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

张芬, 李睿, 赵淑琦, 王彦娜, 倪志晶, 常秀丽, 周志俊

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

基金项目: 国家自然科学基金项目(81872643)
详细信息
    作者简介:

    张芬(1996—),女,硕士生;E-mail:19211020032@fudan.edu.cn

    通讯作者:

    周志俊,E-mail:zjzhou@fudan.edu.cn

  • 中图分类号: R114

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

Funds: This study was funded.
More Information
  • 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.

     

  • 氟咯草酮(flurochloridone, FLC)作为新型除草剂,自上市以来在欧盟和北美国家中广泛应用。欧洲食品安全局(European Food Safety Authority, EFSA)在对FLC危险度评估报告中指出FLC的靶器官为睾丸和附睾[1]。前期研究结果显示,FLC能够导致精子畸形率增加,支持细胞空泡化,并可诱导支持细胞凋亡或自噬发生[2-3],损伤血睾屏障完整性[4]。FLC可诱导支持细胞产生大量活性氧,损伤内质网结构,破坏内质网钙离子(Ca2+)稳态[3],提示FLC可能会诱导支持细胞发生内质网应激,进而诱导细胞凋亡发生。

    内质网在细胞内承担着重要的生命活动,主要包括蛋白质的正确折叠与合成运输、钙离子的储存与调节等[5]。当内质网中出现未折叠及错误折叠蛋白质累积,会引起内质网应激[6],同时启动未折叠蛋白反应[7-8],通过降低蛋白质转录和翻译、上调内质网伴侣蛋白葡萄糖调节蛋白78(glucose regulated protein 78, GRP78)水平、加强错误折叠蛋白降解等方式,使细胞恢复正常[9-10]。未折叠蛋白反应的激活涉及三条信号通路的参与,分别由肌醇需酶1α(inosital-requiring enzyme 1α, IRE1α)、蛋白激酶R样内质网激酶(protein kinase R like endoplasmic reticulum kinase, PERK)和活化转录因子6(activating transcription factor 6, ATF6)介导[11-12],它们与内质网伴侣蛋白GRP78解离并通过自身二聚体或磷酸化激活下一级信号通路,引发不同的细胞反应[13]

    越来越多的研究证实,内质网应激与细胞凋亡之间的关系十分密切。未折叠蛋白反应的下游蛋白,如c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)[14],可通过调节B淋巴细胞瘤-2蛋白(B-cell lymphoma-2, Bcl-2)家族蛋白,如Bcl-2、Bcl-2相互作用的死亡介质(Bcl-2 interacting mediator of cell death, Bim)和Bcl-2相关X蛋白(Bcl-2 associated X protein, Bax)等的表达水平来促进细胞凋亡的发生[15-16]

    在FLC诱导睾丸组织及支持细胞损伤的过程中,是否启动了内质网应激和未折叠蛋白反应,以及未折叠蛋白反应的哪些信号通路在细胞凋亡的过程发挥作用还有待进一步研究证实。本研究通过对FLC染毒的C57BL/6小鼠睾丸组织中细胞凋亡发生情况的研究,利用小鼠睾丸支持细胞系TM4细胞模型,围绕内质网应激及未折叠蛋白反应IRE1α-JNK信号通路,观察其在不同染毒浓度下的改变情况,并且通过相应的干预实验探索其在FLC诱导支持细胞凋亡中的作用,为FLC诱导睾丸组织细胞凋亡的机制研究提供新思路。

    本研究利用了实验室先前完成的6~8周龄雄性C57BL/6小鼠28 d经口染毒FLC的标本。FLC染毒剂量分别为0(0.5%羧甲基纤维素钠)、3、15、75和375 mg·(kg·d)−1[2]。观察睾丸组织中细胞凋亡发生情况,检测睾丸组织中的凋亡蛋白表达水平。在体外实验中,使用小鼠睾丸支持细胞系TM4细胞进行FLC(40、80和160 μmol·L−1)染毒6 h,检测细胞凋亡情况及内质网应激相关蛋白水平。干预实验中使用IRE1α磷酸化抑制剂4μ8C(上海Selleck)、JNK磷酸化抑制剂SP600125(上海碧云天)预处理TM4细胞6 h后再进行160 μmol·L−1 FLC染毒6 h,检测细胞活力、细胞凋亡情况及内质网应激相关蛋白水平。根据文献报道,选择4μ8C的处理浓度为25、50 μmol·L−1[17],SP600125的处理浓度为10、20 μmol·L−1[18-19]

