为探讨何首乌饮介导基因表观遗传(DNA甲基化)的改变对大鼠睾丸间质细胞(Leydig)分泌睾酮的影响,选用12、18月龄 Wistar雄性大鼠睾丸组织,甲基化芯片筛选出受何首乌饮调控并与精子发生功能密切的基因AXL、Ttc29、wnt2b、MAPK10、C-myb,甲基化特异性PCR(MSP)与实时荧光定量PCR(RT-qPCR)技术检测基因甲基化水平及mRNA表达水平;BrdU荧光染色观察细胞增殖情况;慢病毒转染敲低Leydig细胞DNMT1,分析wnt2b、C-myb甲基化程度和睾酮分泌水平是否受DNMT1影响.结果显示:何首乌饮可调控衰老大鼠睾丸组织中关键基因DNA甲基化和mRNA水平,促进细胞增殖和睾酮分泌;敲低Leydig细胞DNMT1后,细胞分泌睾酮水平降低,同时wnt2b和C-myb DNA甲基化水平也明显下降.这表明何首乌饮可能介导基因DNA甲基化程度促进大鼠Leydig细胞分泌睾酮,进而提高生精功能.
To investigate the effect of Heshouwuyin-mediated gene epigenetic(DNA methylation)changes on testosterone secretion in rat Leydig cells, the testicular tissues of 12-month-old and 18-month-old Wistar male rats were selected. The genes AXL, Ttc29, wnt2b, MAPK10 and C-myb regulated by Heshouwuyin and closely related to spermatogenesis were screened by methylation chip. MSP and RT-qPCR were used to detect gene methylation level and mRNA expression level. BrdU fluorescence staining- DOI:10.3969/j.issn.1000-1565.2024.03.009何首乌饮介导DNMT1调控DNA甲基化促进大鼠Leydig细胞功能石炳烨1,2,刘鑫如2,吴恬2,杨玉娇2,甄晓兰3,牛嗣云2(1.河北大学附属医院 超声诊断科,河北 保定 071000;2.河北大学 基础医学院, 河北 保定 071000;3.河北省药品医疗器械检验研究院 体外诊断试剂室,河北 石家庄 050000)摘 要:为探讨何首乌饮介导基因表观遗传(DNA甲基化)的改变对大鼠睾丸间质细胞(Leydig)分泌睾酮的影响,选用12、18月龄 Wistar雄性大鼠睾丸组织,甲基化芯片筛选出受何首乌饮调控并与精子发生功能密切的基因AXL、Ttc29、wnt2b、MAPK10、C-myb,甲基化特异性PCR(MSP)与实时荧光定量PCR(RT-qPCR)技术检测基因甲基化水平及mRNA表达水平;BrdU荧光染色观察细胞增殖情况;慢病毒转染敲低Leydig细胞DNMT1,分析wnt2b、C-myb甲基化程度和睾酮分泌水平是否受DNMT1影响.结果显示:何首乌饮可调控衰老大鼠睾丸组织中关键基因DNA甲基化和mRNA水平,促进细胞增殖和睾酮分泌;敲低Leydig细胞DNMT1后,细胞分泌睾酮水平降低,同时wnt2b和C-myb DNA甲基化水平也明显下降.这表明何首乌饮可能介导基因DNA甲基化程度促进大鼠Leydig细胞分泌睾酮,进而提高生精功能.关键词:何首乌饮;睾丸组织;DNA甲基化;睾酮中图分类号:Q291 文献标志码:A 文章编号:1000-1565(2024)03-0301-11Heshouwuyin mediates DNMT1 to regulate DNA methylation to promote rat Leydig cell functionSHI Bingye1,2, LIU Xinru2, WU Tian2, YANG Yujiao2, ZHEN Xiaolan3, NIU Siyun2(1. Department of Ultrasound Diagnosis, Afiliated Hospital of Hebei University,Baoding 071000, China;2. School of Basic Medical Sciences, Hebei University, Baoding 071000, China;3. In Vitro Diagnostic Reagent Room, Hebei Pharmaceutical and Medical Device Inspection and Research Institute, Shijiazhuang 050000, China)Abstract: To investigate the effect of Heshouwuyin-mediated gene epigenetic(DNA methylation)changes on testosterone secretion in rat Leydig cells, the testicular tissues of 12-month-old and 18-month-old Wistar male rats were selected. The genes AXL, Ttc29, wnt2b, MAPK10 and C-myb regulated by Heshouwuyin and closely related to spermatogenesis were screened by methylation chip. MSP and RT-qPCR were used to detect gene methylation level and mRNA expression level. BrdU fluorescence staining- 收稿日期:2023-08-11;修回日期:2023-11-10 基金项目:国家自然科学基金资助项目(81673714);河北省重点研发计划项目-中医药创新专项(223777134D);河北省高等学校科学技术研究项目(ZD2022060);河北省中医药管理局科研计划项目(2022115) 第一作者:石炳烨(1983—),男,河北大学附属医院主治医师,河北大学在读硕士研究生,主要从事男性生殖衰老研究.E-mail:2435231348@qq.com 通信作者:牛嗣云(1967—),女,满族,河北大学教授,博士,博士生导师,主要从事生殖衰老方向研究.E-mail:nsy1688@163.com甄晓兰(1976—),女,河北省药品医疗器械检验研究院副主任药师,主要从事生殖衰老方向研究.E-mail:zhenxlan2007@163.com第3期石炳烨等:何首乌饮介导DNMT1调控DNA甲基化促进大鼠Leydig细胞功能was used to observe cell proliferation. DNMT1 was knocked down by lentivirus transfection in Leydig cells, and the effects of DNMT1 on wnt2b and C-myb methylation and testosterone secretion were analyzed. The results showed that Heshouwuyin could regulate the DNA methylation and mRNA levels of key genes in testicular tissue of aging rats, and promote cell proliferation and testosterone secretion. After knocking down DNMT1 in Leydig cells, the level of testosterone secretion was decreased, and the levels of wnt2b and C-myb DNA methylation were also significantly decreased, indicating that Heshouwuyin may improve the ability of rat testicular cells to secrete testosterone by regulating the level of gene DNA methylation, thereby improving the spermatogenic function.
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