[C8mim]Cl对HepG2细胞凋亡和内质网应激通路的影响

张榜军,屠振鹏,冯伊伊,刘 洋,2,李效宇*

[C8mim]Cl对HepG2细胞凋亡和内质网应激通路的影响

张榜军1,屠振鹏1,冯伊伊1,刘 洋1,2,李效宇1*

(1.河南师范大学生命科学学院,河南 新乡 453007；2.河南师范大学学报编辑部,河南 新乡 453007)

为研究离子液体氯化1-辛基-3-甲基咪唑([C8mim]Cl)是否通过内质网应激(ERS)通路诱导细胞凋亡,在MTT法检测细胞活力的基础上,用0, 50, 100, 200μmol/L[C8mim]Cl处理HepG2细胞24h后,采用流式细胞仪检测细胞凋亡,western blot检测ERS通路相关蛋白表达.结果显示:[C8mim]Cl处理后HepG2细胞凋亡呈浓度依赖性增高.ERS相关蛋白葡萄糖调节蛋白78(GRP78)、磷酸化RNA依赖的蛋白激酶样内质网激酶(p-PERK)、磷酸化真核起始因子2α(p-eIF2α)、磷酸化肌醇需求酶-1(p-IRE1)、激活转录因子4(ATF4)和ATF6显著上调. [C8mim]Cl还显著诱导了C/EBP同源蛋白(CHOP)和半胱氨酸天冬氨酸蛋白酶4(caspase 4)蛋白表达,促进了caspase 9和caspase 3活性升高.因此,[C8mim]Cl可通过ERS通路诱导HepG2细胞凋亡.

[C8mim]Cl；凋亡；内质网应激；CHOP; caspase 4

离子液体(ILs)是由有机阳离子和有机或无机阴离子组成的一类低熔点有机盐类化合物[1-2].近年来,由于其极低的蒸汽压、高热稳定性和化学稳定性以及良好的溶解性而受到广泛关注[3],在许多工业领域具有很大的潜在应用[4-7].目前生产的ILs种类超过350种,其产量和需求继续增加[8].虽然当前环境中还未检测到ILs的存在,但其大规模应用后将不可避免地释放到环境中.由于ILs具有高水溶性和低生物降解性,当其释放到水生态系统中时会长期存在并沿食物链累积,对水生生物甚至人类健康构成威胁[3].

本课题组前期试验发现,氯化1-辛基-3-甲基咪唑([C8mim]Cl)暴露可引起PC12细胞DNA损伤,细胞内钙离子和活性氧升高,三磷酸腺苷含量耗竭,促进半胱氨酸天冬氨酸蛋白酶3(caspase 3)活性升高,说明线粒体功能障碍参与[C8mim]Cl诱导的细胞凋亡[9-10].此外,线粒体和死亡受体途径也参与了[C8mim]Br诱导的HepG2细胞凋亡[11-12].

研究证实,细胞内外各种刺激因子可激活内质网应激(ERS),触发未折叠蛋白反应(UPR)[13].UPR激活RNA 依赖的蛋白激酶样内质网激酶(PERK)、肌醇需求酶1(IRE-1)和激活转录因子6(ATF6)3种内质网蛋白来恢复正常内质网稳态.然而,严重或持续的UPR会通过IRE1、ATF6和PERK途径启动c-jun N-末端激酶(JNK)或C/EBP同源蛋白(CHOP)等凋亡信号分子,最终导致细胞死亡[14-16].ERS还可以通过caspase依赖途径触发凋亡[16-17].目前,ERS是否也参与ILs诱导的细胞凋亡尚不清楚.因此,本文采用流式细胞仪和western blot方法,研究了[C8mim]Cl对HepG2细胞凋亡和ERS相关信号通路蛋白的影响,旨在为探讨ILs细胞毒性的分子机制提供参考.

1 材料与方法

1.1 试验试剂

[C8mim]Cl 购自北京华威锐科化工有限公司;细胞凋亡检测试剂盒购自美国BD公司;3-(4,5二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)、caspase 3和9试剂盒购自碧云天生物技术有限公司;Roswell Park Memorial Institute 1640(RPMI 1640)培养基购自北京索莱宝科技有限公司;胎牛血清购自浙江天杭生物科技股份有限公司;细胞裂解液、蛋白酶和磷酸酶抑制剂、双色蛋白上样缓冲液和二喹啉甲酸(BCA)蛋白定量试剂盒购自博士德生物工程有限公司.

