濕熱應(yīng)激誘導(dǎo)小鼠心肌細(xì)胞凋亡的機(jī)制

王曉武，袁彬彬，林 曦，王顯悅，董文鵬，楊永超，張衛(wèi)達(dá)

(廣州軍區(qū) 廣州總醫(yī)院 心血管外科中心，廣東 廣州 510010)

濕熱應(yīng)激誘導(dǎo)小鼠心肌細(xì)胞凋亡的機(jī)制

王曉武，袁彬彬，林 曦，王顯悅，董文鵬，楊永超，張衛(wèi)達(dá)*

(廣州軍區(qū) 廣州總醫(yī)院 心血管外科中心，廣東 廣州 510010)

目的探討濕熱應(yīng)激誘導(dǎo)心肌細(xì)胞凋亡的作用機(jī)制。方法構(gòu)建濕熱應(yīng)激小鼠模型，分為濕熱應(yīng)激組(42 ℃，RH 65%)(H組)和對(duì)照組(C組)；TUNEL法原位標(biāo)記凋亡的心肌細(xì)胞，EIISA檢測(cè)Ang Ⅱ的表達(dá)水平；體外培養(yǎng)小鼠心肌細(xì)胞，分別加入caspase-3抑制劑Z-DEVD-FMK和P38 MAPK抑制劑SB203580共培養(yǎng)24 h，隨后加入Ang Ⅱ共培養(yǎng)24 h；Annexin V-FITC凋亡試劑盒檢測(cè)細(xì)胞凋亡率；Western blot檢測(cè)P38 MAPK及caspase-3的表達(dá)水平。結(jié)果濕熱應(yīng)激條件導(dǎo)致小鼠心肌細(xì)胞凋亡率明顯高于常規(guī)組，且伴隨有大量Ang Ⅱ的生成(Plt;0.05)；體外實(shí)驗(yàn)證實(shí)，Ang Ⅱ能夠劑量依賴性的誘導(dǎo)心肌細(xì)胞凋亡，同時(shí)誘導(dǎo)caspase-3和P38 MAPK的活化；Z-DEVD-FMK預(yù)處理明顯抑制Ang Ⅱ誘導(dǎo)的心肌細(xì)胞凋亡，表明Ang Ⅱ主要通過(guò)caspase-3活化途徑來(lái)誘導(dǎo)心肌細(xì)胞凋亡；此外，SB203580可抑制Ang Ⅱ誘導(dǎo)的caspase-3的表達(dá)。結(jié)論濕熱應(yīng)激誘發(fā)心肌細(xì)胞凋亡主要是通過(guò)Ang Ⅱ誘導(dǎo)的P38 MAPK-caspase-3通路來(lái)實(shí)現(xiàn)的。

濕熱應(yīng)激；血管緊張素Ⅱ；細(xì)胞凋亡；P38 MAPK；caspase-3

高濕熱環(huán)境對(duì)機(jī)體各個(gè)系統(tǒng)均有不同程度的損害，極端濕熱環(huán)境可以導(dǎo)致某些蛋白結(jié)構(gòu)和功能改變，導(dǎo)致體能減弱甚至死亡，也是誘發(fā)心血管疾病的重要原因[1-2]。研究顯示，濕熱應(yīng)激能夠?qū)е滦募〖?xì)胞凋亡，且在溫度升高過(guò)程中細(xì)胞凋亡速率增加[3]。

細(xì)胞生長(zhǎng)、分裂、衰老、凋亡等改變受多種因素影響，血管緊張素Ⅱ(AngⅡ)在影響細(xì)胞改變的因素中占最主要地位。越來(lái)越多的證據(jù)表明，濕熱應(yīng)激條件可能引發(fā)腎素-血管緊張素系統(tǒng)活性被激活，進(jìn)而導(dǎo)致心肌細(xì)胞凋亡[4, 5]。但是，濕熱應(yīng)激導(dǎo)致心肌細(xì)胞凋亡的分子機(jī)制目前尚不清楚。