    小鼠睾丸支持细胞系TM4细胞(购自国家细胞资源平台,编号:1101MOU-PUMC000298)的培养条件为5%二氧化碳、37 ℃恒温,完全培养基为含有10%胎牛血清(美国Gibco)、1%青霉素-链霉素(美国Sigma)的高糖培养基(美国Gibco)。FLC(上海笃玛)固体粉末溶解于二甲基亚砜中制成不同浓度的染毒液用于后续实验。

    小鼠睾丸组织石蜡切片置于二甲苯中浸洗进行脱蜡处理,再依次置于100%、95%、85%、75%乙醇中浸洗,根据TUNEL凋亡检测试剂盒(上海碧云天)的说明书配制并在切片上滴加适量TUNEL检测液,最后滴加抗荧光淬灭封片液[含4',6-二脒基-2-苯基吲哚(DAPI)]进行封片处理,尽快在荧光显微镜下观察,每张切片随机选择3个独立视野进行观察。

    将细胞接种至6孔板中,待细胞贴壁后进行染毒处理,处理结束后,收集培养液,每孔中加入500 μL胰酶消化液消化细胞,收集细胞悬液,1000×g离心5 min,弃上清,随后加入195 μL膜联蛋白V(Annexin V)-异硫氰酸荧光素(FITC)结合液重悬细胞,再分别加入5 μL Annexin V-FITC和10 μL碘化丙啶(PI),混匀后室温避光孵育20 min,立即上机检测。使用FlowJo软件(版本号v10.0.7r2)进行凋亡细胞分析。

    将细胞接种至96孔板中,待细胞贴壁后进行染毒处理,处理结束后,每孔中加入10 μL CCK-8溶液,继续置于培养箱中培养1.5 h,取出96孔板于酶标仪中在波长为450 nm处测定光密度,将处理组与对照组光密度的比值作为相对细胞活力值。

    采用Western blotting检测睾丸组织匀浆及TM4细胞内抗凋亡蛋白BCL-2、促凋亡蛋白Bim和Bax的蛋白水平,进一步检测TM4细胞中内质网应激标记蛋白GRP78、未折叠蛋白反应信号通路蛋白磷酸化PERK(p-PERK)、ATF6和磷酸化IREIα(p-IRE1α)以及内质网应激诱导凋亡关键蛋白p-JNK的表达水平。具体条件如下:蛋白电泳条件为80 V,1 h,待蛋白样本进入分离胶后转至100 V,1 h。转膜条件为100 V,1.5 h。将膜置于5%脱脂奶粉溶液中,室温封闭2 h。随后与含0.05% Tween20的三羟甲基氨基甲烷缓冲液(TBST)稀释的一抗(Bcl-2,1∶500稀释;Bim,1∶1000稀释;Bax,1∶500稀释;GRP78,1∶1000稀释;p-PERK,1∶500稀释;ATF6,1∶1000稀释;p-IRE1α,1∶1000稀释;p-JNK,1∶1000稀释)在4 °C孵育过夜。一抗孵育结束后,将蛋白条带与辣根过氧化物酶结合的二抗置于室温中孵育1 h,滴加适量增强化学发光法(ECL)超敏检测化学发光液,在ECL化学发光检测系统(美国Syngene)中检测目的蛋白条带的发光情况。使用ImageJ软件(版本号1.52v)分析蛋白条带灰度值,以β-actin或甘油醛-3-磷酸脱氢酶(GAPDH)作为内参蛋白,计算目的蛋白灰度值/内参灰度值的数值。