PERK(多克隆抗体)和GRP78(多克隆抗体)抗体购自武汉赛维尔;CHOP(多克隆抗体)购自武汉三鹰;p-PERK(多克隆抗体),p-IRE1(单克隆抗体)购自武汉华美生物;eIF2α(多克隆抗体)、IRE1(多克隆抗体)、XBP1(多克隆抗体)、ATF4 (多克隆抗体)和ATF6(多克隆抗体)购自沈阳万类生物科技有限公司;caspase 4(多克隆抗体)和GAPDH(单克隆抗体)购自博士德工程有限公司;p-eIF2α(单克隆抗体)和HRP标记的羊抗兔IgG(H+L)二抗购自碧云天生物技术有限公司.

1.2 细胞培养和活力测定

HepG2细胞由新乡医学院提供,在含10%胎牛血清和青链霉素的RPMI 1640培养基中培养,培养条件为37℃、5% CO2.采用MTT法测定细胞活力,根据预试验结果,用不同浓度[C8mim]Cl(0,125,250, 500,750,1000,1500μmol/L)处理HepG2细胞,每个浓度设5个重复.24h后,小心去除培养基,每孔加100μL新鲜培养基,同时加10μL MTT(0.5mg/mL), 37°C孵育4h.用150μL二甲基亚砜溶解沉淀物,并在570nm用酶标仪读数.

1.3 [C8mim]Cl处理

细胞转接到6孔板中生长12h,用[C8mim]Cl(0, 50, 100, 200μmol/L)处理细胞24h,收集细胞进行以下生化分析.

1.4 细胞凋亡测定

细胞凋亡测定参考Li等[14]操作进行.主要步骤如下:收取细胞,用PBS冲洗,在Binding buffer中重新悬浮,加Annexin V-异硫氰酸荧光素(Annexin V –FITC)和碘化丙啶(Propidium iodide,PI)后室温避光染色15min,流式细胞仪检测.

1.5 Western blot

Western blot试验过程按照卜玲玲等[18]和Feng等[19]操作进行.主要步骤如下:用含有蛋白酶和磷酸酶抑制剂的细胞裂解液提取总蛋白.BCA蛋白定量试剂盒对提取的总蛋白进行定量后加双色蛋白上样缓冲液,100℃下煮沸5min.通过SDS-PAGE分离变性蛋白质并转移到PVDF膜上.加相应的一抗(PERK:1:1000稀释;p-PERK:1:600稀释;eIF2α:1:500稀释;p-eIF2α:1:1000稀释;GRP78:1:500稀释; CHOP:1:1000稀释;p-IRE1:1:2000稀释;IRE1:1:2000稀释;XBP1:1:1500稀释;ATF4:1:500稀释;ATF6:1: 1000稀释;caspase 4:1:1000稀释;GAPDH:1:500),置4℃孵育过夜,TBST清洗3次,加二抗(1:2000稀释)室温孵育1h.TBST清洗3次后加ECL化学发光试剂,经ChemiDoc-MP成像系统(Bio-Rad)成像,并使用ImageJ软件进行灰度扫描.

1.6 caspase酶活性测定

caspase 3和9活性测定按照试剂盒说明书进行操作.试验过程参考Li等[14]步骤.

1.7 数据分析

使用SPSS 23.0软件对数据进行方差分析,然后进行Dunnett-t检验,数值以平均值±标准差(SD)表示.*代表处理组和对照组之间差异显著(*<0.05, **<0.01).

2 结果与分析

2.1 [C8mim]Cl对细胞活力的影响

图1 [C8mim]Cl对HepG2细胞活力的影响

如图1所示,[C8mim]Cl处理后细胞活力显著降低,说明[C8mim]Cl影响HepG2细胞的生长.根据试验获得[C8mim]Cl对HepG2细胞的24h IC50为1051.0(944.1～1202.3)µmol/L.