本實(shí)驗(yàn)通過(guò)研究濕熱應(yīng)激誘導(dǎo)小鼠心肌細(xì)胞凋亡的機(jī)理，為探討高濕熱環(huán)境下心肌細(xì)胞凋亡分子機(jī)制的研究奠定基礎(chǔ)，同時(shí)為高濕熱環(huán)境下作業(yè)人群心臟功能的防護(hù)提供理論依據(jù)。

1 材料與方法

1.1 材料

清潔級(jí)BALB/c小鼠60只 (5月齡)，體質(zhì)量20～23 g，雌雄不限，由南方醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供 (合格證號(hào)：2005A050)。Annexin V-FITC凋亡試劑盒(南京凱基生物有限公司)，小鼠Ang Ⅱ EIISA酶聯(lián)免疫試劑(自上海研卉生物科技有限公司)，TUNEL(Roche公司)，DMEM培養(yǎng)基(上海高創(chuàng)化學(xué)科技有限公司)，兔抗鼠P38 MAPK、P-P38 MAPK和caspase-3抗體(Bioworld公司)。

1.2 方法

1.2.1 熱應(yīng)激小鼠模型構(gòu)建：BALB/c小鼠60 只，隨機(jī)分為高溫組(42 ℃，RH 65%)(H組)、常溫組(C組)，各30只。置動(dòng)物于模擬氣候艙，H組小鼠置于高濕熱環(huán)境中；C組在室溫條件下進(jìn)行相同強(qiáng)度訓(xùn)練。兩組小鼠在培養(yǎng)期間，每天進(jìn)行高中等訓(xùn)練3次，每次30min，持續(xù)14 d，運(yùn)動(dòng)后自由進(jìn)食。

1.2.2 EIISA檢測(cè)Ang Ⅱ的表達(dá)水平： 腹腔注射50 mg/kg戊巴比妥鈉(3%)麻醉小鼠后，取出心臟，4 ℃預(yù)冷的0.9%氯化鈉注射液逆行灌洗主動(dòng)脈，然后用濾紙吸干。切取左室心尖部分心肌組織3塊。其中2塊分別編號(hào)放入-70 ℃冰箱保存待測(cè)，另外一塊在處理干凈后快速冷凍，切碎，然后加入冰乙酸(1 mol/L)靜置過(guò)夜，4 ℃，15 000r/min離心40 min，取上清。加入乙醚振蕩混勻后繼續(xù)蒸干，加入等量PBS完全溶解后，用EIISA試劑盒測(cè)定Ang Ⅱ濃度。具體操作步驟按照操作說(shuō)明，簡(jiǎn)述為甲醇洗脫，蒸發(fā)后，用100 μL EIA稀釋兩遍，加入96孔板37 ℃孵育1 h，棄去上清，洗滌后加入IgG避光孵育6～8 h，再次棄去上清，洗滌后加入Ellman氏試劑，避光靜置1 h，用分光光度儀405 nm波長(zhǎng)測(cè)定Ang Ⅱ含量，1式3份。

1.2.3 TUNEL染色：取樣操作同1.2.2，用4%多聚甲醛室溫固定40 min，PBS清洗后70%乙醇在-20 ℃條件下靜置1 h，PBS清洗后加入3% H2O2的甲醇室溫放10 min，PBS清洗后置于1% Triton X-100+0.1%檸檬酸鈉4 ℃靜置3 min，PBS洗滌后加入TUNEL反應(yīng)混合物，37 ℃放置90 min，PBS洗后加入POD 37 ℃放置40 min，PBS清洗后DAB 100 μL室溫靜置10 min，加入蘇木精進(jìn)行對(duì)比染色2 min，得到棕色沉淀， 100倍顯微鏡下分析結(jié)果。

