张芬, 李睿, 赵淑琦, 王彦娜, 倪志晶, 常秀丽, 周志俊. 氟咯草酮通过IRE1α-JNK信号通路介导小鼠睾丸组织细胞及TM4细胞凋亡[J]. 环境与职业医学, 2022, 39(9): 996-1003. DOI: 10.11836/JEOM22098
引用本文: 张芬, 李睿, 赵淑琦, 王彦娜, 倪志晶, 常秀丽, 周志俊. 氟咯草酮通过IRE1α-JNK信号通路介导小鼠睾丸组织细胞及TM4细胞凋亡[J]. 环境与职业医学, 2022, 39(9): 996-1003. DOI: 10.11836/JEOM22098
ZHANG Fen, LI Rui, ZHAO Shuqi, WANG Yanna, NI Zhijing, CHANG Xiuli, ZHOU Zhijun. Flurochloridone-induced apoptosis via IRE1α-JNK signaling pathway in mice testicular cells and TM4 cells[J]. Journal of Environmental and Occupational Medicine, 2022, 39(9): 996-1003. DOI: 10.11836/JEOM22098
Citation: ZHANG Fen, LI Rui, ZHAO Shuqi, WANG Yanna, NI Zhijing, CHANG Xiuli, ZHOU Zhijun. Flurochloridone-induced apoptosis via IRE1α-JNK signaling pathway in mice testicular cells and TM4 cells[J]. Journal of Environmental and Occupational Medicine, 2022, 39(9): 996-1003. DOI: 10.11836/JEOM22098

氟咯草酮通过IRE1α-JNK信号通路介导小鼠睾丸组织细胞及TM4细胞凋亡

Flurochloridone-induced apoptosis via IRE1α-JNK signaling pathway in mice testicular cells and TM4 cells

  • 摘要: 背景

    氟咯草酮(FLC)可诱导睾丸支持细胞凋亡,但其具体机制尚未阐明。

    目的

    通过体内动物及体外细胞系构建FLC染毒模型,探究FLC染毒后小鼠睾丸组织中细胞凋亡以及小鼠睾丸支持细胞系TM4细胞凋亡和内质网应激活化情况,并通过干预实验探究肌醇需酶1α(IRE1α)-c-Jun氨基末端激酶(JNK)信号通路在FLC诱导TM4细胞凋亡过程中的作用。

    方法

    利用C57BL/6雄性小鼠28 d经口染毒3、15、75和375 mg·(kg·d)−1 FLC结束后留取的睾丸标本,通过TUNEL染色观察睾丸组织中细胞凋亡发生情况,通过Western blotting检测睾丸组织中的凋亡蛋白B淋巴细胞瘤-2(Bcl-2)、Bcl-2相互作用介质(Bim)、Bcl-2相关X蛋白(Bax)表达水平。在体外细胞实验中,使用不同浓度(40、80和160 μmol·L−1)的FLC对TM4细胞染毒6 h,采用流式细胞术检测细胞凋亡率,Western blotting检测凋亡蛋白(Bcl-2、Bim、Bax)及内质网应激相关蛋白葡萄糖调节蛋白78(GRP78)、磷酸化-蛋白激酶R样内质网激酶(p-PERK)、活化转录因子6(ATF6)、磷酸化-肌醇需酶1α(p-IRE1α)、磷酸化-JNK(p-JNK)水平。随后使用IRE1α磷酸化抑制剂4μ8C(25、50 μmol·L−1)、JNK磷酸化抑制剂SP600125(10、20 μmol·L−1)预处理TM4细胞6 h后再使用160 μmol·L−1 FLC染毒6 h,采用Western blotting检测凋亡及内质网应激相关蛋白水平,通过CCK-8法检测细胞活力。

    结果

    雄性C57BL/6小鼠经口染毒FLC 28 d后,睾丸组织TUNEL染色结果显示生精小管间质及基底部有细胞凋亡发生。Bcl-2家族抗凋亡蛋白Bcl-2水平在FLC染毒75、375 mg·(kg·d)−1组中较对照组下降(P<0.05);而促凋亡蛋白Bim水平在FLC染毒75 mg·(kg·d)−1组中,Bax水平在FLC染毒375 mg·(kg·d)−1组中较对照组上升(P<0.05)。0、40、80和160 μmol·L−1 FLC染毒组的细胞凋亡率分别为2.7%±0.2%、4.8%±1.3%、9.4%±0.3%、13.2%±0.2%,FLC染毒组细胞凋亡率均明显上升(P<0.05)。TM4细胞中Bcl-2蛋白水平在160 μmol·L−1 FLC染毒时下降(P<0.05),而Bim和Bax蛋白水平在80、160 μmol·L−1 FLC染毒时均上升(P<0.05),内质网应激相关蛋白(GRP78、p-PERK、ATF6、p-IRE1α和p-JNK)水平在FLC染毒后均上升(P<0.05)或呈上升趋势。4μ8C(25、50 μmol·L−1)和SP600125(10、20 μmol·L−1)预处理后,由FLC染毒引起的GRP78、p-IRE1α、p-JNK以及Bim、Bax蛋白表达水平上调幅度降低(P<0.05)或呈降低的趋势;预先使用两种抑制剂处理细胞后,FLC对细胞活力的损伤得到明显缓解(P<0.05)或呈缓解的趋势。

