Study On The Homocysteine-induced Injury Of Mouse Ovarian Granulosa Cells And Its Mitigation Mechanism By Betaine

Previous studies have shown that high levels of homocysteine(Hcy)can induce the occurrence of reproductive disorders in female animals and human women of childbearing age,but its pathogenesis and mitigation measures are still unclear.In this study,C57BL/6mice were used as experimental subjects.The experimental animal model was constructed by adding 2 g/L Hcy to drinking water.At the same time,200μM Hcy was used to treat mouse primary ovarian granulosa cells(GCs)and human ovarian granulosa cells(KGN)in vitro to analyze the effects of high Hcy treatment on maternal ovarian function,reproductive performance and granulosa cells,and to explore its potential molecular mechanism in combination with transcriptome and in vitro cell remission test.The results showed that:(1)After 42 days of 2 g/L Hcy drinking water feeding(21 days of pregnancy,21 days of lactation),the concentration of Hcy in maternal plasma increased significantly,the number of medium and large diameter follicles increased significantly,the body weight of maternal rats increased significantly,and the litter weight of weaned offspring decreased significantly(P<0.05).(2)Transcriptome analysis showed that 119 differentially expressed genes were identified after high Hcy treatment of mouse primary GCs(P<0.05).GO analysis showed that these genes were mainly enriched in hormone metabolism,vascular system development and G protein-coupled receptor binding signaling pathways.Cyp1a1 and Cyp1b1 genes were reported to play a key role in maternal estrogen synthesis and regulation of cell proliferation and apoptosis.(3)In vitro cell experiments showed that:compared with the control group,the addition of 200μM Hcy to the medium significantly increased the production of mitochondrial reactive oxygen species(ROS)in KGN cells,accompanied by the significant down-regulation of antioxidant genes SOD,CAT,proliferation-promoting genes PCNA and anti-apoptotic genes Bcl-2,and the significant up-regulation of pro-apoptotic genes Bax and Caspase 3.And the activity of Caspase 3,a key enzyme of apoptosis,was significantly increased(P<0.05).On the contrary,the addition of 25 m M Betaine(Bet)significantly(P<0.05)alleviated the increase of ROS content,the expression of pro-apoptotic genes and the activity of Caspase 3 induced by high Hcy treatment,and significantly(P<0.05)up-regulated the expression of SOD,CAT,PCNA and Bcl-2 genes.At the same time,Me RIP-PCR assay showed that the high m~6A methylation level on the transcripts of Cyp1a1 and Cyp1b1 genes was significantly decreased under Bet treatment(P<0.05),indicating that Bet alleviated KGN injury induced by high Hcy may be related to the abnormal increase of m~6A methylation level on these two genes.The above results explain the pathogenesis and mitigation measures of high Hcy-induced reproductive disorders in female animals and humans to a certain extent,and provide important scientific ideas and experimental basis for human prevention and treatment of such diseases.