1.2.4 心肌細(xì)胞培養(yǎng)及處理：取出生半天的小鼠，無(wú)菌操作取出心臟，迅速放入D-Hanks溶液中，剪取心尖部分的心室組織，用預(yù)冷的0.9%氯化鈉注射液清洗，除去血污。剪碎后加入消化液(0.1%分散酶、0.05%胰蛋白酶、0.1% Ⅱ型膠原蛋白酶)，37 ℃振蕩培養(yǎng)消化15 min。棄上清重復(fù)3次。離心棄上清，沉淀細(xì)胞重懸計(jì)數(shù)后，培養(yǎng)于含血清的培養(yǎng)液(DMEM培養(yǎng)基10%馬血清，5%的胎牛血清)，差速貼壁的方法富集心肌細(xì)胞。計(jì)數(shù)后按照細(xì)胞1×105個(gè)/mL濃度，每皿2 L細(xì)胞懸液培養(yǎng)于培養(yǎng)皿。原代培養(yǎng)心肌細(xì)胞，每3孔為一組接種于24孔板中，每孔細(xì)胞約為1×108個(gè)/mL。將細(xì)胞隨機(jī)分為3組：A組，用分別含有1×10-6、1×10-7和1×10-8mol/L濃度梯度 Ang Ⅱ的DNEM培養(yǎng)液培養(yǎng)；B組，1×10-6mol/L Ang Ⅱ和SB203580處理組；C組，1×10-6mol/L Ang Ⅱ和Z-DEVD-FMK處理組；DNEM培養(yǎng)液加DMSO作為對(duì)照。均培養(yǎng)24 h后觀察結(jié)果。

1.2.5 AnnexinV-FITC檢測(cè)細(xì)胞凋亡，按照說(shuō)明書操作。

1.2.6 Western blot：提取各組細(xì)胞總蛋白：棄培養(yǎng)基后，用4 ℃預(yù)冷的PBS緩沖液洗滌細(xì)胞，洗2次后加入200 μL預(yù)冷的裂解液，冰上放置20 min，4 ℃，12 000r/min離心10 min，取上清測(cè)即為總蛋白。200 μg總蛋白加入上樣緩沖液總體積至30 μL，樣品處理后進(jìn)行SDS-PAGE電泳。電泳結(jié)束后，采用半干轉(zhuǎn)法將蛋白轉(zhuǎn)移至PVDF膜上，用5%脫脂奶粉4 ℃過(guò)夜孵育，TBST洗滌3次后加一抗37 ℃孵育1 h，再次用TBST洗3次，加入二抗。ECL底物顯色，紅外線燈照射顯影。

1.3 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1濕熱應(yīng)激對(duì)心肌細(xì)胞凋亡率及小鼠AngⅡ蛋白表達(dá)水平的影響

與對(duì)照組相比，H組誘導(dǎo)檢測(cè)到大量棕色沉淀信號(hào)(圖1)。濕熱應(yīng)激條件下Ang Ⅱ蛋白含量約為3 500 pg/mL，對(duì)照組約為1 400 pg/mL。

圖1 濕熱應(yīng)激對(duì)小鼠心肌細(xì)胞凋亡的影響Fig 1 The role of damp-heat on the myocardial apoptosis (×100)

2.2 Ang Ⅱ?qū)π募〖?xì)胞凋亡的影響

Ang Ⅱ能夠誘導(dǎo)大量陽(yáng)性信號(hào)的產(chǎn)生(圖2A)。定量分析數(shù)據(jù)顯示，Ang Ⅱ 濃度為1×10-7和1×10-6mol/L時(shí)誘導(dǎo)的心肌細(xì)胞凋亡率顯著高于對(duì)照組 (Plt;0.05)(圖2B)。

A.apoptosis of myocardial cell detected by Annexin V; B.quantitative analysis of myocardial cell apoptosis rate； *Plt;0.05, **Plt;0.01 compared with control圖2 Ang Ⅱ預(yù)處理對(duì)心肌細(xì)胞的影響Fig 2 The role of Ang Ⅱ on apoptosis of myocardial cell

