| Background Bisphenol A(BPA)is used as a synthetic monomer for a variety of raw materials in industry,and it exists in large quantities in the plastic products used by modern people.It is a kind of environmental endocrine disruptors(EDCs)that are currently widely exposed by humans.As the production and use of BPA increase year by year,the population is increasingly exposed to BPA through occupation or the environment.Studies have shown that environmental BPA exposure is positively correlated with the incidence of metabolic diseases such as obesity,insulin resistance(IR),and type 2 diabetes(T2D).Animal experiments also showed that obese mice induced by long-term exposure to high fat diet(HFD)with BPA showed more rapid weight gain and increased serum insulin levels,impaired glucose tolerance,hyperlipidemia,IR and other symptoms.It is suggested that BPA promotes mouse obesity and IR induced by HFD.The liver is the body’s more sensitive target organ during the occurrence of IR.Long-term BPA exposure may cause irreversible liver damage in the occurrence of obesity.The obesity process is accompanied by liver steatosis and inflammatory damage,which can lead to abnormal liver function,cirrhosis,and eventually liver cancer.The activation of NLRP3 inflammasomes and mediating the secretion of related inflammatory factors are the key to the study of inflammatory and immune responses in liver tissues.However,in BPA-induced HFD-induced IR-related liver damage,whether the upregulation of liver IL-1βsecretion is related to the activation of NLRP3 inflammasome is still unclear,and further investigation is needed.Objective Whole animal experiments were used to detect the IR phenotype,liver damage,NLRP3 activation and inflammatory factor IL-1β expression in the liver of HFD-fed mice exposed to BPA,and to explore the effect of BPA exposure on HFD-induced IR-related liver damage and NLRP3 inflammasome Relationship.And use the NLRP3 activation inhibitor MCC950 to inhibit the activation of NLRP3 inflammasomes to further verify the regulatory role of NLRP3 inflammasome activation in BPA promoting HFD-induced IR-related liver injury.Method Three-week-old male wild-type C57BL/6J mice with specific pathogen free(SPF)were selected,a total of 60 mice.After one week of adaptation to feeding in the animal room,they were randomly divided into 5 groups,including NCD group(low-fat diet,normal drinking water),HFD group(high-fat diet,normal drinking water),and HFD + 1000 nM BPA exposure group(high-fat diet),BPA drinking water exposure),HFD + NLRP3 inhibitor group(high-fat feed,normal drinking water),HFD + 1000 nM BPA + NLRP3 inhibitor group(high-fat feed,BPA drinking water exposure).Each group includes two for 8 weeks and 16 weeks treatment time,each group has 6 mice at each time point.The mice were exposed to 1000 nM BPA via drinking water.HFD +NLRP3 inhibitor group mice and HFD + 1000 nM BPA + NLRP3 inhibitor group mice were injected intraperitoneally with 10 mg/kg NLRP3 activation inhibitor MCC950 every other day from week 5 to week 8 and week 13 to week 16.The weight,water consumption and food consumption of the mice were weighed at a fixed time every week.At the end of 8 weeks and 16 weeks of rearing,blood was collected from the tail and eyeballs of the mice.FG and FI were detected with mouse serum,and IR was evaluated.Perform GTT and ITT experiments.The liver function of mice was detected by AST and ALT experiments.The mice were sacrificed,and the middle part of the hepatic lobe of the liver was taken for histopathological examination,and the histopathological changes of the liver of the mice were observed.Immunohistochemistry was used to detect the expression of IL-1β,IL-18,TNF-α and NLRP3 in liver tissues.Immunofluorescence was used to detect the expression of NLRP3 protein in mouse liver macrophages.WB was used to detect the expression of related proteins in the IR signaling pathway in mouse liver tissues and the cleavage of related proteins in the activation pathway of NLRP3 inflammasomes.QRT-PCR analysis of mouse liver NLRP3 inflammasome activation related factors IL-1β,ASC,NLRP3 and caspase-1 mRNA expression.Results1 General conditions of mice1.1 Water and diet of mice During the experiment,the mice were in good condition,and no adverse phenomena such as death occurred.There were no statistically significant differences in the average weekly water intake and food intake of mice in each group at the two time