[C8mim]Cl對HepG2細胞凋亡和內質網應激通路的影響

張榜軍,屠振鵬,馮伊伊,劉 洋,2,李效宇*

[C8mim]Cl對HepG2細胞凋亡和內質網應激通路的影響

張榜軍1,屠振鵬1,馮伊伊1,劉 洋1,2,李效宇1*

(1.河南師范大學生命科學學院,河南 新鄉 453007；2.河南師范大學學報編輯部,河南 新鄉 453007)

為研究離子液體氯化1-辛基-3-甲基咪唑([C8mim]Cl)是否通過內質網應激(ERS)通路誘導細胞凋亡,在MTT法檢測細胞活力的基礎上,用0, 50, 100, 200μmol/L[C8mim]Cl處理HepG2細胞24h后,采用流式細胞儀檢測細胞凋亡,western blot檢測ERS通路相關蛋白表達.結果顯示:[C8mim]Cl處理后HepG2細胞凋亡呈濃度依賴性增高.ERS相關蛋白葡萄糖調節蛋白78(GRP78)、磷酸化RNA依賴的蛋白激酶樣內質網激酶(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)、激活轉錄因子4(ATF4)和ATF6顯著上調. [C8mim]Cl還顯著誘導了C/EBP同源蛋白(CHOP)和半胱氨酸天冬氨酸蛋白酶4(caspase 4)蛋白表達,促進了caspase 9和caspase 3活性升高.因此,[C8mim]Cl可通過ERS通路誘導HepG2細胞凋亡.

[C8mim]Cl；凋亡；內質網應激；CHOP; caspase 4

離子液體(ILs)是由有機陽離子和有機或無機陰離子組成的一類低熔點有機鹽類化合物[1-2].近年來,由于其極低的蒸汽壓、高熱穩定性和化學穩定性以及良好的溶解性而受到廣泛關注[3],在許多工業領域具有很大的潛在應用[4-7].目前生產的ILs種類超過350種,其產量和需求繼續增加[8].雖然當前環境中還未檢測到ILs的存在,但其大規模應用后將不可避免地釋放到環境中.由于ILs具有高水溶性和低生物降解性,當其釋放到水生態系統中時會長期存在并沿食物鏈累積,對水生生物甚至人類健康構成威脅[3].

本課題組前期試驗發現,氯化1-辛基-3-甲基咪唑([C8mim]Cl)暴露可引起PC12細胞DNA損傷,細胞內鈣離子和活性氧升高,三磷酸腺苷含量耗竭,促進半胱氨酸天冬氨酸蛋白酶3(caspase 3)活性升高,說明線粒體功能障礙參與[C8mim]Cl誘導的細胞凋亡[9-10].此外,線粒體和死亡受體途徑也參與了[C8mim]Br誘導的HepG2細胞凋亡[11-12].

研究證實,細胞內外各種刺激因子可激活內質網應激(ERS),觸發未折疊蛋白反應(UPR)[13].UPR激活RNA 依賴的蛋白激酶樣內質網激酶(PERK)、肌醇需求酶1(IRE-1)和激活轉錄因子6(ATF6)3種內質網蛋白來恢復正常內質網穩態.然而,嚴重或持續的UPR會通過IRE1、ATF6和PERK途徑啟動c-jun N-末端激酶(JNK)或C/EBP同源蛋白(CHOP)等凋亡信號分子,最終導致細胞死亡[14-16].ERS還可以通過caspase依賴途徑觸發凋亡[16-17].目前,ERS是否也參與ILs誘導的細胞凋亡尚不清楚.因此,本文采用流式細胞儀和western blot方法,研究了[C8mim]Cl對HepG2細胞凋亡和ERS相關信號通路蛋白的影響,旨在為探討ILs細胞毒性的分子機制提供參考.

1 材料與方法

1.1 試驗試劑

[C8mim]Cl 購自北京華威銳科化工有限公司;細胞凋亡檢測試劑盒購自美國BD公司;3-(4,5二甲基噻唑-2)-2,5-二苯基四氮唑溴鹽(MTT)、caspase 3和9試劑盒購自碧云天生物技術有限公司;Roswell Park Memorial Institute 1640(RPMI 1640)培養基購自北京索萊寶科技有限公司;胎牛血清購自浙江天杭生物科技股份有限公司;細胞裂解液、蛋白酶和磷酸酶抑制劑、雙色蛋白上樣緩沖液和二喹啉甲酸(BCA)蛋白定量試劑盒購自博士德生物工程有限公司.

PERK(多克隆抗體)和GRP78(多克隆抗體)抗體購自武漢賽維爾;CHOP(多克隆抗體)購自武漢三鷹;p-PERK(多克隆抗體),p-IRE1(單克隆抗體)購自武漢華美生物;eIF2α(多克隆抗體)、IRE1(多克隆抗體)、XBP1(多克隆抗體)、ATF4 (多克隆抗體)和ATF6(多克隆抗體)購自沈陽萬類生物科技有限公司;caspase 4(多克隆抗體)和GAPDH(單克隆抗體)購自博士德工程有限公司;p-eIF2α(單克隆抗體)和HRP標記的羊抗兔IgG(H+L)二抗購自碧云天生物技術有限公司.