    结论

    FLC能够诱导小鼠睾丸组织发生细胞凋亡,并且通过激活内质网应激及IRE1α-JNK信号通路诱导TM4细胞凋亡发生。

     

    Abstract: Background

    Flurochloridone (FLC) can induce apoptosis in Sertoli cells, but the specific mechanism remains unknown.

    Objective

    To investigate the testicular cell apoptosis in mice as well as apoptosis and activation of endoplasmic reticulum stress in TM4 cell line induced by FLC through in vivo and in vitro study designs respectively, and study the role of inosital-requiring enzyme 1α (IRE1α)-c-Jun N-terminal kinase (JNK) signaling pathway in the process of FLC-induced apoptosis in TM4 cells through intervention study design.

    Methods

    Testicular tissues were collected from male C57BL/6 mice which were treated with 3, 15, 75, and 375 mg·(kg·d)−1 FLC by oral perfusion for 28 d. Apoptosis was observed by TUNEL staining, and the levels of apoptosis-related proteins were detected by Western blotting, including B-cell lymphoma-2 (Bcl-2), Bcl-2 interacting mediator of cell death (Bim), and Bcl-2 associated X protein (Bax). In the in vitro study, TM4 cells were treated with different concentrations of FLC (40, 80, and 160 μmol·L−1) for 6 h, then apoptosis rate was detected by flow cytometry, and the levels of apoptosis-related proteins (Bcl-2, Bim, and Bax) and endoplasmic reticulum stress-related proteins glucose regulated protein 78 (GRP78), phosphorylated-protein kinase R like endoplasmic reticulum kinase (p-PERK), activating transcription factor 6 (ATF6), phosphorylated-inosital-requiring enzyme 1α (p-IRE1α), and phosphorylated-JNK (p-JNK) were measured by Western blotting. In the intervention study, TM4 cells were pretreated with IRE1α phosphorylation inhibitor 4μ8C and JNK phosphorylation inhibitor SP600125 for 6 h, then treated with 160 μmol·L−1 FLC for 6 h. The levels of apoptosis-related proteins and endoplasmic reticulum stress-related proteins were measured by Western blotting, and cell viability was detected by cell counting kit-8.

    Results

    After the male C57BL/6 mice orally exposed to FLC for 28 d, apoptosis occurred in the seminiferous tubule. The protein expression level of Bcl-2, apoptosis inhibitor, was decreased in the 75 and 375 mg·(kg·d)−1 groups (P<0.05), and the protein expression levels of Bim and Bax, apoptosis promoters, were increased in the 75 and 375 mg·(kg·d)−1 groups respectively (P<0.05). The percentages of apoptotic cells in the 0, 40, 80, and 160 μmol·L−1 FLC groups were 2.7%±0.2%, 4.8%±1.3%, 9.4%±0.3%, and 13.2%±0.2%, respectively, increased significantly compared with the control group (P<0.05). The protein expression level of Bcl-2 also was decreased in the 160 μmol·L−1 FLC group (P<0.05), while the levels of Bim and Bax were increased in both of the 80 and 160 μmol·L−1 groups (P<0.05). The expression levels of endoplasmic reticulum stress-related proteins (GRP78, p-PERK, ATF6, p-IRE1α, and p-JNK) were increased (P<0.05) or showed a rising trend in TM4 cells. Pre-treatment with 4μ8C (25 and 50 μmol·L−1) and SP600125 (10 and 20 μmol·L−1) significantly down-regulated the protein expression levels of GRP78, p-IRE1α, p-JNK, and Bax induced by FLC (P<0.05) or in a downward trend. Both of the inhibitors alleviated the decreased cell viability induced by FLC (P<0.05) or in alleviating fashion.

    Conclusion

    FLC could induce apoptosis in mice testis and TM4 cell apoptosis through activating endoplasmic reticulum stress and IRE1α-JNK signaling pathway.

     

/

返回文章
返回