points of 8 weeks and 16 weeks(P > 0.05).1.2 Mouse body weight changes Compared with the NCD group,as the age of the mice increased,the body weight of the mice in the HFD group increased at 8 weeks and 16 weeks,and the difference was statistically significant(P < 0.05).Compared with the HFD group,the weight of the mice in the HFD + 1000 nM BPA group increased,and the difference was statistically significant from the 5th week(P < 0.05).The weight gain of the mice was suppressed after using the NLRP3 inhibitor.Compared with the HFD group,the mice in the HFD + NLRP3 inhibitor group lost weight,and the difference was statistically significant from the 7th week(P < 0.05).Compared with the HFD + 1000 nM BPA group,the mice in the HFD + 1000 nM BPA + NLRP3 inhibitor group decreased significantly,and the difference was statistically significant from the 8th week(P <0.05).2 Mouse insulin resistance2.1 Mice FG,FI levels and HOMA-IR Compared with the NCD group,the FG,FI levels and HOMA-IR levels of the mice in the 8-week and 16-week HFD groups increased,and the difference was statistically significant(P < 0.05).Compared with the HFD group,the FG,FI levels and HOMA-IR of the mice in the 8 and 16 weeks HFD + 1000 nM BPA exposure group were significantly increased.The FG and FI levels of the mice in the HFD +NLRP3 inhibitor group at 8 and 16 weeks were significantly higher.Both the level and HOMA-IR decreased significantly,and the difference was statistically significant(P <0.05).Compared with the HFD + 1000 nM BPA exposure group,the 8 and 16 weeks HFD + 1000 nM BPA + NLRP3 inhibitor group mice FG,FI levels and HOMA-IR were significantly lower,the difference was statistically significant(P < 0.05).2.2 Mice GTT and ITT GTT experiments were performed on mice.After intraperitoneal injection of glucose,the blood glucose levels of mice at 8 and 16 weeks increased rapidly.The blood glucose levels of mice in each group increased to the maximum concentration at the 15 th minute,and then gradually recovered.The blood glucose of the mice in the NCD group returned to the initial level at 2h,while the blood glucose of the mice in the HFD group and the HFD + 1000 nM BPA exposure group was still higher than the initial level.The results of the area under the curve of GTT showed that the area under the curve of mice in the 8-week and 16-week HFD group was higher than that in the NCD group(P < 0.05),and the area under the curve in the HFD + 1000 nM BPA exposure group was higher than that of the HFD group.It is statistically significant(P< 0.05).The blood glucose of mice in the HFD + NLRP3 inhibitor group and HFD +1000 nM BPA + NLRP3 inhibitor group returned to the initial level at 2h.The area under the curve of mice in the HFD + NLRP3 inhibitor group at 8 and 16 weeks was significantly lower than that in the HFD group(P < 0.05).The area under the curve of mice in the HFD + 1000 nM BPA + NLRP3 inhibitor group at 8 and 16 weeks was lower than that in the HFD + 1000 nM BPA exposure group,and the difference was statistically significant(P < 0.05).In the ITT experiment,after the mice were injected with insulin intraperitoneally,the blood glucose levels of the mice at 8 and 16 weeks decreased rapidly.The blood glucose levels of the mice in each group decreased to the lowest concentration at the30 th minute,and then gradually recovered.The blood glucose of the mice in the NCD group returned to the initial level at 2h,while the blood glucose of the mice in the HFD group and the HFD + 1000 nM BPA exposure group was still higher than the initial level.The results of the area under the curve of ITT showed that the area under the curve in the 8-week and 16-week HFD group was higher than that in the NCD group,and the difference was statistically significant(P < 0.05).The area under the curve in the HFD + 1000 nM BPA exposure group higher than the HFD group(P < 0.05).The blood glucose of mice in the HFD + NLRP3 inhibitor group and HFD + 1000 nM BPA+ NLRP3 inhibitor group returned to the initial level at 2h.The area under the curve of mice in the HFD + NLRP3 inhibitor group at 8 and 16 weeks was significantly lower than that in the HFD group(P < 0.05).The area under the curve of mice in the HFD +1000 nM BPA + NLRP3 inhibitor group at 8 and 16 weeks was lower than that in the HFD + 1000 nM BPA exposure group,and the difference was statistically significant(P < 0.05).3 Liver damage in mice3.1 Mice liver coefficient Compared with