    使用GraphPad Prism软件(版本号8.3.0)进行统计分析,不同处理组数据间的统计分析采用单因素方差分析,随后使用LSD进行两两组间比较。Western blotting及CCK-8实验每组包含6个平行数据,流式细胞术实验每组包含3个平行数据。检验水准α=0.05。

    雄性小鼠经口染毒FLC 28 d后,睾丸组织TUNEL染色结果(图1A)显示,375 mg·(kg·d)−1染毒组发生明显的细胞凋亡,主要集中在睾丸间质及生精小管基底部,提示有间质细胞及支持细胞凋亡发生。对凋亡蛋白水平的检测发现(图1B图1C),Bcl-2蛋白水平呈现随染毒剂量增加而降低的趋势,Bim蛋白、Bax蛋白水平分别在75、375 mg·(kg·d)−1组中较对照组增加(P<0.05)。

    图  1  FLC诱导小鼠睾丸组织损伤及凋亡发生
    A:睾丸组织TUNEL染色,蓝色荧光为细胞核,绿色荧光为凋亡细胞,白色箭头表示FLC引起的支持细胞凋亡;B:Western blotting检测凋亡蛋白的特征性条带图;C:Western blotting条带量化图(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。
    Figure  1.  FLC-induced testis injury and apoptosis in mice
    A: TUNEL staining of mice testis tissue, cell nuclei are stained blue and apoptotic cells are stained green, apoptotic cells induced by FLC are pointed by white arrows; B: The representative blots of apoptotic proteins determinated by Western blotting; C: The quantitative data of Western blotting (n=6). Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001.

    细胞凋亡检测发现(图2A图2B),40、80和160 μmol·L−1 FLC染毒组的细胞凋亡率分别为4.8%±1.3%、9.4%±0.3%、13.2%±0.2%,均高于对照组(2.7%±0.2%)(P<0.05)。检测Bcl-2家族凋亡蛋白的表达水平(图2C图2D),与对照组相比,抗凋亡蛋白Bcl-2水平在160 μmol·L−1下降(P<0.05),而促凋亡蛋白Bim和Bax的水平在80和160 μmol·L−1时均出现上调(P<0.05)。

      2  FLC诱导TM4凋亡发生
    A:流式细胞术检测细胞凋亡特征图;B:细胞凋亡率(n=3);C:Western blotting检测凋亡蛋白水平特征性条带图;D:Western blotting条带量化图(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。
      2.  FLC-induced apoptosis in TM4 cells
    A: Apoptotic cells detected by flow cytometry; B: Apoptotic rate (n=3); C: The representative blots of apoptotic proteins determinaed by Western blotting; D: The quantitative data of Western blotting (n=6). Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001.

    对内质网应激相关蛋白水平的检测结果显示(图3),与对照组相比,GRP78蛋白表达水平在80、160 μmol·L−1染毒组中上升,p-PERK蛋白表达水平在40 μmol·L−1时即出现上调(P<0.05),ATF6和p-IRE1α蛋白表达水平在160 μmol·L−1时上升(P<0.05)。同时,p-JNK蛋白表达水平也在40和160 μmol·L−1时上升(P<0.05),在80 μmol·L−1时上升不明显,呈“U型”效应曲线。

    图  3  FLC诱导TM4细胞内质网应激及未折叠蛋白反应发生
    A:Western blotting检测内质网应激及未折叠蛋白反应蛋白特征性条带图;B1~B5:Western blotting条带量化图(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。
    Figure  3.  FLC-activated endoplasmic reticulum stress and unfolded protein response in TM4 cells
    A: The representative blots of endoplasmic reticulum stress- and unfolded protein response-related proteins determinated by Western blotting; B1-B5: The quantitative data of Western blotting (n=6). Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001.