2.2 [C8mim]Cl对HepG2细胞凋亡的影响

如图2所示,流式细胞仪检测结果显示50, 100, 200μmol/L [C8mim]Cl处理HepG2细胞后凋亡细胞从4.35%升高到11.26%、20.76%和52.9%,表明[C8mim]Cl诱导的细胞凋亡呈现浓度依赖性.

2.3 [C8mim]Cl 对内质网应激相关蛋白的影响

如图3所示,GRP78、CHOP和caspase 4作为ERS相关的生物标志物,在[C8mim]Cl处理24h后显著增加.此外,[C8mim]Cl处理显著增加p-eIF2α和p-IRE1的表达;200μmol/L [C8mim] Cl处理后,p-PERK、ATF4和ATF6水平也显著增加(图4).然而,在[C8mim]Cl处理组中, PERK、eIF2α、IRE1和XBP1的蛋白质水平普遍降低(图4).表明ERS参与了[C8mim]Cl介导的细胞凋亡.

图3 [C8mim]Cl对HepG2细胞GRP78、CHOP和caspase 4蛋白的影响

2.4 [C8mim]Cl对caspase 3和9酶活性的影响

如图5所示,[C8mim]Cl处理可显著促进HepG2细胞caspase 3和caspase 9的活性,提示caspase 3和caspase 9可能在[C8mim]Cl引起的细胞凋亡中起重要作用.

3 讨论

研究表明,ILs能显著抑制体外细胞的细胞活力,包括PC12[9],Hela[20],CCO[21],9[22]和Caco-2细胞株[23].本文发现[C8mim]Cl也显著降低了HepG2细胞活力,表明离子液体暴露对细胞有毒害作用.

细胞凋亡是一种受到严格控制的细胞死亡形式,在调节许多细胞生物学过程、维持正常发育和组织内环境稳定方面起关键作用[24].越来越多的证据表明,诱导细胞凋亡是许多环境毒物细胞毒性的重要机制[25-27].Ma等[12]研究发现,离子液体[C8mim]Br可激活线粒体通路,诱导细胞凋亡,表明细胞凋亡可能是离子液体的细胞毒性机制之一.本文中,流式细胞检测也证实[C8mim]Cl诱导了细胞凋亡.然而,ILs诱导细胞凋亡的通路还需深入研究.近年来,ERS介导的细胞凋亡受到越来越多的关注[28-29].ILs是否会通过激活ERS来诱导细胞凋亡目前还未见报道.

GRP78是ER定位的伴侣蛋白,与PERK、IRE1和ATF6等跨膜蛋白结合,在正常生理条件下保持非活性状态.内质网应激发生时,其结合态解离,GRP78表达水平升高,3种跨膜蛋白(ATF6、PERK和IRE1)被激活,启动内质网介导的UPR,防止蛋白质聚集的形成和驱动错误折叠的蛋白质降解来维持内质网的内稳态[30].因此,GRP78被用作内质网应激的指标[31-32].在本文中,[C8mim]Cl暴露可显著提高GRP78蛋白水平,提示[C8mim]Cl可能破坏HepG2细胞内质网稳态,诱导内质网应激产生.

CHOP是C/EBP家族转录因子.研究表明, CHOP过度表达加剧了人肺癌来源的RERF-LC Ad2细胞和肾小管上皮细胞的凋亡,而敲除CHOP则抑制了内质网应激诱导的凋亡[33-34].因此,CHOP在ERS介导的细胞凋亡中起着重要作用,已被广泛认为是内质网应激的生物标志物[30,35].本试验发现[C8mim]Cl暴露后HepG2细胞中CHOP表达显著增加,说明CHOP参与了[C8mim]Cl诱导的细胞凋亡过程.

PERK-eIF2α-ATF4轴是内质网应激诱导CHOP表达的主要途径[14].在UPR条件下,PERK通过自身磷酸化激活,然后磷酸化eIF2α的α亚单位,从而通过抑制一般蛋白翻译来降低内质网应激.然而,磷酸化的eIF2α也可以激活ATF4[14].同时,ATF6与GRP78分离后转移到高尔基体,被切割为有活性的ATF6[13].ATF4和 ATF6又转移到细胞核并调节内质网应激反应基因的表达,包括GRP78和CHOP,这是内质网应激介导的凋亡途径中的一个重要通路[36].本文发现[C8mim]Cl处理后,HepG2细胞的PERK和eIF2α显著降低,而磷酸化PERK和eIF2α升高.此外,在[C8mim]Cl处理的细胞中,ATF4和ATF6也显著增加.因此,我们推测PERK和ATF6可能通过激活其下游成分CHOP参与[C8mim]Cl诱导的凋亡.