2.3AngⅡ通過(guò)活化caspase-3誘導(dǎo)心肌細(xì)胞凋亡

Ang Ⅱ誘導(dǎo)心肌細(xì)胞caspase-3蛋白大量活化(圖3A)。Caspase-3抑制劑Z-DEVD-FMK預(yù)處理后Ang Ⅱ誘導(dǎo)的細(xì)胞凋亡率明顯低于對(duì)照組(圖3B)。

2.4AngⅡ通過(guò)P38MAPK信號(hào)通路誘導(dǎo)caspase-3的活化

Ang Ⅱ明顯誘導(dǎo)心肌細(xì)胞P38 MAPK的活化(圖4A)，Ang Ⅱ蛋白表達(dá)量與對(duì)照組相比約為1.7倍(圖4B)。此外，P38 MAPK抑制劑SB203580預(yù)處理后，caspase-3的表達(dá)下降(圖4C)，約為對(duì)照組的0.3倍(圖4D)。

3 討論

高濕熱能誘導(dǎo)多種細(xì)胞凋亡，對(duì)于在高濕熱環(huán)境工作人員健康造成極大威脅。本實(shí)驗(yàn)通過(guò)構(gòu)建濕熱應(yīng)激小鼠模型，檢測(cè)心肌細(xì)胞凋亡率，發(fā)現(xiàn)濕熱應(yīng)激誘導(dǎo)小鼠心肌細(xì)胞大量凋亡，同時(shí)伴有Ang Ⅱ蛋白的大量表達(dá)。Ang Ⅱ在細(xì)胞血管平滑肌細(xì)胞(VSMC)生長(zhǎng)、凋亡、細(xì)胞遷移、炎性反應(yīng)和纖維化等多種變化中占主要地位[6-7]，但是， Ang Ⅱ是否參與濕熱應(yīng)激誘導(dǎo)的心肌細(xì)胞凋亡及其分子機(jī)制尚不清楚。

*Plt;0.05 compared with control; #Plt;0.05 compared with Ang Ⅱ圖3 Ang Ⅱ通過(guò)活化caspase-3誘導(dǎo)心肌細(xì)胞凋亡Fig 3 Ang Ⅱ induced cardiomyocytes apoptosis by activating caspase-3

進(jìn)一步體外實(shí)驗(yàn)證實(shí)，Ang Ⅱ能夠劑量依賴的誘導(dǎo)小鼠心肌細(xì)胞大量凋亡。Caspase-3是多種細(xì)胞凋亡途徑中重要的效應(yīng)蛋白酶，是細(xì)胞凋亡的主要執(zhí)行者[8]。Caspase-3與染色體凝聚和DNA片斷化等細(xì)胞凋亡的特征性標(biāo)志均密切相關(guān)，同時(shí)是caspase家族介導(dǎo)的細(xì)胞凋亡的線粒體通路和死亡受體通路的交匯點(diǎn)[8]。本研究發(fā)現(xiàn)，心肌細(xì)胞與AngⅡ共培養(yǎng)可誘導(dǎo)caspase-3蛋白的活化，caspase-3抑制劑Z-DEVD-FMK預(yù)處理能夠顯著降低AngⅡ誘導(dǎo)的細(xì)胞凋亡，表明AngⅡ可通過(guò)誘導(dǎo)caspase-3蛋白的活化介導(dǎo)心肌細(xì)胞凋亡。P38 MAPK通路是MAPK信號(hào)通路的一種，在細(xì)胞多種信號(hào)傳導(dǎo)通路中起重要作，用參與應(yīng)激介導(dǎo)的細(xì)胞凋亡反應(yīng)[9-10]。進(jìn)一步機(jī)制分析表明，Ang Ⅱ 可誘導(dǎo)P38 MAPK通路的活化，加入P38 MAPK抑制劑SB203580后caspase-3的表達(dá)明顯降低，表明濕熱應(yīng)激誘導(dǎo)心肌細(xì)胞凋亡主要是通過(guò)Ang Ⅱ 誘導(dǎo)的P38 MAPK-caspase-3通路來(lái)實(shí)現(xiàn)的，提示Ang Ⅱ 可作為預(yù)防和治療濕熱環(huán)境誘導(dǎo)的心血管疾病的潛在靶標(biāo)，對(duì)高濕熱環(huán)境作業(yè)的人群心臟功能的防護(hù)具有重要意義。