    单纯的4μ8C能够降低p-IRE1α的蛋白表达水平,同时降低GRP78及促凋亡蛋白Bax的表达水平(图4A图4B图4C图4D),但是在一定程度上可抑制细胞活力(图4E)。在经过FLC染毒的细胞中,4μ8C能够降低GRP78、p-IRE1α、p-JNK以及Bax的表达水平,同时可以改善FLC诱导的细胞活力损伤。

    图  4  IRE1α在FLC诱导细胞凋亡中的作用
    A:Western blotting检测内质网应激蛋白水平特征性条带图;B:Western blotting条带量化图(n=6);C:Western blotting检测凋亡蛋白水平特征性条带图;D:Western blotting条带量化图(n=6);E:CCK-8检测细胞活力(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。与160 μmol·L−1 FLC且0 μmol·L−1 4μ8C组相比,#:P<0.05,##:P<0.01,###:P<0.001。
    Figure  4.  The role of IRE1α in cell apoptosis induced by FLC
    A: The representative blots of endoplasmic reticulum stress-related proteins determinated by Western blotting; B: The quantitative data of Western blotting (n=6); C: The representative blots of apoptotic proteins determinated by Western blotting; D: The quantitative data of Western blotting (n=6); E: Cell viability detected by CCK-8 assay. Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001. Compared with the 160 μmol·L−1 FLC and 0 μmol·L−1 4μ8C group, #: P<0.05, ##: P<0.01, ###: P<0.001.

    SP600125预处理细胞后的内质网应激相关蛋白水平的检测结果显示,SP600125能够降低JNK的磷酸化水平,并呈浓度依赖性下调(图5A图5B)。同时SP600125在高浓度时能够下调p-IRE1α、GRP78的蛋白表达水平,浓度依赖性抑制Bax的蛋白表达水平(图5C图5D),并且可以缓解FLC诱导的细胞活力损伤(图5E)。

    图  5  JNK在FLC诱导细胞凋亡中的作用
    A:Western blotting检测内质网应激蛋白水平特征性条带图;B:Western blotting条带量化图(n=6);C:Western blotting检测凋亡蛋白水平特征性条带图;D:Western blotting条带量化图(n=6);E:CCK-8检测细胞活力(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。与160 μmol·L−1 FLC且0 μmol·L−1 SP600125组相比,#:P<0.05,##:P<0.01,###:P<0.001。
    Figure  5.  The role of JNK in cell apoptosis induced by FLC
    A: The representative blots of endoplasmic reticulum stress-related proteins determinated by Western blotting; B: The quantitative data of Western blotting (n=6); C: The representative blots of apoptotic proteins determinated by Western blotting; D: The quantitative data of Western blotting (n=6); E: Cell viability detected by CCK-8 assay. Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001. Compared with the 160 μmol·L−1 FLC and 0 μmol·L−1 SP600125 group, #: P<0.05, ##: P<0.01, ###: P<0.001.

    FLC可诱导小鼠睾丸组织中发生细胞凋亡,改变Bcl-2家族凋亡蛋白(Bcl-2、Bim、Bax)的表达水平,并且诱导TM4细胞活力损伤及细胞凋亡发生,在该过程中存在内质网应激发生,并且激活未折叠蛋白反应信号通路之一IRE1α通路,进一步针对IRE1α及其下游JNK通路进行抑制剂干预实验,证实了FLC通过激活IRE1α-JNK信号通路介导TM4细胞凋亡发生。

    关于FLC的毒性研究主要集中在其对雄性动物生殖系统的损伤,其可损伤血睾屏障完整性[4],通过细胞外调节蛋白激酶1/2(extracellular regulated protein kinases 1/2, ERK1/2)信号通路导致支持细胞内质网Ca2+稳态失调[3],并通过线粒体途径诱导支持细胞凋亡[4],激活磷脂酰肌醇3-激酶(phosphoinositide 3-kinase, PI3K)/丝氨酸/苏氨酸激酶(serine/threonine kinases, Akt)/雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)信号通路引起自噬发生[2],还可激活促分裂素原活化蛋白激酶(mitogen-activated protein kinases, MAPK)信号通路[20],同时FLC还可激活内质网应激,通过PERK通路诱导细胞凋亡发生[21]。本研究补充了内质网应激与细胞凋亡发生之间可能存在其他的信号通路,进一步完善了FLC诱导凋亡发生的机制网络。

