基于Notch信号通路探索...卵巢生殖干细胞的作用及机制_杨一博.pdf

第 29 卷第 5 期2023年 3 月中国实验方剂学杂志Chinese Journal of Experimental Traditional Medical FormulaeVol.29，No.5Mar.，2023基于 Notch信号通路探索菟丝子总黄酮配伍雷公藤多苷对生理小鼠卵巢生殖干细胞的作用及机制杨一博，徐颖*，王璐，邹钊，徐盼瑜，杜寒倩，李佳珊，林娜*（中国中医科学院 中药研究所，北京 100700）摘要 目的：探讨菟丝子总黄酮配伍雷公藤多苷片对雌性生理小鼠卵巢生殖干细胞的作用，并从神经源性位点缺口同源蛋白（Notch）信号通路探讨其作用机制。方法：将 60只 5周龄雌性昆明种小鼠随机分为正常组，1、2倍临床等效剂量雷公藤多苷片组（低、高剂量组 13.65、27.3 mg kg-1d-1），菟丝子总黄酮（低、高剂量组 150、300 mg kg-1d-1），配伍组（雷公藤多苷片低剂量+菟丝子总黄酮低剂量组），每组 10只，连续给药 3周后取材，计算子宫、卵巢脏脑指数，光镜下观察卵巢组织病理形态学改变；酶联免疫吸附测定法（ELISA）检测血清中雌二醇含量；免疫荧光法观察卵巢上皮的卵巢生殖干细胞标志物果蝇 VASA基因同源类似物（Mvh）、八聚体结合转录因子 4（Oct4）、酪氨酸激酶受体（c-kit）、同源框蛋白质（Nanog）及 Notch信号通路分子神经源性基因 Notch同源蛋白 1（Notch1）、Hes家族 BHLH 转录因子 1（Hes1）、锯齿典型 Notch配体 1（Jagged1）的表达。结果：与正常组比较，雷公藤多苷片低剂量组和高剂量组对小鼠子宫、卵巢的脏脑系数及形态学差异无统计学意义，仅可致小鼠闭锁卵泡数显著增加（P0.01）；雷公藤多苷片低剂量对卵巢生殖干细胞标志物和 Notch信号通路分子的表达有降低趋势，雷公藤多苷片高剂量下 Mvh、c-kit、Nanog的表达水平明显降低（P0.05，P0.01），Notch1、Hes1表达水平显著降低（P0.01）；菟丝子总黄酮低剂量组上述指标均明显升高（P0.05，P0.01）；与雷公藤多苷片低剂量组比较，配伍组闭锁卵泡数显著降低（P0.01），Mvh、Nanog表达水平显著升高（P0.01），Notch1、Hes1表达水平明显升高（P0.05，P0.01）。结论：卵巢生殖干细胞是雷公藤多苷片生殖毒性的源头靶点，菟丝子总黄酮可参与调控卵巢生殖干细胞途径缓解雷公藤多苷片所致的生殖损伤，其作用机制可能与 Notch信号通路相关。关键词 雷公藤多苷片；菟丝子总黄酮；配伍；卵巢生殖干细胞；神经源性位点缺口同源蛋白（Notch）信号通路中图分类号 R2-0；R33；R289；R593.22 文献标识码 A 文章编号 1005-9903（2023）05-0009-07doi 10.13422/ki.syfjx.20220944 网络出版地址 https:/ 2022-04-06 15:22:09Effect and Mechanism of Cuscutae Semen Total Flavonoids Combined with Tripterygium wilfordii Polyglycoside Tablets on Ovarian Germline Stem Cells of Physiological Mice by Notch Signaling PathwayYANG Yibo，XU Ying*，WANG Lu，ZOU Zhao，XU Panyu，DU Hanqian，LI Jiashan，LIN Na*（Institute of Chinese Materia Medica，China Academy of Chinese Medical Sciences，Beijing 100700，China）Abstract Objective：To observe the effect of Cuscutae Semen total flavonoids combined with Tripterygium wilfordii polyglycoside tablets（TWPT）on ovarian germline stem cells of female physiological mice through neurogenic locus notch homolog（Notch）signaling pathway.Method：Sixty female Kunming mice（5 weeks old）were randomly divided into normal group，Tripterygium wilfordii polyglycoside tablets 收稿日期 2022-01-07基金项目 国家自然科学基金项目（81974537，82004248）；中央级公益性科研院所基本科研业务费专项（ZXKT20011）；中国中医科学院科技创新工程项目（CI2021A03802）第一作者 杨一博，硕士，从事中药药理学研究，E-mail：通信作者 *徐颖，研究员，博士生导师，从事中药生殖药理学和中药药性研究，E-mail：；*林娜，研究员，博士生导师，从事中药药理学及中药药性研究，E-mail：9第 29 卷第 5 期2023年 3 月中国实验方剂学杂志Chinese Journal of Experimental Traditional Medical FormulaeVol.29，No.5Mar.