| Objectives Bisphenol A(BPA)is one of the main organic chemical raw materials,which is widely used in the production of polymer materials and daily consumer goods.BPA,as a common environmental endocrine disruptor,will have adverse effects on all biological systems.Current studies in vivo and in vitro indicate that BPA may act as one of obesogens,disrupt the balance of lipid metabolism and induce lipid deposition in the body.Currently,there are limited studies on the effects of BPA on lipid metabolism,and its mechanism of action is still unclear.Thus,in our study,an animal model of BPA exposure during pregnancy was established to observe the general toxicity indexes of male fetal mice,and biochemical and molecular biological methods were used to detect the levels of serum lipids and liver lipids in fetal mice,as well as the expression of lipid metabolism-related pathways and genes in liver,so as to further investigate the possible mechanism of BPA’s effect on liver lipid metabolism.This study aims to provide a theoretical basis for the regulatory mechanism of BPA effects on progeny lipid metabolism and provide new clues forfurther targeted therapy.Methods The experimental animals were SPF healthy Sprague-Dawley rats(40 females,20 males)aged 9 weeks.After a week of adaptive feeding,female and male rats were caged in a ratio of 2:1 every night.The vaginal smear test was performed the next morning and the date for observing sperm under the microscope was set as Gestation day 0(GD0).Pregnant rats were randomly divided into 4 groups,which were given gavage for 15 consecutive days from GD5 to GD19(once a day),and the doses of BPA were 0,0.05,0.5 and 5 mg/kg,respectively,which were recorded as control group and BPA exposure group.After natural delivery and lactation,The offspring male mice were sacrificed at weaning stage(PND21)and sexual maturity(PND56)respectively,and their serum and tissue were collected for the subsequent detection of experimental indicators.1.The diet,body weight and the weight of each organ of the offspring male rats in the two stages were observed,and the organ coefficient was calculated to analyze the general development.2.Determination of triglycerides and total cholesterol in serum and liver of male rats at two stages of offspring by ELISA.3.The expression levels of fatty acid synthesis-related genes and oxidative metabolism-related genes as well as the expression levels of key genes in the fatty acid synthesis pathway in the liver of offspring male rats at two stages were detected by Real-time PCR and Western-blot.Results1.Effects of BPA exposure during pregnancy on general indexes of offspring male rats1)At PND21 stage,compared with the control group,the body weight of offspring male rats in all BPA exposure groups increased,but the difference was significant only in 0.05 mg/kg group(P<0.05).There was no significant change in kidney wet weight,but liver and testicle weight increased in BPA exposure groups,and there were significant differences in liver weight in 0.5 mg/kg group and testicle weight in 0.05 and 0.5 mg/kg groups(P<0.05).The liver organ coefficients were not changed,but the kidney and testis organ coefficients were significantly increased in the 5 mg/kg BPA exposure group(P<0.05).2)At PND56 stage,compared with the control group,the body weight of offspring male rats in BPA exposure groups was decreased,but that in 0.5 mg/kg group was significantly decreased(P<0.05).There were no significant changes in liver and testis weight,and kidney weight in 5 mg/kg group was significantly decreased(P<0.05).There was no significant difference in testicular organ coefficient among all groups.Liver organ coefficient in 0.05 and 0.5 mg/kg groups was significantly increased(P<0.05),and kidney organ coefficient in 0.05 and 5 mg/kg groups was significantly increased(P<0.05).2.Effects of BPA exposure during pregnancy on serum and liver lipid levels in offspring male rats1)At PND21 stage,compared with the control group,triglyceride(TG)levels in serum and liver of BPA exposed groups were significantly increased(P<0.05).Serum total cholesterol(TC)in all groups exposed to BPA was significantly increased(P<0.05),TC content in liver was significantly increased in 0.5 and 5 mg/kg groups(P<0.05),and significantly decreased in 0.05 mg/kg group(P<0.05).2)At PND56 stage,compared with the control group,TG levels in serum and liver of BPA exposed groups were significantly increased(P<0.05).Serum TC levels were significantly increased in all groups exposed to BPA(P<0.05),but there was no significant difference in TC content in liver.3.Effects of BPA exposure during pregnancy on the expression of fatty acid oxidation related genes in the liver of offspring male rats1)At PND21 stage,compared with control group,PPARα m RNA expression in BPA exposed groups was significantly decreased(P<0.05),and PPARα protein expression level in 5 mg/kg group was also significantly decreased(P<0.05).The relative expression of CPT1α m RNA and protein was significantly decreased in the 5mg/kg group(P<0.05).2)At the PND56 stage,compared with the control group,the m RNA expression and protein levels of PPARα and CPT1α in the BPA exposed groups showed a decreased trend.The m RNA expression and protein level of PPARα in 0.5 and 5mg/kg groups were significantly decreased(P<0.05),and the protein level of CPT1αin 5 mg/kg group was significantly decreased(P<0.05).4.Effects of BPA exposure during pregnancy on the expression of genes related to fatty acid synthesis in the liver of offspring male rats1)At PND21 stage,compared with the control group,the m RNA expressions of SREBP-1,FAS and SCD-1 in BPA exposed groups were significantly increased,and the protein expressions of SREBP-1 and SCD-1 were significantly increased in all groups(P<0.05).The m RNA expression of ACC1 in 5 mg/kg group was significantly increased(P<0.05).2)At PND56 stage,compared with the control group,m RNA expressions of SREBP-1,ACC1,FAS and SCD-1 in BPA exposed groups were significantly increased(P<0.05),and protein levels of SREBP-1 and SCD-1 were significantly increased(P<0.05).5.Effects of BPA exposure during pregnancy on expression of factors related to fatty acid synthesis pathway in liver of offspring male rats1)At PND21 stage,compared with control group,m TOR m RNA expression in0.05 mg/kg group was significantly increased(P<0.05).The m RNA expression of CRTC2 was significantly increased in all groups exposed to BPA(P<0.05).The expression of m TOR protein in BPA exposed groups was significantly increased compared with the control group(P<0.05),but the level of phosphorylated protein was not significantly changed.There was no significant change in CRTC2 protein level,but phosphorylated protein level in 0.5 and 5 mg/kg groups was significantly increased(P<0.05).2)At PND56 stage,compared with control group,m TOR m RNA expression level in 5 mg/kg group was significantly increased(P<0.05),but no significant change was found in other two groups.The m RNA expression level of CRTC2 in 0.05 and 0.5 mg/kg groups was significantly increased(P<0.05),but there was no significant change in 5 mg/kg group.m TOR protein expression was significantly increased in 0.5 and 5 mg/kg groups,but phosphorylated protein level was not significantly changed.There was no significant change in m TOR protein expression in 0.05 mg/kg group,but the phosphorylated protein level was significantly increased(P<0.05).The total protein level of CRTC2 had no significant change in the two periods,but the phosphorylation level of CRTC2 was significantly increased at 0.5and 5 mg/kg(P<0.05).Conclusion1.BPA exposure during pregnancy can affect body weight,organs and organ coefficient of offspring male mice to varying degrees;It can increase the levels of TG and TC in serum and liver of offspring male mice;2.BPA exposure during pregnancy affects lipid levels in the liver of male offspring by inhibiting the expression of fatty acid oxidation-related genes PPARα and CPT1α,and promoting the expression of fatty acid synthesis-related genes SREBP-1,ACC1,FAS and SCD-1.That is,the regulation of SREBP-1 mediated m TOR pathway by CRTC2 has adverse effects on lipid metabolism in offspring male mice. |
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