the NCD group,the organ coefficients of the liver tissues of mice in the 8-week and 16-week HFD groups were significantly increased(P < 0.05).Compared with the HFD group,the organ coefficients of the liver tissues of the mice exposed to HFD + 1000 nM BPA at 8 and 16 weeks were significantly higher(P < 0.05).Comparing the same treatment group at different time points,the organ coefficient of the liver tissue of the 16-week HFD group and the HFD + 1000 nM BPA exposure group was significantly higher than that of the 8-week mouse(P < 0.05).Compared with the HFD group,the HFD + NLRP3 inhibitor group had no significant difference in the organ coefficient of liver tissue at 8 weeks,while the organ coefficient at 16 weeks decreased significantly(P < 0.05).Compared with the HFD + 1000 nM BPA exposure group,the organ coefficients of the liver tissue of mice in the 8-week and16-week HFD + 1000 nM BPA + NLRP3 inhibitor groups decreased,and the 16-week difference was statistically significant(P < 0.05).3.2 Mice liver function The ALT and AST experiments on mice showed that compared with the NCD group,the AST of the mice in the HFD group at 8 weeks was significantly higher(P <0.05),and there was no difference in ALT.The ALT and AST of the mice in the HFD group at 16 weeks were both higher.The difference was statistically significant(P <0.05).Compared with the HFD group,the ALT and AST of the mice in the 8-week and16-week HFD + 1000 nM BPA exposure groups increased,and the difference was statistically significant(P < 0.05).Compared with the HFD group,the AST of the mice in the HFD + NLRP3 inhibitor group at 8 weeks was significantly lower(P < 0.05),while the ALT and AST of the mice in the HFD + NLRP3 inhibitor group at 16 weeks were significantly lower(P < 0.05).Compared with the HFD + 1000 nM BPA exposure group,the 8-week and 16-week HFD + 1000 nM BPA + NLRP3 inhibitor groups were significantly lower in ALT and AST,and the difference was statistically significant(P <0.05).3.3 Histopathological changes in mouse liver tissue The liver tissues of mice were examined by histopathology,and the HE staining results showed that compared with NCD,the liver tissue cells of mice in the HFD group at 8 and 16 weeks showed slight swelling,bullous steatosis,and inflammatory cell infiltration,and at 16 weeks Time changes are more obvious.Compared with the HFD group,the liver tissue cells of the mice exposed to HFD + 1000 nM BPA at 8 and16 weeks were arranged disorderly,cell swelling,bullous steatosis,and inflammatory cell infiltration were more serious,and the changes were more obvious at 16 weeks.Compared with the HFD group,the mice in the HFD + NLRP3 inhibitor group had reduced liver tissue cell damage,degeneration,and infiltration at 8 weeks and 16 weeks.Compared with the HFD + 1000 nM BPA exposure group,the liver tissue cell damage,degeneration,and infiltration of the mice in the HFD + 1000 nM BPA +NLRP3 inhibitor group at the two treatment times were also significantly reduced.3.4 Expression of inflammatory factors in mouse liver tissue According to the IHC observation results,the average integrated optical density(IOD)was calculated.The results showed that compared with the NCD group,the IL-1β,IL-18,and TNF-α in the liver tissues of the HFD group at 8 and 16 weeks The positive expressions all increased,and the difference was statistically significant(P <0.05).Compared with the HFD group,the positive expressions of IL-1β,IL-18,and TNF-α in the liver tissues of mice exposed to HFD + 1000 nM BPA at 8 and 16 weeks increased,and the difference was statistically significant(P < 0.05).Compared with the HFD group,the positive expressions of IL-1β,IL-18,and TNF-α in the liver tissue of the mice in the HFD + NLRP3 inhibitor group decreased significantly at the two treatment times,and the difference was statistically significant(P < 0.05).In addition,the positive expression of IL-1β,IL-18,and TNF-α in the liver tissue of mice in the HFD + 1000 nM BPA + NLRP3 inhibitor group at 8 and 16 weeks were significantly lower than those in the HFD + 1000 nM BPA exposure group,and the difference was statistically significant(P < 0.05).4 NLRP3 inflammasome activation in mouse liver tissue4.1 Mice liver tissue NLRP3 protein expression Immunofluorescence was used to detect the expression of F4/80 and NLRP3 in the macrophages of mouse liver tissue.The results showed that the expression of F4/80 and NLRP3 in the HFD group at 8 and 16 