和GRP78解离后,激活的IRE1α催化XBP-1剪接,以诱导多个参与UPR基因的表达,以恢复内质网稳态[13-14].在本文中,p-IRE1α显著增加,而剪接的XBP-1在[C8mim]Cl处理24h后显著下调,表明[C8mim]Cl暴露可能破坏了XBP-1介导的保护途径.研究表明IRE1可招募ASK1和TRAF2,形成IRE1- TRAF2-ASK1复合物后激活JNK途径诱导细胞凋亡[37].因此,需要进一步研究IRE1α相关信号通路在[C8mim]Cl诱导细胞凋亡中的作用.

Caspase家族负责调控许多环境毒物介导的细胞凋亡[38].小鼠caspase-12是一个内质网驻留的caspase家族成员,对caspase-12缺陷小鼠的研究结果表明,它在内质网应激介导的细胞凋亡中被特异性激活[39].但是,人类caspase-12蛋白在进化过程中由于若干突变而处于失活状态,在内质网应激诱导的细胞凋亡中不起作用[40].然而,先前的研究表明,人类caspase-4在内质网应激诱导的细胞死亡途径中与小鼠caspase 12具有相同的功能[17].同时,caspase 12可通过caspase 9/caspase 3途径诱导细胞凋亡[41].本研究发现,caspase 4蛋白在[C8mim]Cl处理后显著上调,caspase 9和caspase 3活性也显著升高,提示[C8mim]Cl 能够激活caspase 4,可能通过caspase9/ caspase-3通路在凋亡诱导中发挥作用.

4 结论

4.1 [C8mim]Cl处理可诱导HepG2细胞凋亡.

4.2 [C8mim]Cl处理能激活HepG2细胞PERK、IRE1和ATF6信号通路, 诱导细胞UPR反应.

4.3 CHOP和caspase 4信号通路在[C8mim]Cl诱导的HepG2细胞凋亡中可能起重要作用.

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Effects of [C8mim]Cl on apoptosis and endoplasmic reticulum stress pathway in HepG2 cells.

ZHANG Bang-jun1,TU Zhen-peng1, FENG Yi-yi1, LIU Yang1, 2,LIXiao-yu1*

(1.College of Life Science, Henan Normal University, Xinxiang 453007, China；2.Journal of Henan Normal University, Henan Normal University, Xinxiang 453007, China)., 2021,41(6)：2932～2938

In order to explore whether ionic liquid 1-octyl-3-methylimidazolium chloride ([C8mim]Cl) can induce apoptosis through endoplasmic reticulum stress(ERS) pathway, HepG2 cells were treated with 0,50,100,200μmol/L [C8mim]Cl for 24h based on the MTT cell viability assay. The apoptosis was detected by flow cytometry and western blot was used to assay the expressions of ERS related proteins. The results showed that [C8mim]Cl increased the apoptosis in HepG2 cells with a concentration-dependent manner. [C8mim]Cl significantly increased the expressions of ERS related proteins, including glucose regulated protein 78 (GRP78), phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), phosphorylated inositol-requiring enzyme 1 (p-IRE1), activating transcription factor 4 (ATF4) and ATF6. [C8mim]Cl also significantly induced the expressions of C/EBP homologous protein (CHOP) and caspase 4 protein, lead to the increase of caspase 9 and caspase 3 activities. Therefore, [C8mim]Cl could induce apoptosis in HepG2 cells through ERS pathway.

[C8mim]Cl；apoptosis；ERS；CHOP；caspase 4

X171.5

A

1000-6923(2021)06-2932-07

张榜军(1978-),男,河南林州人,讲师,博士,研究方向为环境毒理学.发表论文20余篇.

2020-11-20

河南省高等学校重点科研项目(19zx011);河南师范大学博士启动经费资助项目(qd16147)

* 责任作者, 教授, lixiaoyu65@263.net