The Role And Mechanism Of FTO-NRF2 Axis-mediated Testicular Leydig Cells(TM3)damage By Bisphenol F

Objective: In recent years,Bisphenol F(BPF),an emerging bisphenol pollutant,has been widely used in the manufacture of epoxy resins and coatings as an alternative to Bisphenol A(BPA),which poses a new public health risk.With the in-depth study of bisphenols,it has been found that bisphenols can regulate cell growth,proliferation,migration and apoptosis by binding to nuclear receptors and membrane receptors,leading to steroid hormone disorders in animals and humans,thus inducing a variety of cell or tissue damage.BPF can induce a series of hazards,such as tumor formation,inflammatory response,oxidative stress,carbohydrate metabolism disorders and reproductive damage.More and more attention has been paid to the male reproductive injury induced by BPF.Male reproductive damage mainly includes the decrease of sperm count,the decrease of motility,the change of testicular morphology,and the decrease of related hormone secretion.BPF exposure was negatively correlated with estrogen and total testosterone in men.In addition,BPF disrupted testosterone biosynthesis in TM3 cells,interfered with germ cell proliferation,and disrupted spermatogenesis.However,the specific mechanism of BPF-induced reproductive injury is still unclear.Epigenetic modification is an important mechanism of reproductive damage caused by environmental toxicants.Epigenetics includes RNA methylation,DNA methylation and histone modification.The m6 A modification is the most important RNA methylation modification,which is composed of three parts,including RNA methylases such as Methyltransferase like 3(METTL3),Methyltransferase like 14(METTL14),etc.,RNA demethylases such as Fat mass and obesity associated gene(FTO),alpha-ketoglutarate-dependent dioxygenase alk B homolog 5(ALKBH5),etc.,and RNA-binding protein such as a family of RNA-binding protein YT521-B homology(YTH)proteins,YTH N6-methyladenosine RNA-binding protein 1(YTHDF1),YTH N6-methyladenosine RNA-binding protein 2(YTHDF2),etc.FTO,the first RNA demethylase,is involved in various physiological processes by regulating hormone secretion,reactive oxygen species production,autophagy,and male gonadal development.The expression of Fto m RNA was positively correlated with the total number of spermatozoa,fertilization rate and embryo egg rate,and the variation of Fto gene was associated with the decline of semen quality.YTHDF2,an m6A-binding protein that regulates m RNA stability,is involved in biological processes that are closely related to cell proliferation and apoptosis.YTHDF2 plays different biological functions by promoting the degradation of m RNA and regulating the expression of related genes.YTHDF2 can modify m6 A to promote the degradation of m RNA and regulate spermatogenesis,thus affecting cell proliferation.However,whether m6 A RNA methylation is involved in BPF-induced male reproductive impairment is unknown.In recent years,studies also have found that oxidative stress is closely related to epigenetics.Proper levels of reactive oxygen species(ROS)in germ cells can promote cell growth,proliferation,hormone secretion,and germ cell related functions,but when ROS is excessive,it can lead to germ cell membrane peroxidation,cellular inflammation,and testicular dysfunction.To maintain cellular homeostasis,nuclear factor erythroid 2 related factor 2(NRF2),a key regulator of oxidative stress,maintains cellular oxidative stress balance by activating antioxidant enzymes.BPF exposure can reduce the levels of Catalase and Superoxide dismutase,increase lipid peroxidation,and reduce the secretion of testosterone,luteinizing hormone and follicle-stimulating hormone,ultimately leading to male reproductive damage.However,it is unclear whether RNA methylation affects oxidative stress to regulate BPF-induced reproductive toxicity.In this study,the model of TM3 Leydig cells exposed to BPF(0,20,40,80 μM)was established to explore the mechanism of reproductive injury induced by BPF in mouse.To explore the role of epigenetics in BPF induced reproductive damage by establishing a differential expression model of Fto gene.In this study,we explored a novel signaling pathway and molecular mechanism in mouse TM3 cells,and revealed the toxic effects of BPF on TM3 cells through oxidative stress and m6 A RNA methylation modification,aiming to explore the effects of bisphenol pollutants on male reproduction.To sum up,it provides a new insight into the role and mechanism of bisphenol in male reproductive damage.Methods:1.Effect of BPF on the phenotype of TM3 Cells(1)TM3 cells were treated with BPF with final concentrations of 0 μM,20μM,40 μM and 80 μM for 72 h.Morphological changes of cells were observed under microscope,cell survival was detected by CCK-8 method,and apoptosis were detected by flow cytometry.