Mechanisms Of Female Reproductive Toxicity Due To Excessive Autophagy Of Ovarian Granulosa Cells Induced By Plasticizer DEHP Through Its Metabolite MEHP

Background In recent years,the pollution and proliferation of plastics have gradually become a worldwide problem.The plasticizer DEHP is widely used in various plastic PVC products.DEHP is quickly metabolized by enzymes in the body to MEHP,which is the main substance involved in many diseases in the body such as cancer,diabetes,premature ovarian failure and endometriosis.In the past,most studies on DEHP reproductive toxicity have focused on ovarian estrogen and granulosa cell apoptosis.Although some attention has been paid to the role of cellular autophagy and oxidative stress in the ovary,The connection between the type of autophagy and oxidative stress is not clear.Besides,the molecular mechanism of regulating oxidative stress and whether there is a correlation with DEHP-induced ovarian reproductive toxicity is still unknown.Therefore,it is urgent to provide new insights and theoretical basis for the research to investigate the mechanism of ovarian damage by DEHP.Objective Construct and evaluate the reproductive toxicity model of plasticizer(DEHP)damage in female mic.Determine whether DEHP and its metabolite MEHP can cause excessive autophagy and oxidative stress in ovarian granulosa cells.Exploring the potential relationship between the two and the potential mechanisms that may regulate autophagy in ovarian granulosa cells through oxidative stress pathways.Methods 1.Female 4-week-old ICR mice were gavaged with DEHP(500,1500mg/kg)for 30 consecutive days.Their body weight was recorded weekly and gvaginal cell exfoliation was used to detect the estrous cycle of female mice.Ovarian organ coefficients were calculated.Serum estrogen levels were measured by Elisa and ovarian structures were examined by histomorphological HE.2.Western blotting(WB)and immunohistochemistry were used to detect the expression,distribution,and localization of ovarian autophagy-related genes Beclin-1 and LC3-?.CCK-8 was used to detect the survival rate of GCs and KGN cells treated by MEHP Evaluate whether DEHP and its metabolite MEHP can cause granulosa cell autophagy by in this way.3.The changes of oxidative stress indicators MDA,SOD,and GSH in ovarian and KGN cells were detected;the content of endogenous ROS in KGN cells was detected by DCFH-DA staining;the expression of autophagy-related genes was detected by Wb,As a way to investigate the relationship between oxidative stress and autophagy in granulosa cells caused by MEHP.4.Western blotting and RT-PCR were used to detect the expression of the Keap1-nrf2 gene in KGN cells.Nrf2 immunofluorescence,nucleoplasmic separation,co-immunoprecipitation and LC3-immunofluorescence assay were used to detect whether the Nrf2 into the nucleus,To evaluate the relationship between the antioxidant pathway Keap1-Nrf2 and autophagy.Results Through our study,we found that 500mg/kg and 1500mg/kg DEHP exposure in female mice resulted in a decrease in the ratio of late to interestrus and a disruption of the estrous cycle in mice.Decreased the level of estrogen levels.Ddecreased the ovarian organ coefficients,Decreased the number of ovarian primary and secondary Besides.The results of western blot and immunohistochemistry showed that DEHP exposure increased the expression of autophagy-related genes Beclin-1 and LC3-?,But they had no specific distribution in the ovary.Metabolomics revealed the presence of large amounts of the DEHP metabolite MEHP in the intestine.After treating mouse GCs and human KGN cells with 200?M MEHP for 24h,The number and viability of cells were significantly reduced and the expression of microtubule-associated protein light chain L3-II was significantly increased.In addition to this,The application of the autophagic agonist rapamycin(Rapa)and LY294002 pretreatment of KGN cells aggravated the cell damage caused by MEHP.This indicates that DEHP and its metabolite MEHP can cause damaging autophagy in granulosa cells.To investigate the mechanism of MEHP-induced autophagy in granulosa cells,we examined the changes in the viability of oxidative index MDA and antioxidant index SOD and GSH after MEHP treatment of granulosa cells.The results showed that MEHP can significantly reduce the levels of intracellular SOD and GSH and increase the levels of MDA.After pretreatment of human ovarian KGN cells with the antioxidant NAC,It can reverse the damage of MEHP to granular cells to a certain extent and remove excess intracellular ROS,This indicated that MEHP could lead to damaging autophagy of granulosa cells through oxidative stress.Our research also found that exposing KGN cells with 200?M MEHP can significantly increase the expression of the antioxidant pathway Nrf2 and its downstream HO-1genes,Promote the dissociation of Nrf2 from Keap1.At the same time,pretreatment of KGN cells with Nrf2 inhibitor ML385 can aggravate the damage of MEHP to granulosa cells and cause the up-regulation of autophagy-related genes Beclin-1 and LC3-?,activating autophagy.In contrast,Using Keap1-Nrf2 binding inhibitor ML334 reversed this MEHP-induced granulocyte damage and protected cell survival.Conclusion This study shows that DEHP can destroy the structure and function of the ovary and cause ovarian reproductive damage.DEHP and its metabolite MEHP can activate ovarian granulosa cell autophagy and oxidative stress.MEHP can cause excessive granulosa cell autophagy by inducing granulosa cells to produce large amounts of ROS.DEHP and its active metabolite MEHP can regulate excessive autophagy of granulosa cells through the Keap1-Nrf2 signaling pathway.