| The main function of Leydig cells is to synthesize and secrete androgens,which plays an important role in regulating the growth and development of male reproductive organs and spermatogenesis.In production,environmental and physical factors can produce oxidative stress on the animal organism,and the imbalance between the oxidative and antioxidant effects of the organism can lead to functional and even structural damage to tissues and cells,thus affecting the reproductive performance of animals and ultimately the development of the animal husbandry.Oxidative stress can induce damage to Leydig cell,but whether it leads to necroptosis and its mechanisms are unclear.The aim of this experiment was to investigate the effect of oxidative stress on necroptosis of bovine Leydig cells and to investigate the role of m6A methylation in it.Bovine Leydig cells were used as the study subjects and H2O2was used to induce oxidative stress in bovine Leydig cells.Effects of H2O2on necroptosis and oxidative stress in Leydig cells by DCFH-DA fluorescent probe assay,flow cytometry,q RT-PCR and western blot.Screening of differentially expressed target genes by m RNA-seq and Me RIP-seq in bovine Leydig cells under oxidative stress injury model conditions,and screening of signaling pathways that may be involved in regulation by KEGG analysis and GO enrichment analysis,verification of sequencing results by q RT-PCR and western blot.Flow cytometry,q RT-PCR and western blot were performed to detect the mechanism of action of m6A modification and target genes in oxidative stress-induced necroptosis in bovine Leydig cells.The results of the study were as follows:1.Leydig cells were treated with different concentrations（0,200,400,600,800 and 1000μM）of H2O2for 12 h.The subsequent treatment concentrations were screened and the levels of necroptosis and ROS were detected.the results of CCK8 showed that 400μM H2O2significantly reduced cell viability（P<0.05）;the results of flow cytometry as well as transmission electron microscopy showed that H2O2treatment increased the level of necroptosis in Leydig cells（P<0.05）;real-time quantitative polymerase chain reaction,western blot and DCFH-DA fluorescent probe results showed that the m RNA and protein expression of necroptosis-related genes RIPK1,RIPK3 and MLKL were significantly increased and ROS level was elevated（P<0.05）,when ROS scavenger NAC was added,ROS levels decreased,while the expression of necroptosis-related genes was significantly downregulated（P<0.05）.The results suggested that oxidative stress could induce necroptosis in bovine Leydig cells.2.mRNA-seq and MeRIP-seq were performed to screen the differentially expressed target genes under oxidative stress model in bovine Leydig cells,and the target gene expression levels were verified by q RT-PCR and western blot.q RT-PCR results showed that H2O2treatment caused significant upregulation in ALKBH5 expression.The m RNA-seq results showed that there are significant differences in the control group and the H2O2group,screened 696 different expressed genes,including 270 up-regulated genes and 426 down-regulated genes.GO function is enriched to cell cycles,positive regulation of cell division,DNA binding,positive regulation of gene expression,and positive regulation of cell migration,KEGG is enriched to cell cycle,PI3K/AKT signaling pathway,oxide phosphoric acidization,P53 signaling pathway,TNF signaling pathway.The combined analysis of the m RNA-seq and Me RIP-seq,screened 226different expression genes,of which 74 genes m RNA expression levels were up-regulated,and the m6A modification level was down-regulated,filtering the differences related to the necroptosis expressions gene SQSTM1,GO function enrichment and KEGG analysis found that this process involved PI3K/AKT signal pathway.It was found that the m6A modification level of SQSTM1 was decreased by Me RIP-RT-q PCR.Western blot results showed that si-ALKBH5transfection could reduce H2O2-induced SQSTM1 expression increase.3.The functions of the target genes SQSTM1 and ALKBH5 were validated.The results showed that si-ALKBH5 transfection attenuated oxidative stress-induced necroptosis in bovine Leydig cells.When co-treated with si-ALKBH5 transfection and SQSTM1 overexpression,overexpression of SQSTM1 reversed the inhibitory effect of transfection with si-ALKBH5 on necroptosis in bovine Leydig cells,p-PI3K and p-AKT protein expression was significantly up-regulated and p-PI3K/PI3K and p-AKT/AKT were significantly increased activating the PI3K/AKT signaling pathway by transfection with si-SQSTM1.The results suggested that ALKBH5 could upregulate SQSTM1 and inhibited PI3K/AKT signaling pathway,thus promoting necroptosis in bovine Leydig cells.In summary,oxidative stress upregulated SQSTM1 expression in an ALKBH5-dependent m6A modification and promoted necroptosis in bovine Leydig cells by inhibiting the PI3K/AKT signaling pathway. |
