楊羽菲，趙叢，楊平珍，王先寶，鄧意，陳愛華

(南方醫(yī)科大學(xué)珠江醫(yī)院，廣州510280)

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·論著·

治療性低溫對(duì)缺氧期心肌細(xì)胞自噬的影響及機(jī)制

楊羽菲，趙叢，楊平珍，王先寶，鄧意，陳愛華

(南方醫(yī)科大學(xué)珠江醫(yī)院，廣州510280)

目的觀察治療性低溫(TH)在缺氧期對(duì)心肌細(xì)胞自噬活性及自噬流的影響，并探討其作用機(jī)制。方法取大鼠心肌細(xì)胞株H9c2，分為對(duì)照組、缺氧組、TH干預(yù)組、雷帕霉素干預(yù)組、雷帕霉素+TH干預(yù)組，對(duì)照組置于37 ℃培養(yǎng)箱中培養(yǎng)，不作處理；缺氧組和TH干預(yù)組構(gòu)建缺氧模型后，分別置于37、32 ℃培養(yǎng)箱中培養(yǎng)；雷帕霉素干預(yù)組和雷帕霉素+TH干預(yù)組于構(gòu)建模型前2 h加入雷帕霉素0.1 μmol/L，構(gòu)建模型后分別置于37、32 ℃培養(yǎng)箱中培養(yǎng)。采用Western blot法檢測(cè)對(duì)照組、缺氧組和TH干預(yù)組自噬相關(guān)蛋白自噬微管相關(guān)蛋白輕鏈3B(LC3B)、p62、Beclin-1蛋白表達(dá)，以及應(yīng)用雷帕霉素前后哺乳動(dòng)物雷帕霉素靶蛋白(mTOR)、核糖體蛋白S6(S6)的表達(dá)差異；采用腺病毒GFP-mRFP-LC3熒光瞬時(shí)轉(zhuǎn)染技術(shù)檢測(cè)自噬流。結(jié)果①對(duì)照組、缺氧組和TH干預(yù)組LC3B、p62、Beclin-1蛋白表達(dá)：缺氧組和TH干預(yù)組LC3B、Beclin-1高于對(duì)照組(P均<0.05)，缺氧組p62降低(P<0.05)。TH干預(yù)組LC3B明顯低于缺氧組，p62增高(P均<0.05)。缺氧組與TH干預(yù)組Beclin-1無統(tǒng)計(jì)學(xué)差異。②雷帕霉素處理后自噬蛋白LC3B、p62及mTOR通路相關(guān)蛋白p-mTOR、p-S6表達(dá)：TH干預(yù)組與缺氧組比較LC3B降低，而p62、p-mTOR和p-S6均升高(P均<0.05)；雷帕霉素干預(yù)組與缺氧組比較LC3B升高，p62降低(P均<0.05)，而p-mTOR和p-S6差異無統(tǒng)計(jì)學(xué)意義；雷帕霉素+TH干預(yù)組與TH干預(yù)組比較LC3B升高，p62、p-mTOR和p-S6均降低，結(jié)果均有統(tǒng)計(jì)學(xué)差異(P均<0.05)；雷帕霉素+TH干預(yù)組與雷帕霉素干預(yù)組比較LC3B降低，p62升高(P均<0.05)，而p-mTOR和p-S6差異無統(tǒng)計(jì)學(xué)意義。③雙熒光自噬流：缺氧組和TH干預(yù)組的黃點(diǎn)和紅點(diǎn)數(shù)多于對(duì)照組(P均<0.05)，TH干預(yù)組的黃點(diǎn)與紅點(diǎn)數(shù)少于缺氧組(P均<0.05)。結(jié)論 TH能夠抑制缺氧期心肌細(xì)胞自噬，其機(jī)制可能與促進(jìn)mTOR信號(hào)通路相關(guān)蛋白表達(dá)有關(guān)。

治療性低溫；心肌細(xì)胞；缺氧；自噬；雷帕霉素

治療性低溫(TH)能夠減少活性氧族(ROS)產(chǎn)生，改善細(xì)胞內(nèi)離子水平，保持細(xì)胞內(nèi)pH平衡[1～3]，影響細(xì)胞能量代謝，減輕細(xì)胞損害，對(duì)缺氧缺血的心肌細(xì)胞有一定的保護(hù)作用，是臨床治療缺血性疾病的有效方法。自噬是指細(xì)胞質(zhì)內(nèi)大分子物質(zhì)被包裹并傳遞至溶酶體進(jìn)行降解，維持細(xì)胞內(nèi)的物質(zhì)與能量平衡。研究認(rèn)為，自噬是心肌細(xì)胞存活與否的關(guān)鍵調(diào)節(jié)因素之一。雷帕霉素靶蛋白(mTOR)是在哺乳動(dòng)物細(xì)胞中負(fù)向調(diào)控自噬的關(guān)鍵蛋白，其活化對(duì)自噬具有抑制作用[4]。mTOR通路是缺血期調(diào)控自噬的兩條經(jīng)典通路之一。磷酸化的mTOR(p-mTOR)是mTOR通路活化的標(biāo)志。核糖體蛋白6(S6)是mTOR下游的一個(gè)效應(yīng)蛋白，磷酸化S6(p-S6)被認(rèn)為是mTOR激活程度的重要標(biāo)記蛋白。2014年1月～2016年1月，我們觀察了TH在大鼠心肌細(xì)胞缺氧期對(duì)H9c2細(xì)胞自噬活性相關(guān)蛋白[自噬微管相關(guān)蛋白輕鏈3B(LC3B)、p62及Beclin-1]表達(dá)的影響，并采用雷帕霉素抑制mTOR信號(hào)通路，觀察心肌細(xì)胞自噬活性相關(guān)蛋白和mTOR通路相關(guān)蛋白p-mTOR、p-S6的變化，探討TH治療心肌細(xì)胞缺氧損傷的作用機(jī)制。

