFD There was no significant difference in the expression of proteins IRS-1,AKT and PI3K in adipose tissue of mice between the groups at 8 and 16 weeks of exposure(P>0.05).Compared with the NCD group,the expression levels of p-IRS-1,p-AKT and p-PI3 K in the adipose tissue of the HFD group and BPA-exposed group were down-regulated.Compared with the HFD group,the protein expression level of the BPA-exposed group was dose-dependent.The expression of protein in adipose tissue of HFD+1000 nmol/L BPA group was significantly different at 8 and 16 weeks(P<0.05).4.Effects of BPA exposure on inflammatory cell infiltration in epididymis adipose tissue of mice with HFD The results of HE staining showed that compared with the NCD group,the cells in HFD group and BPA-exposed group became significantly larger,and the infiltration of inflammatory cells in the interstitial space increased in a dose-and time-dependent manner.5.Effect of BPA exposure on the expression levels of related inflammatory factors in serum of mice with HFD Compared with the NCD group,the expression levels of inflammatory factors IL-1β,IL-6 and TNF-α in the serum of HFD group and BPA-exposed group increased with the increase of BPA exposure dose,and IL-10 showed a time-and dose-dependent decrease.Compared with the HFD group,the expression levels of related inflammatory factors in the serum of HFD+1000 nmol/L BPA group were statistically significant at 8 and 16 weeks(P<0.05).6.Effects of BPA exposure on the expression of inflammatory factors IL-1β and NLRP3 in epididymis adipose tissue of mice with HFD6.1 Immunohistochemical detection the expression of IL-1β and NLRP3 in epididymis adipose tissue of mice Immunohistochemical observation showed that the expression of inflammatory factor IL-1β and inflammasome NLRP3 in the HFD group and BPA-exposed group were significantly increased in adipose tissue compared with the NCD group.Analysis of cumulative optical density values showed that there was a statistically significant difference in the IOD of IL-1β and NLRP3 in the adipose tissue of the BPA-exposed group compared with the NCD group at 8 weeks and 16 weeks(P<0.05).Compared with the HFD group,the IOD of NLRP3 in the BPA-exposed group was statistically significant at 8 weeks and 16 weeks(P<0.05).6.2 Effect of BPA exposure on NLRP3 protein expression in ATMs of mice The immunofluorescence assay of mice ATMs aggregation showed that the expression of F4/80 and NLRP3 in HFD group and BPA-exposed group was significantly increased in a dose-and time-dependent manner compared with the NCD group.7.Effect of BPA exposure on the expression of related m RNA in epididymis adipose tissue of mice with HFD Compared with the NCD group,the expression levels of NLRP3,ASC,caspase-1and IL-1β m RNA in the adipose tissue of the HFD group and BPA-exposed group were up-regulated.Compared with the HFD group,the BPA-exposed mice increased with the increase of BPA exposure dose,and the m RNA expression levels of adipose tissue in the HFD+1000 nmol/L BPA group were statistically different at 8 and 16 weeks(P<0.05).8.Effect of BPA exposure on the expression of related proteins in epididymis adipose tissue of mice with HFD Compared with the NCD group,the expression levels of NLRP3,caspase-1 and IL-1β in the adipose tissue of the HFD group and BPA-exposed group were up-regulated.Compared with the HFD group,the average gray value analysis showed that the protein expression levels in the BPA-exposed group increased with increasing BPA exposure dose.The protein expression level of HFD+1000 nmol/L BPA group mice was significantly higher at 8 and 16 weeks(P<0.05).There was no significant difference in the expression of pro-caspase-1 and pro-IL-1β between the groups at 8and 16 weeks of exposure(P>0.05).Conclusions1.BPA exposure alters the IR phenotype of HFD-induced obese mice and reduces insulin sensitivity in mice adipose tissue.2.BPA exposure can promote HFD-induced mice obesity and adipose tissue inflammation.3.BPA exposure promoted the expression of NLRP3 in adipose tissue of mice by HFD-induced,and was associated with increased IL-1β secretion.4.Long-term exposure to BPA can activate NLRP3 inflammasome by HFD-induce,up-regulates IL-1β secretion,and aggravates obesity-derived IR through interference with insulin signaling pathways. |
