北京大学学报(医学版) ›› 2018, Vol. 50 ›› Issue (4): 607-612.doi: 10.3969/j.issn.1671-167X.2018.04.005

• 论著 • 上一篇    下一篇

雷公藤甲素对TM4细胞氧化应激及PI3K/AKT通路的影响

王昊*,陈亮*△,叶小云   

  1. (北京大学第一医院妇产科生殖与遗传医疗中心,北京100034)
  • 出版日期:2018-08-18 发布日期:2018-08-18
  • 通讯作者: 陈亮 E-mail: bdyychenliang@163.com
  • 基金资助:
    北京市自然科学基金(7142158)资助

Triptolide induces oxidative stress and apoptosis and activates PIK3/Akt signaling pathway in TM4 sertoli cells

WANG Hao, CHEN Liang, YE Xiao-yun   

  1. (Medical Center of Reproductive and Genetics,Department of Obstetrics and Gynecology,Peking University First Hospital, Beijing 100034, China)
  • Online:2018-08-18 Published:2018-08-18
  • Contact: CHEN Liang E-mail: bdyychenliang@163.com
  • Supported by:
    Supported by the Beijing Nature Science Foundation (7142158)

摘要: 目的:探讨雷公藤甲素(triptolide ,TP)对TM4小鼠睾丸支持细胞氧化应激、凋亡的影响及相关分子信号调控机制。方法:不同浓度雷公藤甲素对TM4细胞染毒24 h;细胞增殖实验检测雷公藤甲素对TM4细胞增殖的抑制作用;通过2′,7′二氯荧光黄双乙酸盐(6-carboxy-2′,7′dichlorofluorescein diacetate,DCFH-DA)探针检测TM4细胞内活性氧水平的变化;Annexin V/PI双染检测雷公藤甲素诱导TM4细胞凋亡变化; Western blot免疫印迹法分别检测凋亡标志蛋白cleavedPARP和PI3K/Akt通路相关蛋白表达情况。结果:细胞增殖实验提示雷公藤甲素对TM4细胞有显著抑制作用,呈浓度梯度上升[10 nmol/L:(73.77±20.95)%, 100 nmol/L:(51.60±10.43)%, 500 nmol/L:(44.34±5.78)%];雷公藤甲素处理使细胞内活性氧水平上调(P<0.01),呈浓度依赖性;给药组TM4细胞早期凋亡和晚期凋亡比例均上调[对照组:(3.84±1.50)%, 100 nmol/L:(13.04±2.03)%, 200 nmol/L:(16.24±1.34)%, 400 nmol/L:(18.76±3.45)%],各给药组cleavedPARP表达上调(P<0.01);给药组TM4细胞中Akt、p70S6K磷酸化水平上调(P<0.01),mTOR磷酸化水平未见显著变化(P>0.05)。结论:在体外培养条件下,雷公藤甲素可抑制TM4睾丸支持细胞增殖,并诱导TM4细胞凋亡增加,呈浓度依赖性;雷公藤甲素同时可引起TM4细胞中氧化应激水平上调,这可能是其细胞毒性机制之一;雷公藤甲素可导致Akt和p70S6K的激活,磷酸化水平升高,提示PI3K/Akt信号通路可能参与雷公藤甲素诱导的TM4细胞氧化应激反应,激活PI3K/Akt信号通路可能是其分子调控机制之一。

关键词:  , 雷公藤甲素, 睾丸支持细胞, 氧化应激, 细胞凋亡, PI3K/Akt信号通路

Abstract: Objective: To investigate the effect of triptolide (TP) on oxidative stress and apoptosis in TM4 sertoli cells and related molecular mechanism. Methods: TM4 cells were incubated with different concentrations of triptolide for 24 h, then collected for further experiments. Cell proliferation analysis was used to measure the inhibitive effect of triptolide on proliferation of TM4 cells; DCFH-DA (6-carboxy-2′,7′dichlorofluorescein diacetate) probe was used to stain the TM4 cells, the level change of intracellular ROS was discovered through flow cytometry; the TM4 cells were stained by Annexin V-FITC/PI to detect whether triptolide induced apoptosis in the TM4 cells; Protein was extracted from the TM4 cells in control and triptolide group. Western blot was performed to determine the expression of apoptosis marker protein cleaved-PARP and PI3K/Akt signaling pathway-related proteins [p-Akt (Ser473), Akt, p-mTOR (Ser2448), mTOR, p-p70S6K (Thr389), p70S6K]. Results: Cell proliferation analysis revealed that triptolide reduced the TM4 cells viability significantly compared with control group in a dosage-dependent manner [10 nmol/L: (73.77±20.95)%, 100 nmol/L: (51.60±10.43)%, 500 nmol/L: (44.34±5.78)%]; The level of intracellular ROS in the TM4 cells was significantly induced in a dosage-depen-dent manner (P<0.01); triptolide remarkably induced earlystage and late-stage apoptosis in the TM4 cells [control: (3.84±1.50)%, 100 nmol/L: (13.04±2.03)%, 200 nmol/L: (16.24±1.34)%, 400 nmol/L: (18.76±3.45)%]; The expression of cleaved-PARP was significantly upregulated in the TM4 cells after incubation with triptolide (P<0.01); The expression levels of p-Akt/Akt and p-p70S6K/p70s6k were significantly increased compared with control group (P<0.01). No significant change was observed among the expression levels of p-mTOR/mTOR (P>0.05). Conclusion: In vitro studies showed that triptolide could effectively suppress the proliferation and induce apoptosis of TM4 sertoli cells. The oxidative stress was upregulated after incubation with triptolide, which may be one of the mechanisms of cytotoxicity in TM4 cells. Treatment of triptolide led to activation of Akt and p70S6K, indicating that the PI3K/Akt signaling pathway may be involved in response to oxidative stress in TM4 cells. The activation of PI3K/Akt signaling pathway was one of the molecular mechanisms involved in triptolide-mediated oxidative stress in TM4 cells. Our study provides insight into alleviating reproductive toxicity of triptolide in clinical and developing male contraceptive.

Key words: Triptolide, Sertoli cell, Oxidative stress, Apoptosis, PI3K/Akt signaling pathway

中图分类号: 

  • R698
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