A.the role of Ang Ⅱ on the expression of P38; B.the role of SB203580 on the expression of caspase-3; *Plt;0.05 compared with control圖4 Ang Ⅱ通過(guò)P38MAPK信號(hào)通路誘導(dǎo)caspase-3的活化Fig 4 Ang Ⅱ activate caspase-3 by way of P38 MAPK

此外，有研究表明Ang Ⅱ可通過(guò)NADPH誘導(dǎo)的活性氧(ROS)的生成誘導(dǎo)多種血管效應(yīng)[11]。一方面ROS可通過(guò)改變細(xì)胞內(nèi)氧化還原狀態(tài)和對(duì)蛋白質(zhì)的氧化修飾影響甚至損害細(xì)胞膜、線粒體膜和DNA等的結(jié)構(gòu)和功能，激發(fā) P38MAPK等信號(hào)通路活化促使細(xì)胞凋亡；另一方面過(guò)量的ROS可作為信號(hào)分子，并通過(guò)信號(hào)級(jí)聯(lián)反應(yīng)，參與并調(diào)控細(xì)胞凋亡程序的啟動(dòng)[12-13]。濕熱應(yīng)激條件下，Ang Ⅱ是否通過(guò)誘導(dǎo)ROS產(chǎn)生來(lái)調(diào)節(jié)P38 MAPK介導(dǎo)的細(xì)胞凋亡還有待于進(jìn)一步研究。

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Mechanism involved in damp-heat induced mice myocardial cell apoptosis

WANG Xiao-wu, YUAN Bin-bin, LIN Xi, WANG Xian-yue, DONG Wen-peng,YANG Yong-chao, ZHANG Wei-da*

( Centre of Cardiovascular Surgery, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, China)

ObjectiveTo explore the mechanism of damp-heat induced mice myocardial cell apoptosis.MethodsIn this study, mice was exposed to damp-heat (42 ℃, RH 65%) (H group) environment or room temperature (C group). The myocardial cell apoptosis rates in tissues were analyzed by TUNEL staining. The expression levels of Ang Ⅱ were detected by EIISA assay. Cardiomyocytes of rats with 1 day were pretreated with caspase-3 inhibitor Z-DEVD-FMK, or P38 MAPK inhibitor SB203580, for 24 h, followed by culturing with the indicated dose of Ang Ⅱ. AnnexinV-FITC was used to analyze cell apoptosis ratio. In addition, the expression of caspase-3 and P38 MAPK was assessed by Western blot.ResultsThe rates of cell apoptosis and Ang Ⅱ expression levels in H group were significantly higher than those in C group (Plt;0.05).Invitro, Ang Ⅱ dose-dependently induced cardiomyocytes apoptosis, accompany with the up-regulation of caspase-3 and P38 MAPK expression. When preconditioning with Z-DEVD-FMK, the apoptotic ratio of cardiomyocytes was significantly attenuated in Ang Ⅱ-treated group, implying that Ang Ⅱ triggered cell apoptosis in caspase-3-depedent manner. Additionally, pretreatment with SB203580 dramatically abrogated caspase-3 expression induced by Ang Ⅱ.ConclusionsDamp-heat environment induced cardiomyocytes apoptosis by Ang Ⅱ-activated P38 MAPK-caspase-3 pathway. Consequently, Ang Ⅱ may be a potential target for innovative strategies against cardiovascular diseases induced by Damp-heat environment.

damp-heat stress; angiotensin Ⅱ; apoptosis; P38 MAPK; caspase-3

2013-08-21

2013-12-23

*通信作者(correspondingauthor)： weidazhanggz@163.com

1001-6325(2014)04-0531-05

研究論文

R 852.51

A