    FLC的靶细胞为支持细胞,鉴于TM4细胞系在众多研究中作为小鼠睾丸支持细胞体外模型[22-24],本研究选择TM4细胞系进行体外实验,因此在睾丸组织中观察到凋亡发生后,进一步检测了TM4支持细胞凋亡发生情况,其凋亡率随染毒浓度增加而增加,凋亡蛋白水平也发生改变,提示FLC诱导TM4细胞凋亡发生。

    细胞凋亡可由死亡受体依赖途径、线粒体途径及内质网途径介导[25],其中线粒体途径和内质网途径均依赖Bcl-2家族蛋白介导凋亡发生[26],通过促凋亡蛋白Bim与线粒体膜上表达的蛋白Bax结合,导致线粒体外膜通透性增加,促进半胱天冬酶和细胞色素c释放到胞浆中,引发细胞凋亡[27]。相比于线粒体途径诱导的凋亡,内质网应激诱导的凋亡还涉及未折叠蛋白反应三条信号通路的活化,分别由PERK、ATF6和IRE1α介导。前期研究已经探究了FLC可通过PERK通路激活CCAAT增强子结合蛋白同源蛋白(CCAAT enhancer binding protein homologous protein, CHOP),诱导凋亡发生[21],而许多研究表明,IRE1α信号通路也可以诱导细胞凋亡的发生[28]

    过量或持续的内质网应激可激活IRE1α的磷酸激酶活性,使下游凋亡信号调节激酶1(apoptosis signal regulating kinase-1, ASK1)活化,激活的ASK1可增加下游蛋白JNK的表达[29]。IRE1α还可与肿瘤坏死因子受体相关因子-2(tumor necrosis factor receptor associated factor-2, TRAF2)相互作用激活JNK。JNK激活后可与凋亡相关蛋白相互作用,如促进Bax的表达诱导线粒体凋亡途径[30]。因此本研究通过抑制剂体外干预TM4细胞着重探究了IRE1α和JNK在FLC诱导细胞凋亡中的作用。

    4μ8C通过抑制IRE1α的RNase结构域限制x盒结合蛋白1(X-box binding protein 1, XBP1)的剪切,从而阻断未折叠蛋白反应的激活[31]。在本研究中,使用不同浓度的4μ8C预处理TM4细胞6 h后,可以看到4μ8C可降低磷酸化IRE1α的水平,其下游蛋白JNK的磷酸化水平也降低,并且促凋亡蛋白Bax在高浓度4μ8C的预处理组中发生下调,提示IRE1α参与了FLC诱导的内质网应激以及JNK的活化,能够通过抑制IRE1α的磷酸化缓解FLC诱导的细胞凋亡,证实FLC可能通过IRE1α-JNK-Bax信号通路诱导TM4细胞凋亡发生。为了进一步验证JNK在连接IRE1α和凋亡之间的作用,使用了JNK磷酸化抑制剂SP600125进行干预实验[32],SP600125能够浓度依赖性地抑制JNK的磷酸化,进而导致Bax蛋白表达水平下降,提示FLC诱导的TM4细胞凋亡受到JNK活化的影响,p-JNK参与了IRE1α介导细胞凋亡的过程,进一步证实了IRE1α-JNK-Bax信号通路在FLC诱导TM4细胞凋亡中的作用。同时两种抑制剂能够显著缓解FLC诱导的细胞活力受损,进一步证实了IRE1α/JNK通路参与了FLC诱导的TM4细胞活力受损。

    本研究结果进一步证实了FLC能够诱导小鼠睾丸组织发生细胞凋亡,同时诱导TM4细胞凋亡发生,并且诱导内质网应激发生,激活未折叠蛋白反应IRE1α通路,通过抑制剂干预实验发现,FLC通过IRE1α-JNK信号通路介导凋亡发生。本研究为FLC诱导凋亡发生的机制网络补充了新的信号通路,但是该通路与其他通路之间是否存在关联尚需进一步研究,如内质网应激与氧化应激之间的关系、内质网途径诱导凋亡与线粒体途径诱导凋亡之间的关系等,还需再进一步研究中进行探究。

