Effects And Mechanism Research Of Exposure To Bisphenol AF On Cardiomyocytes Apoptosis And Autophagy

Background and objectives: Bisphenol AF(BPAF)is a kind of bisphenol A(BPA)analogue with endocrine disrupting properties and is widely used in the production of important chemicals such as fluoroelastomers and polyesters.In recent years,BPAF has been monitored in environmental media.The study found that BPA is a of kind of risk factors for cardiovascular disease,while BPAF has not been reported on cardiovascular disease.In this study,ICR mice and AC16 human cardiomyocytes were used as subjects.Parental mice were exposed to BPAF,and their cardiotoxicity to offspring mice was observed.The potential molecular biological mechanism of BPAF on cardiotoxicity was further explored.Methods:1.Pregnant ICR mice were exposed to sodium carboxymethylcellulose(control),0.4 mg/kg(low dose)and 4.0 mg/kg(high dose)BPAF daily during pregnancy and lactation.The offspring mice were sacrificed 56 days after birth,and the heart tissues were taken.The following experiments were conducted: the expression of Caspase-3,Bcl-2,Bax,LC3 and BNIP3 were measured by Western blot analysis and immunohistochemistry staining,as well as the expression of upstream related factors Akt,AMPK and mTOR.2.AC16 human cardiomyocytes were cultured and the effects of BPAF(1nM)on cardiomyocytes were compared after adding PI3K/Akt antagonist(LY294002)and mTOR inhibitor(rapamycin).The following experiments were conducted: the expression of Bcl-2,Bax,LC3 and BNIP3 were measured by Western blot analysis,as well as the level of upstream related factors Akt,AMPK and mTOR.And cell assays were performed to determine whether PI3K/Akt or AMPK/mTOR signaling pathway was involved in the BPAF induced apoptosis and autophagy on myocardial.Results:1.The results of animal experimentation:(1)Low-dose BPAF significantly inhibited the expression levels of myocardial apoptosis-related factors Bax and Caspase-3 and the regulatory factor mTOR,promoted the expression levels of myocardial anti-apoptotic factor Bcl-2 and the regulatory factors Akt and AMPK in progeny mice,thereby having inhibited apoptosis,however,the effects of high-dose BPAF were contrary to the effects of low-dose BPAF,and promoted myocardial apoptosis.(2)Low-dose BPAF upregulated the protein expression of myocardial autophagy-related factors LC3,Beclin-1 and BNIP3 and regulatory factors Akt and AMPK in progeny mice,downregulated the protein expression of the regulatory factor mTOR,thereby having promoted myocardial autophagy.The effects of high-dose BPAF to progeny mouse heart were contrary to the effects of low-dose BPAF,and inhibited myocardial autophagy.(3)Low-dose BPAF promoted the expression of AMPK and Akt and inhibited the expression of mTOR in progeny mouse cardiomyocytes.The effects of high-dose BPAF on regulatory factors were opposite to those of low dose.2.The results of AC16 human cardiomyocyte experimentation:(1)After exposure to 1nM BPAF for 24 hours,AC16 human cardiomyocytes increased the content of apoptosis-related factor Bcl-2 protein and decreased the Bax protein content,indicating that BPAF promoted the anti-apoptotic effect of cardiomyocytes.Meanwhile,the autophagy-related factors LC3 and BNIP3 protein content Elevated,indicating that BPAF promotes autophagy in cardiomyocytes.(2)After adding PI3 K / Akt antagonist(LY294002),Akt content in cardiomyocytes decreased,Bcl-2 protein content decreased,Bax protein content was not affected,indicating that BPAF inhibited cardiomyocyte anti-apoptosis;PI3K/Akt was blocked After the disruption,the content of mTOR protein was unchanged,and the contents of LC3 and BNIP3 were also unaffected,indicating that the autophagy effect of BPAF on cardiomyocytes was not affected by PI3K/Akt antagonists.(3)After adding mTOR inhibitor(rapamycin),the content of mTOR protein in cardiomyocytes decreased,and the content of Bax protein also decreased,but the expression of Bcl-2 protein was not changed,indicating that the anti-apoptotic effect of BPAF on cardiomyocytes was enhanced;After inhibition,both LC3 and BNIP3 protein levels increased,indicating that BPAF enhanced autophagy in cardiomyocytes.Conclusions:1.Low-dose BPAF induced autophagy and inhibited apoptosis in cardiomyocytes.And the effects of high-dose BPAF were contry to those of low-dose in cardiomyocytes.2.mTOR was a key regulator of BPAF-induced autophagy and apoptosis in cardiomyocytes.3.BPAF induced cardiomyocyte apoptosis via PI3K/Akt and AMPK/mTOR signaling pathway,and induced autophagy in cardiomyocytes via AMPK/mTOR signaling pathway.