基于Notch信号通路研究雷公藤多苷对体外培养小鼠卵巢生殖干细胞的毒性作用及机制研究

Toxic effect and mechanism of Tripterygium glycosides on ovarian germline stem cells of mice in vitro by Notch signaling pathway

以优化培养体系中生长状态良好的卵巢生殖干细胞(ovarian germline stem cells,OGSCs)为研究对象,观察雷公藤多苷(Tripterygium glycosides,TG)对OGSCs的影响,并从Notch信号通路探讨其生殖毒性的作用机制。采用cell counting kit⁃8(CCK⁃8)检测3.75、7.5、15μg·mL^(-1) TG对体外培养小鼠OGSCs活力的影响;免疫荧光技术和逆转录聚合酶链式反应(reverse transcription⁃polymerase chain reaction,RT⁃PCR)检测3.75μg·mL^(-1) TG给药后OGSCs标志物VASA基因同源类似物(mouse vasa homologue,MVH)、八聚体结合转录因子4(octamer⁃binding transcription factor 4,Oct4)蛋白和基因的表达;RT⁃PCR检测Notch信号通路关键分子神经源性基因Notch同源蛋白1(neurogenic locus Notch homolog protein 1,Notch1)、Hes家族BHLH转录因子1(Hes family BHLH transcription factor 1,Hes1)、锯齿典型Notch配体1(jagged canonical Notch ligand 1,Jagged1)基因表达;提取RNA进行转录组学分析TG干预OGSCs的作用机制。3.75μg·mL^(-1) TG配伍40 ng·mL^(-1) Notch信号通路γ⁃促分泌酶激动剂jagged canonical Notch ligand(Jagged)联合给药,采用CCK⁃8检测OGSCs活力水平;免疫荧光双标法检测MVH与Hes1、Notch1、Jagged1蛋白共表达。结果显示,与空白组比较,TG给药后均显著抑制OGSCs活力(P<0.01或P<0.001);显著降低OGSCs标志物MVH、Oct4蛋白和基因表达(P<0.05,P<0.01或P<0.001);显著抑制Notch1、Hes1、Jagged1基因表达(P<0.001)。转录组学分析提示,TG通过干预Notch信号通路相关配体Jagged1影响OGSCs的生长增殖。实验验证结果表明,配伍Notch信号通路γ⁃促分泌酶激动剂Jagged可显著缓解TG所致OGSCs活力的降低(P<0.001);并与TG组比较,显著升高MVH/Hes1、MVH/Notch1、MVH/Jagged1蛋白共表达(P<0.01或P<0.001)。综上可知,TG可发挥γ⁃促分泌酶抑制剂样作用,通过下调OGSCs标志物MVH、Oct4和Notch信号通路分子Notch1、Hes1、Jagged1参与OGSCs途径介导Notch信号通路诱发生殖毒性。

The ovarian germline stem cells(OGSCs)cultured in the optimized culture system were used as the research object to observe the effect of Tripterygium glycosides(TG)on OGSCs and explore the mechanism of reproductive toxicity by the Notch signaling pathway.Cell counting kit⁃8(CCK⁃8)was used to observe the viability level of OGSCs in mice cultured in vitro by TG of 3.75,7.5,and 15μg·mL^(-1).Immunofluorescence technology and reverse transcription⁃polymerase chain reaction(RT⁃PCR)were used to detect the protein and gene expression level of OGSCs marker mouse vasa homologue(MVH)and octamer⁃binding transcription factor 4(Oct4)by TG of 3.75μg·mL^(-1).RT⁃PCR detected the gene expression of neurogenic locus Notch homolog protein 1(Notch1),Hes family BHLH transcription factor 1(Hes1),and jagged canonical Notch ligand 1(Jagged1).The RNA was extracted for transcriptome analysis to analyze the mechanism of action of TG intervention on OGSCs.3.75μg·mL^(-1) of TG was combined with 40 ng·mL^(-1) Notch signaling pathwayγ⁃secretagocin agonist jagged canonical notch ligand(Jagged)for administration.CCK⁃8 was used to detect the viability level of OGSCs.Double immunofluorescence technology was used to detect the protein co⁃expression of MVH with Hes1,Notch1,and Jagged1.The results showed that compared with the blank group,the TG administration group significantly inhibited the activity of OGSCs(P<0.01 or P<0.001).It could reduce the protein and gene expression of OGSC markers,namely MVH and Oct4(P<0.05,P<0.01,or P<0.001).It could significantly inhibit the gene expression of Notch1,Hes1,and Jagged1(P<0.001).Transcriptomic analysis showed that TG affected the growth and proliferation of OGSCs by intervening Jagged1,a ligand associated with the Notch signaling pathway.The experimental results showed that the combination of Notch signaling pathwayγ⁃secretagorein agonist Jagged could significantly alleviate the decrease in OGSC viability induced by TG(P<0.001)and significantly increased the OGSC viability compared with the TG group(P<0.001).It also could significantly increase the co⁃expression of MVH/Jagged1,MVH/Hes1,and MVH/Notch1 proteins(P<0.01 or P<0.001).It suggested that TG play the role ofγ⁃secretagorease inhibitors by downregulating the OGSC markers including MVH and Oct4 and Notch signaling pathway molecules such as Notch1,Hes1,and Jagged1,participate in the OGSC pathway,and mediate reproductive toxicity caused by the Notch signaling pathway.