  • 图  1   FLC诱导小鼠睾丸组织损伤及凋亡发生

    A:睾丸组织TUNEL染色,蓝色荧光为细胞核,绿色荧光为凋亡细胞,白色箭头表示FLC引起的支持细胞凋亡;B:Western blotting检测凋亡蛋白的特征性条带图;C:Western blotting条带量化图(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。

    Figure  1.   FLC-induced testis injury and apoptosis in mice

    A: TUNEL staining of mice testis tissue, cell nuclei are stained blue and apoptotic cells are stained green, apoptotic cells induced by FLC are pointed by white arrows; B: The representative blots of apoptotic proteins determinated by Western blotting; C: The quantitative data of Western blotting (n=6). Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001.

    2   FLC诱导TM4凋亡发生

    A:流式细胞术检测细胞凋亡特征图;B:细胞凋亡率(n=3);C:Western blotting检测凋亡蛋白水平特征性条带图;D:Western blotting条带量化图(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。

    2.   FLC-induced apoptosis in TM4 cells

    A: Apoptotic cells detected by flow cytometry; B: Apoptotic rate (n=3); C: The representative blots of apoptotic proteins determinaed by Western blotting; D: The quantitative data of Western blotting (n=6). Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001.

    图  3   FLC诱导TM4细胞内质网应激及未折叠蛋白反应发生

    A:Western blotting检测内质网应激及未折叠蛋白反应蛋白特征性条带图;B1~B5:Western blotting条带量化图(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。

    Figure  3.   FLC-activated endoplasmic reticulum stress and unfolded protein response in TM4 cells

    A: The representative blots of endoplasmic reticulum stress- and unfolded protein response-related proteins determinated by Western blotting; B1-B5: The quantitative data of Western blotting (n=6). Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001.

    图  4   IRE1α在FLC诱导细胞凋亡中的作用

    A:Western blotting检测内质网应激蛋白水平特征性条带图;B:Western blotting条带量化图(n=6);C:Western blotting检测凋亡蛋白水平特征性条带图;D:Western blotting条带量化图(n=6);E:CCK-8检测细胞活力(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。与160 μmol·L−1 FLC且0 μmol·L−1 4μ8C组相比,#:P<0.05,##:P<0.01,###:P<0.001。

    Figure  4.   The role of IRE1α in cell apoptosis induced by FLC

    A: The representative blots of endoplasmic reticulum stress-related proteins determinated by Western blotting; B: The quantitative data of Western blotting (n=6); C: The representative blots of apoptotic proteins determinated by Western blotting; D: The quantitative data of Western blotting (n=6); E: Cell viability detected by CCK-8 assay. Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001. Compared with the 160 μmol·L−1 FLC and 0 μmol·L−1 4μ8C group, #: P<0.05, ##: P<0.01, ###: P<0.001.

    图  5   JNK在FLC诱导细胞凋亡中的作用

    A:Western blotting检测内质网应激蛋白水平特征性条带图;B:Western blotting条带量化图(n=6);C:Western blotting检测凋亡蛋白水平特征性条带图;D:Western blotting条带量化图(n=6);E:CCK-8检测细胞活力(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。与160 μmol·L−1 FLC且0 μmol·L−1 SP600125组相比,#:P<0.05,##:P<0.01,###:P<0.001。

    Figure  5.   The role of JNK in cell apoptosis induced by FLC

    A: The representative blots of endoplasmic reticulum stress-related proteins determinated by Western blotting; B: The quantitative data of Western blotting (n=6); C: The representative blots of apoptotic proteins determinated by Western blotting; D: The quantitative data of Western blotting (n=6); E: Cell viability detected by CCK-8 assay. Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001. Compared with the 160 μmol·L−1 FLC and 0 μmol·L−1 SP600125 group, #: P<0.05, ##: P<0.01, ###: P<0.001.

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  • 收稿日期:  2022-03-17
  • 录用日期:  2022-07-15
  • 网络出版日期:  2022-11-20
  • 刊出日期:  2022-09-24

目录

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