B(a)P Induces Ovarian Granulosa Cell Apoptosis Via The TRAF2-NF?B-Caspase 1 Axis During Early Pregnancy

Objective:Benzopyrene(B(a)P),one of the representative polycyclic aromatic hydrocarbons(PAHs),belongs to endocrine disruptors and has ovarian toxicity.Our previous results showed that B(a)P affected the embryo implantation and decidualization during early pregnancy.Those processes were all regulated by the synthesis and secretion of estrogen and progesterone of ovarian granulosa cells.However,the mechanism of B(a)P-induced ovarian toxicity during early pregnancy is poor understood.This study explored the impacts of B(a)P on ovarian function and the specific toxic mechanism during early pregnancy.Methods:1.The female mice with vaginal plug were randomly divided into the control group and the B(a)P group.The first day of pregnancy was recorded as"D1".Mice in B(a)P group were given 0.2 mg/kg B(a)P by gavage every morning from D1 to D7,while the mice in the control group recevied corn oil at 0.1m L/10g animal body weight every morning from D1 to D7.Serum and ovarian were collected on D4 and D7,respectively.2.The human ovarian granulosa cell line KGN was divided into the control group,h CG group and BPDE group.The cell in control group was treated with DMSO,while the cell in h CG group was treated with 1.0IU/m L h CG for 24h to induce luteinization.The KGN cell in BPDE group was exposed to BPDE(0.5?mol/L)combined with h CG(1.0 IU/m L)for24h.BPDE is the main metabolite of B(a)P,which exerts toxic effects at the cellular level in vivo.3.Establishment of oxidative stress positive control cell model:KGN cell was pre-treated with h CG(1.0 IU/m L)for 20h to induce luteinize,and then treated with different concentrations of H₂O₂plus h CG for 4h.4.Establishment of NAC-treated cell model:KGN cell was treated with5m M NAC for 2h in advance,and then incubated with h CG(1.0 IU/m L)plus BPDE(0.5?mol/L)or H₂O₂(50?M).5.Establishment of TRAF2 overexpression cell model:The overexpressionplasmidvectors(pc DNA3.1(+)-vectorand pc DNA3.1(+)-TRAF2)were constructed and used to transfect KGN cells for 72h,respectively.6.Establishment of Bet A agonist cell model:KGN cell was treated with Bet A(1?M)in co-with h CG(1.0 IU/m L)and BPDE(0.5?mol/L)for24h.7.Establishment of VX-765 inhibitor cell model:KGN cell was treated with VX-765(5?M)in co-with h CG(1.0 IU/m L)and BPDE(0.5?mol/L)for 24h.After the collection of materials,the following experiments were conducted.(1)ELISA was used to detect the estrogen and progesterone levels in serum.RT-q PCR was used to detect the m RNA levels of 3?-HSD,17?-HSD and P450scc in vivo and in vitro.(2)RT-q PCR was used to detect the m RNA levels of oxidative stress indicators CAT and SOD2,while CM-H₂DCFDA was used to detect ROS level in vivo and in vitro.(3)The levels of inflammatory cytokine IL-18,IL-1?and TNF?were detected by ELISA and RT-q PCR in vivo and in vitro.(4)Western blot and IF methods were used to detect the expression of pyroptosis-related proteins in vivo and in vitro.(5)TUNEL was used to detect the apoptosis both in vivo and in vitro.(6)RT-q PCR,Western blot and IF methods were used to detect the expression of TRAF2 in vivo and in vitro.Based on the results,we explored the role of TRAF2 on the ovarian toxicity caused by BPDE via overexpression of TRAF2 in luteinized KGN cell.(7)The nuclear and cytoplasmic proteins were isolated,and the expression of the NF?B signaling pathway-related proteins was detected by western blot in vivo and in vitro.IF was used to detect the location of NF?B protein in KGN cell.Bet A,an NF?B agonist,was used to treat luteinized KGN cell to investigate the role of NF?B on BPDE-induced ovarian toxicity.(8)Western blot was used to detect the expressions of Caspase 1 and t BID in vivo and in vitro.After treatment with the Caspase 1 inhibitor VX-765,the expression of the pro-apoptotic gene t BID was observed in luteinized KGN cell.(9)The BPDE and H₂O₂-infected cell models were treated with the antioxidant NAC,to observe the protective effects of NAC on BPDE or H₂O₂-induced ovarian granulosa cells toxicity.Results:1.B(a)P reduced the serum levels of estrogen and progesterone during early pregnancy.B(a)P and its metabolite BPDE inhibited the m RNA levels of the 3?-HSD,17?-HSD and P450scc2.B(a)P and its metabolite BPDE down-regulated the m RNA levels of oxidative stress indicators CAT and SOD2,promoted the generation of ROS and enhanced oxidative stress.Similar results were observed in H₂O₂treated group.NAC could migrate of oxidative stress caused by BPDE(or H₂O₂).3.B(a)P and its metabolite BPDE promoted the release of inflammatory cytokine IL-18,IL-1?and TNF?,and caused inflammatory reactions.Inflammatory reaction was observed in H₂O₂positive cell model.NAC inhibited inflammation induced by BPDE(or H₂O₂).4.B(a)P and BPDE induced ovarian apoptosis rather than pyroptosis.During this process,BPDE activated Caspase 1.5.B(a)P and BPDE inhibited the expression of TRAF2.Overexpression TRAF2 could inhibited BPDE-induced apoptosis,and down-regulated the m RNA levels of the 3?-HSD,17?-HSD,and P450scc in luteinized KGN cells.NAC alleviated the inhibitory effect of BPDE(or H₂O₂)on TRAF2expression.6.B(a)P and BPDE inhibited the phosphorylation of I?B,and blocked NF?B signals from the cytoplasm into the nucleus.Besides,NF?B signaling pathway was located downstream of TRAF2 and regulated by TRAF2.Using Bet A(a NF?B agonist)or overexpression of TRAF2resulted in a recovery of BPDE-induced apoptosis and down-regulation m RNA levels of the 3?-HSD,17?-HSD and P450scc in luteinized KGN cell.7.B(a)P and BPDE activated Caspase 1 and up-regulated the expression of pro-apoptotic gene t BID.Overexpression of TRAF2 and using Bet A inhibited Caspase 1 activation.Using VX-765,a Caspase 1 inhibitor,salvaged the BPDE-induced abnormal expression of t BID.Conclusion:B(a)P and its metabolite BPDE enhanced oxidative stress,induced inflammatory responses,cleaved the pro-apoptotic gene BID on mitochondria and promoted the apoptosis of ovarian granulosa cell via the TRAF2-NF?B-Caspase 1 axis.As the consequence,it inhibited the levels of estrogen and progesterone during early pregnancy.