，2023low-，high-dose groups（13.65 mg kg-1d-1 and 27.3 mg kg-1d-1，1 and 2 times clinical equivalent dose），Cuscutae Semen total flavonoids low-and high-dose groups（150 mg kg-1d-1 and 300 mg kg-1d-1），and combination group（13.65 mg kg-1d-1 TWPT and 150 mg kg-1d-1 Cuscutae Semen total flavonoids），with 10 in each group.After 3 weeks of continuous administration，the uterus/brain and ovarian/brain indexes were calculated，and the pathological changes of ovarian tissue were observed under light microscope.The content of estradiol in serum was determined by enzyme linked immunosorbent assay（ELISA）.Immunofluorescence assay was performed to observe the expressions of germline stem cell markers in ovarian epithelium，including mouse vasa homologue（Mvh），octamer-binding transcription factor 4（Oct4），tyrosine-protein kinase receptor（c-kit），Nanog，Notch signaling pathway molecules，neurogenic locus notch homolog protein 1（Notch1），hes family BHLH transcription factor 1（Hes1），and jagged canonical Notch ligand 1（JAG1）.Result：Compared with the normal group，low and high doses of TWPT had no significant effect on the uterus/brain and ovary/brain indexes and the uterus and ovary morphologies of mice，while only the number of atretic follicles was increased（P0.01）.The expressions of ovarian germline stem cell markers and Notch signaling pathway molecules had a decreasing trend in TWPT low-dose group，while the expressions of Mvh，c-kit，and Nanog were down-regulated（P0.05，P0.01）and the expressions of Notch1 and Hes1 were also reduced（P0.01）in TWPT high-dose group.However，the above indexes were increased in Cuscutae Semen total flavonoids low-dose group（P0.05，P0.01）.Compared with the low does of TWPT group，the combination group had a decrease in the increased number of atretic follicles（P0.01），an improvement in the down-regulated expressions of Mvh and Nanog（P0.01），and an increase in the expressions of Notch1 and Hes1（P0.05，P0.01）.Conclusion：Ovarian germline stem cells are the source target of the reproductive toxicity of TWPT.Cuscutae Semen total flavonoids participate in the regulation of the germline stem cell pathways to alleviate the reproductive toxicity caused by TWPT，and its mechanism of action may be related to the Notch signaling pathway.Keywords Tripterygium wilfordii polyglycoside tablets（TWPT）；Cuscutae Semen total flavonoids；combination；ovarian germline stem cells；neurogenic locus notch homolog protein（Notch）signaling pathway雷公藤多苷片是雷公藤最常用的代表性制剂，被世界卫生组织（WHO）认定为治疗关节炎“中国首创植物新药”，在治疗类风湿关节炎、慢性肾病、红斑狼疮等多种疾病中应用的日益广泛，但其不良反应尤其是临床首发及最突出的生殖损伤严重限制了雷公藤多苷片的临床应用，女性生殖毒性已成为雷公藤临床用药中遇到的重大挑战之一1。目前对雷公藤所致雌性生殖系统损伤机制研究主要集中在抑制卵泡生长发育、引起卵巢细胞凋亡及干扰生殖内分泌 3个方面。目前雷公藤多苷片毒性