(2)DCFH-DC was used to detect the changes of reactive oxygen species in TM3 cells after exposure.Western blot and RT-q PCR were used to detect the changes of oxidative stress molecules such as NRF2 and KEAP1.(3)M6A modification level detection: cell total m6 A modification level of TM3 were detected.Western blot and RT q PCR were used to detect the molecular level of RNA methylation in TM3 cells,such as FTO,YTDFH1,YTHDF2,YTHDC2,METTL3,etc.(4)Western blot and RT q PCR to detect TM3 cell membrane receptor nuclear receptor related molecules such as ERα,ERβ,AR,Ah R,etc.(5)Ch IP assays were used to detect the regulation mode of Fto gene by Ah R receptor.2.Effect of differentially expressed Fto on the phenotype of TM3 Cells(1)After the overexpression of Fto,cell viability was detected by CCK8,cell apoptosis and ROS were detected by flow cytometry.The expression of NRF2,KEAP1 were detected by Western blot and RT-q PCR;(2)After the interfering with Fto,cell apoptosis were detected by flow cytometry.The expression of NRF2,KEAP1 were detected by Western blot and RT-q PCR;(3)After the differential expression of Fto,a BPF exposure TM3 cell model was established to detect the changes of cell activity and apoptosis.3.Study on the mechanism of Fto on BPF induced TM3 cell injury(1)Me RIP was used to detect the changes of Nrf2 m RNA m6 A modification level in TM3 cells after overexpression of FTO,actinomycin D test was used to detect the changes of Nrf2 m RNA stability after overexpression of Ythdf2,and RIP was used to detect the binding of YTHDF2 and Nrf2.(2)Detect the changes of apoptosis and related molecules after the addition of NRF2 agonist dimethyl fumarate and the differential expression of Fto.Results:1.BPF induces increased apoptosis and imbalance of redox homeostasis of TM3 cells,fianlly leading to reproductive damage.(1)BPF exposure significantly reduced the cell viability of TM3 cells,increased the apoptosis rate in a dose-dependent manner.(2)After treatment of TM3 cells with BPF of 0 μ M,20 μ M,40 μ M and 80 μM for 72 hours,the level of ROS in cells was significantly increased,and the level of NRF2 gene expression was significantly decreased.(3)After treatment with BPF for 72 hours,the total m6 A modification level of TM3 cells increased significantly.The levels of FTO m RNA and protein were significantly reduced,the levels of YTDFH1 and METTL3 m RNA and protein were not significantly changed,and the levels of YTHDF2 and YTHDC2 m RNA were not significantly changed,but the levels of protein were significantly reduced.(4)After exposure to BPF,the protein levels of AR and Ah R in TM3 cells were significantly reduced.(5)Ah R transcriptionally regulated FTO m RNA and affected the expression level of FTO gene.2.Overexpression of FTO gene inhibited the toxic effect of BPF on TM3 cells(1)Over expression of FTO in TM3 cells promoted cell viability,reduced apoptosis,significantly reduced the level of ROS in cells,significantly reduced the levels of P53 and BAX proteins and m RNA,and significantly increased the levels of BCL2,NRF2 proteins and m RNA.(2)On the contrary,interference with FTO inhibited cell viability and promoted cell apoptosis.And the level of ROS in cells increased significantly.The levels of P53 and BAX proteins and m RNA were significantly increased,while the levels of BCL2,NRF2 proteins and m RNA were significantly reduced.(3)FTO aggravated the decrease of cell viability and increase of apoptosis caused by BPF exposed TM3 cells.3.DMF promotes the protection of FTO gene against BPF induced TM3cytotoxicity(1)In TM3 cells,overexpression of FTO significantly reduced the m6 A modification level of NRF2 m RNA,and overexpression of YTHDF2 promoted the stability of NRF2 m RNA.(2)In TM3 cells,NRF2 agonist dimethyl fumarate(DMF)promoted the protective effect of FTO and reduced the apoptosis induced by interfering with FTO gene.Conclusions:1.BPF promoted cell apoptosis and inhibited in TM3 cells,which has reproductive toxicity and induced reproductive damage.2.RNA demethylase FTO played an protective role in BPF induced reproductive damage.3.FTO-NRF2 axis regulated the apoptosis of TM3 cells by methylation modification of YTHDF2-m6 A,which mediated BPF induced reproductive damage.