1.2 細胞培養和活力測定

HepG2細胞由新鄉醫學院提供,在含10%胎牛血清和青鏈霉素的RPMI 1640培養基中培養,培養條件為37℃、5% CO2.采用MTT法測定細胞活力,根據預試驗結果,用不同濃度[C8mim]Cl(0,125,250, 500,750,1000,1500μmol/L)處理HepG2細胞,每個濃度設5個重復.24h后,小心去除培養基,每孔加100μL新鮮培養基,同時加10μL MTT(0.5mg/mL), 37°C孵育4h.用150μL二甲基亞砜溶解沉淀物,并在570nm用酶標儀讀數.

1.3 [C8mim]Cl處理

細胞轉接到6孔板中生長12h,用[C8mim]Cl(0, 50, 100, 200μmol/L)處理細胞24h,收集細胞進行以下生化分析.

1.4 細胞凋亡測定

細胞凋亡測定參考Li等[14]操作進行.主要步驟如下:收取細胞,用PBS沖洗,在Binding buffer中重新懸浮,加Annexin V-異硫氰酸熒光素(Annexin V –FITC)和碘化丙啶(Propidium iodide,PI)后室溫避光染色15min,流式細胞儀檢測.

1.5 Western blot

Western blot試驗過程按照卜玲玲等[18]和Feng等[19]操作進行.主要步驟如下:用含有蛋白酶和磷酸酶抑制劑的細胞裂解液提取總蛋白.BCA蛋白定量試劑盒對提取的總蛋白進行定量后加雙色蛋白上樣緩沖液,100℃下煮沸5min.通過SDS-PAGE分離變性蛋白質并轉移到PVDF膜上.加相應的一抗(PERK:1:1000稀釋;p-PERK:1:600稀釋;eIF2α:1:500稀釋;p-eIF2α:1:1000稀釋;GRP78:1:500稀釋; CHOP:1:1000稀釋;p-IRE1:1:2000稀釋;IRE1:1:2000稀釋;XBP1:1:1500稀釋;ATF4:1:500稀釋;ATF6:1: 1000稀釋;caspase 4:1:1000稀釋;GAPDH:1:500),置4℃孵育過夜,TBST清洗3次,加二抗(1:2000稀釋)室溫孵育1h.TBST清洗3次后加ECL化學發光試劑,經ChemiDoc-MP成像系統(Bio-Rad)成像,并使用ImageJ軟件進行灰度掃描.

1.6 caspase酶活性測定

caspase 3和9活性測定按照試劑盒說明書進行操作.試驗過程參考Li等[14]步驟.

1.7 數據分析

使用SPSS 23.0軟件對數據進行方差分析,然后進行Dunnett-t檢驗,數值以平均值±標準差(SD)表示.*代表處理組和對照組之間差異顯著(*<0.05, **<0.01).

2 結果與分析

2.1 [C8mim]Cl對細胞活力的影響

圖1 [C8mim]Cl對HepG2細胞活力的影響

如圖1所示,[C8mim]Cl處理后細胞活力顯著降低,說明[C8mim]Cl影響HepG2細胞的生長.根據試驗獲得[C8mim]Cl對HepG2細胞的24h IC50為1051.0(944.1～1202.3)μmol/L.

2.2 [C8mim]Cl對HepG2細胞凋亡的影響

如圖2所示,流式細胞儀檢測結果顯示50, 100, 200μmol/L [C8mim]Cl處理HepG2細胞后凋亡細胞從4.35%升高到11.26%、20.76%和52.9%,表明[C8mim]Cl誘導的細胞凋亡呈現濃度依賴性.

2.3 [C8mim]Cl 對內質網應激相關蛋白的影響

如圖3所示,GRP78、CHOP和caspase 4作為ERS相關的生物標志物,在[C8mim]Cl處理24h后顯著增加.此外,[C8mim]Cl處理顯著增加p-eIF2α和p-IRE1的表達;200μmol/L [C8mim] Cl處理后,p-PERK、ATF4和ATF6水平也顯著增加(圖4).然而,在[C8mim]Cl處理組中, PERK、eIF2α、IRE1和XBP1的蛋白質水平普遍降低(圖4).表明ERS參與了[C8mim]Cl介導的細胞凋亡.

圖3 [C8mim]Cl對HepG2細胞GRP78、CHOP和caspase 4蛋白的影響

2.4 [C8mim]Cl對caspase 3和9酶活性的影響

如圖5所示,[C8mim]Cl處理可顯著促進HepG2細胞caspase 3和caspase 9的活性,提示caspase 3和caspase 9可能在[C8mim]Cl引起的細胞凋亡中起重要作用.

3 討論

研究表明,ILs能顯著抑制體外細胞的細胞活力,包括PC12[9],Hela[20],CCO[21],9[22]和Caco-2細胞株[23].本文發現[C8mim]Cl也顯著降低了HepG2細胞活力,表明離子液體暴露對細胞有毒害作用.

