To investigate the effect of oleanolic acid on leydig cells of aging rats, the MTT method was used to determine the optimal drug concentration of oleanolic acid. The aging module of rat leydig cell was built according to the free radical oxidative damage method. Gene and protein expression of the key gene in the insulin/insulin-like growth factor 1(IGF1)signaling pathway was detected by RT-qPCR, immunofluorescence technique(IF), and Western blot. After treated with INSR inhibitor, cell apoptosis was performed by flow cytometry. The results showed that INSR, IRS1, IRS2 and IGF1 mRNA transcription level and protein expression level were significantly downregulated(P<0.05); IGFBP3 mRNA transcription level was significantly upregulated(P<0.05), otherwise, the expression could not be detected by IF and Western blot. Oleanolic acid reversed the above phenomenon, compared to aging group(P<0.05).- DOI:10.3969/j.issn.1000-1565.2022.04.010齐墩果酸对衰老大鼠睾丸间质细胞胰岛素/IGF1通路关键基因表达的影响李少华1,2,牛双2,董子怡2,宋邑诚2,牛嗣云2(1. 河北大学附属医院 新生儿科,河北 保定 071000;2.河北大学 基础医学院,河北 保定 071000)摘 要:为了探讨齐墩果酸对衰老大鼠睾丸间质细胞的作用,采用MTT法确定齐墩果酸最适药物浓度,氧化应激创建大鼠睾丸间质细胞衰老模型,RT-qPCR、免疫荧光和免疫印迹检测胰岛素/胰岛素样生长因子1(insulin-like growth factor 1, IGF1)信号通路关键基因转录情况和蛋白翻译情况,采用INSR抑制剂处理细胞,流式细胞术检测细胞凋亡情况.结果显示:与正常组相比,INSR、IRS1、IRS2、IGF1的mRNA转录水平和蛋白翻译水平显著下降(P<0.05);IGFBP3的mRNA转录水平显著提高(P<0.05),免疫荧光和免疫印迹均未检测到此基因表达;与衰老组相比,齐墩果酸组明显逆转此现象(P<0.05);齐墩果酸能明显抑制衰老组细胞的凋亡(P<0.05),INSR抑制剂处理后,细胞凋亡率明显高于齐墩果酸组(P<0.05).综上可知,齐墩果酸通过调控胰岛素/IGF1信号通路关键基因的转录和翻译延缓Leydig细胞衰老.关键词:齐墩果酸;胰岛素/IGF1;衰老;睾丸间质细胞;RT-qPCR中图分类号:Q291 文献标志码:A 文章编号:1000-1565(2022)04-0403-08Effects of oleanolic acid on the expression of key genes of insulin/IGF1 pathway in leydig cells of aging ratsLI Shaohua1,2, NIU Shuang2, DONG Ziyi2,SONG Yicheng2, NIU Siyun2(1. Department of Neonatology, Affiliated Hospital of Hebei University, Baoding 071000, China;2. School of Basic Medical Sciences, Hebei University, Baoding 071000, China)Abstract: To investigate the effect of oleanolic acid on leydig cells of aging rats, the MTT method was used to determine the optimal drug concentration of oleanolic acid. The aging module of rat leydig cell was built according to the free radical oxidative damage method. Gene and protein expression of the key gene in the insulin/insulin-like growth factor 1(IGF1)signaling pathway was detected by RT-qPCR, immunofluorescence technique(IF), and Western blot. After treated with INSR inhibitor, cell apoptosis was performed by flow cytometry. The results showed that INSR, IRS1, IRS2 and IGF1 mRNA transcription level and protein expression level were significantly downregulated(P<0.05); IGFBP3 mRNA transcription level was significantly upregulated(P<0.05), otherwise, the expression could not be detected by IF and Western blot. Oleanolic acid reversed the above phenomenon, compared to aging group(P<0.05).- 收稿日期:2021-11-02 基金项目:国家自然科学基金资助项目(81673714);河北省中医药管理局科研项目(2016204;20200234);河北大学研究生创新资助项目(HBU2021ss025) 第一作者:李少华(1980—),女,河北定州人,河北大学附属医院主管护师,主要从事生殖衰老方向研究. E-mail:xiaolizhu2007@163.com 通信作者:牛嗣云(1967—),女,河北承德人,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第4期李少华等:齐墩果酸对衰老大鼠睾丸间质细胞胰岛素/IGF1通路关键基因表达的影响Oleanolic acid can delay cell apoptosis in the aging group(P<0.05). It was found that cell apoptosis rate was significantly higher than oleanolic acid group(P<0.05)after treated with INSR inhibitor. In summary, oleanolic acid delays leydig cell senescence by regulating insulin/IGF1 signaling pathway essential gene expression and transcripts.
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