weeks was increased compared with the NCD group.Compared with the HFD group,the expressions of F4/80 and NLRP3 in the 8-week and 16-week HFD groups were further increased.Compared with the HFD group,the F4/80 and NLRP3 protein expressions in the HFD + NLRP3 inhibitor group at 8 and 16 weeks were reduced.Compared with the HFD + 1000 nM BPA exposure group,the 8 and 16 weeks HFD + 1000 nM BPA+NLRP3 inhibitor groups also had lower F4/80 and NLRP3 expressions.4.2 MRNA expression of NLRP3 inflammasome activation related molecules in mouse liver Compared with the NCD group,the liver ASC mRNA expression level of the HFD group increased at 8 weeks(P < 0.05),and the liver NLRP3,ASC,caspase-1,and IL-1β mRNA expression levels of the HFD group were all increased at 16 weeks.And the difference was statistically significant(P < 0.05).Compared with the HFD group,the liver NLRP3,ASC,caspase-1,and IL-1β mRNA expression levels of the mice in the 8-week and 16-week HFD + 1000 nM BPA exposure groups were significantly increased,and the difference was statistically significant(P < 0.05).Compared with the HFD group,the liver NLRP3,ASC,caspase-1 and IL-1β mRNA expression levels of the mice in the HFD + NLRP3 inhibitor group decreased at 16 weeks,and the difference was statistically significant(P < 0.05).Compared with the HFD + 1000 nM BPA exposure group,the liver NLRP3,ASC,caspase-1 and IL-1β mRNA expression levels of the mice in the 8-week and 16-week HFD + 1000 nM BPA+ NLRP3 inhibitor groups were significantly reduced,and the difference was statistically significant.Significance(P < 0.05).4.3 Related protein expression in mouse liver tissue The average gray value analysis results showed that compared with the NCD group,the liver NLRP3,caspase-1,and IL-1β protein expression levels of the mice in the 8-week and 16-week HFD groups increased,and the difference was statistically significant(P < 0.05).Compared with the HFD group,the liver protein NLRP3,caspase-1,and IL-1β expression levels of the mice in the 8-week and 16-week HFD +1000 nM BPA exposure groups were significantly increased,and the difference was statistically significant(P < 0.05).Compared with the HFD group,the liver NLRP3,caspase-1,and IL-1β protein expression levels of mice in the 8-week and 16-week HFD + NLRP3 inhibitor groups were reduced,and the difference was statistically significant(P < 0.05).Compared with the HFD + 1000 nM BPA exposure group,the liver NLRP3,caspase-1,and IL-1β protein expression levels of the mice in the 8-week and 16-week HFD + 1000 nM BPA + NLRP3 inhibitor groups were significantly reduced,and the difference was statistically significant(P < 0.05).5 Detection of insulin sensitivity in mouse liver tissue IB detected and analyzed the expression of IRS-1/p-IRS-1(Tyr612),PI3Kp85/p-PI3Kp85(Tyr458),and AKT/p-AKT(SER473)in liver tissues after 5minutes of intraperitoneal injection of insulin in mice.The results showed that the protein expressions of IRS-1,PI3Kp85 and AKT in the liver of mice at 8 and 16 weeks were not significantly different between the NCD group,the HFD group,and the HFD+ 1000 nM BPA exposure group(P > 0.05).Compared with the NCD group,the expression levels of p-IRS-1,p-PI3Kp85,and p-AKT in the livers of mice in the8-week and 16-week HFD groups were down-regulated(P < 0.05).Compared with the HFD group,the expression levels of p-IRS-1,p-PI3Kp85,and p-AKT in the liver of mice exposed to HFD + 1000 nM BPA at 8 and 16 weeks were all down-regulated.The protein expressions of IRS-1,PI3Kp85 and AKT in the livers of mice at 8 and 16 weeks were not significantly different between the HFD + NLRP3 inhibitor group and the HFD + 1000 nM BPA + NLRP3 inhibitor group(P > 0.05).Compared with the HFD group,the expression levels of p-IRS-1,p-PI3Kp85,and p-AKT in the liver of mice in the HFD + NLRP3 inhibitor group at 8 and 16 weeks were all increased(P <0.05).Compared with the HFD + 1000 nM BPA exposure group,the expressions of p-IRS-1,p-PI3Kp85,and p-AKT in the liver of mice in the HFD + 1000 nM BPA +NLRP3 inhibitor group at 8 and 16 weeks were significantly increased(P <0.05).Conclusion1.BPA exposure promotes IR and liver damage in HFD-fed mice.2.BPA exposure can increase the secretion of IL-1β by activating the NLRP3 inflammasome,and aggravate IR and related liver damage in HFD-fed mice.3.NLRP3 inflammasome activated by BPA exposure activates IRS-1 / PI3Kp85 / AKT signaling pathway to mediate IR and related liver damage. |