細胞凋亡是一種受到嚴格控制的細胞死亡形式,在調節許多細胞生物學過程、維持正常發育和組織內環境穩定方面起關鍵作用[24].越來越多的證據表明,誘導細胞凋亡是許多環境毒物細胞毒性的重要機制[25-27].Ma等[12]研究發現,離子液體[C8mim]Br可激活線粒體通路,誘導細胞凋亡,表明細胞凋亡可能是離子液體的細胞毒性機制之一.本文中,流式細胞檢測也證實[C8mim]Cl誘導了細胞凋亡.然而,ILs誘導細胞凋亡的通路還需深入研究.近年來,ERS介導的細胞凋亡受到越來越多的關注[28-29].ILs是否會通過激活ERS來誘導細胞凋亡目前還未見報道.

GRP78是ER定位的伴侶蛋白,與PERK、IRE1和ATF6等跨膜蛋白結合,在正常生理條件下保持非活性狀態.內質網應激發生時,其結合態解離,GRP78表達水平升高,3種跨膜蛋白(ATF6、PERK和IRE1)被激活,啟動內質網介導的UPR,防止蛋白質聚集的形成和驅動錯誤折疊的蛋白質降解來維持內質網的內穩態[30].因此,GRP78被用作內質網應激的指標[31-32].在本文中,[C8mim]Cl暴露可顯著提高GRP78蛋白水平,提示[C8mim]Cl可能破壞HepG2細胞內質網穩態,誘導內質網應激產生.

CHOP是C/EBP家族轉錄因子.研究表明, CHOP過度表達加劇了人肺癌來源的RERF-LC Ad2細胞和腎小管上皮細胞的凋亡,而敲除CHOP則抑制了內質網應激誘導的凋亡[33-34].因此,CHOP在ERS介導的細胞凋亡中起著重要作用,已被廣泛認為是內質網應激的生物標志物[30,35].本試驗發現[C8mim]Cl暴露后HepG2細胞中CHOP表達顯著增加,說明CHOP參與了[C8mim]Cl誘導的細胞凋亡過程.

PERK-eIF2α-ATF4軸是內質網應激誘導CHOP表達的主要途徑[14].在UPR條件下,PERK通過自身磷酸化激活,然后磷酸化eIF2α的α亞單位,從而通過抑制一般蛋白翻譯來降低內質網應激.然而,磷酸化的eIF2α也可以激活ATF4[14].同時,ATF6與GRP78分離后轉移到高爾基體,被切割為有活性的ATF6[13].ATF4和 ATF6又轉移到細胞核并調節內質網應激反應基因的表達,包括GRP78和CHOP,這是內質網應激介導的凋亡途徑中的一個重要通路[36].本文發現[C8mim]Cl處理后,HepG2細胞的PERK和eIF2α顯著降低,而磷酸化PERK和eIF2α升高.此外,在[C8mim]Cl處理的細胞中,ATF4和ATF6也顯著增加.因此,我們推測PERK和ATF6可能通過激活其下游成分CHOP參與[C8mim]Cl誘導的凋亡.

和GRP78解離后,激活的IRE1α催化XBP-1剪接,以誘導多個參與UPR基因的表達,以恢復內質網穩態[13-14].在本文中,p-IRE1α顯著增加,而剪接的XBP-1在[C8mim]Cl處理24h后顯著下調,表明[C8mim]Cl暴露可能破壞了XBP-1介導的保護途徑.研究表明IRE1可招募ASK1和TRAF2,形成IRE1- TRAF2-ASK1復合物后激活JNK途徑誘導細胞凋亡[37].因此,需要進一步研究IRE1α相關信號通路在[C8mim]Cl誘導細胞凋亡中的作用.

Caspase家族負責調控許多環境毒物介導的細胞凋亡[38].小鼠caspase-12是一個內質網駐留的caspase家族成員,對caspase-12缺陷小鼠的研究結果表明,它在內質網應激介導的細胞凋亡中被特異性激活[39].但是,人類caspase-12蛋白在進化過程中由于若干突變而處于失活狀態,在內質網應激誘導的細胞凋亡中不起作用[40].然而,先前的研究表明,人類caspase-4在內質網應激誘導的細胞死亡途徑中與小鼠caspase 12具有相同的功能[17].同時,caspase 12可通過caspase 9/caspase 3途徑誘導細胞凋亡[41].本研究發現,caspase 4蛋白在[C8mim]Cl處理后顯著上調,caspase 9和caspase 3活性也顯著升高,提示[C8mim]Cl 能夠激活caspase 4,可能通過caspase9/ caspase-3通路在凋亡誘導中發揮作用.

4 結論

4.1 [C8mim]Cl處理可誘導HepG2細胞凋亡.

4.2 [C8mim]Cl處理能激活HepG2細胞PERK、IRE1和ATF6信號通路, 誘導細胞UPR反應.

4.3 CHOP和caspase 4信號通路在[C8mim]Cl誘導的HepG2細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

張榜軍(1978-),男,河南林州人,講師,博士,研究方向為環境毒理學.發表論文20余篇.

2020-11-20

河南省高等學校重點科研項目(19zx011);河南師范大學博士啟動經費資助項目(qd16147)

* 責任作者, 教授, lixiaoyu65@263.net