1　材料與方法

1.1材料大鼠心肌細(xì)胞株H9c2購自美國ATCC公司。自噬蛋白LC3B、p62、Beclin-1相關(guān)一抗、mTOR信號(hào)通路相關(guān)一抗購于美國CST公司，相關(guān)二抗購于武漢博士德公司。雷帕霉素購于美國Sigma-Aldrich公司。

1.2心肌細(xì)胞自噬活性檢測(cè)采用Western blot法。將大鼠心肌細(xì)胞置于含10%胎牛血清的高糖DMEM培養(yǎng)基中，5% CO2、37 ℃培養(yǎng)箱中培養(yǎng)。細(xì)胞融合至70%～90%后用于實(shí)驗(yàn)。將細(xì)胞分為對(duì)照組、缺氧組、TH干預(yù)組,對(duì)照組置于37 ℃培養(yǎng)箱中培養(yǎng)，不作處理；其余兩組向細(xì)胞中加入PBS，移入細(xì)胞缺氧盒，注入含5% CO2和95% N2的混合氣體構(gòu)建缺氧模型，分別置于37、32 ℃培養(yǎng)箱中培養(yǎng)。向細(xì)胞懸液中加入蛋白裂解液，收集上清，加入15% SDS-PAGE分離蛋白，將凝膠蛋白轉(zhuǎn)至PVDF膜上，室溫封閉2 h，加入相應(yīng)的一抗、二抗，4 ℃過夜孵育。以ECL試劑為底物，X線曝光，顯色、定影。采用Quantity One 4.6軟件對(duì)發(fā)光條帶的灰度值進(jìn)行分析，以β-actin作為內(nèi)參，計(jì)算LC3B、p62、Beclin-1蛋白的相對(duì)表達(dá)量。實(shí)驗(yàn)重復(fù)3次。

1.3雷帕霉素對(duì)心肌細(xì)胞自噬及mTOR通路蛋白的影響觀察將細(xì)胞分為缺氧組、TH干預(yù)組、雷帕霉素干預(yù)組、雷帕霉素+TH干預(yù)組。缺氧組、TH干預(yù)組處理同1.2，雷帕霉素干預(yù)組與雷帕霉素+TH干預(yù)組于缺氧造模前2 h加入雷帕霉素0.1 μmol/L，造模后分別置37、32 ℃培養(yǎng)箱中培養(yǎng)。采用Western blot法檢測(cè)LC3B、p62及p-mTOR和p-S6蛋白表達(dá)，加入相應(yīng)抗體，計(jì)算其相對(duì)表達(dá)量，操作及計(jì)算方法參照1.2。

1.4心肌細(xì)胞自噬流檢測(cè)采用GFP-mRFP-LC3熒光瞬時(shí)轉(zhuǎn)染技術(shù)。用腺病毒GFP-mRFP-LC3瞬時(shí)轉(zhuǎn)染心肌細(xì)胞，培養(yǎng)36 h后分為對(duì)照組、缺氧組、TH干預(yù)組，分別處理3 h。由于GFP 綠色熒光和mRFP 紅色熒光在自噬體同時(shí)存在，在紅、綠熒光合成圖像中呈黃色；但在自噬溶酶體酸性環(huán)境中GFP 綠色熒光降解，只剩下紅色熒光。以合成圖像中的黃色亮點(diǎn)(自噬體)和紅色亮點(diǎn)(自噬溶酶體)數(shù)量反映細(xì)胞內(nèi)自噬囊泡的數(shù)量。分組處理細(xì)胞后在400倍熒光顯微鏡下觀察，隨機(jī)采集300 個(gè)細(xì)胞內(nèi)的紅、綠色LC3 熒光圖，計(jì)數(shù)其合成圖像中平均每個(gè)細(xì)胞含有的黃色亮點(diǎn)和紅色亮點(diǎn)的個(gè)數(shù)。實(shí)驗(yàn)重復(fù)3次。

2　結(jié)果

2.1各組LC3B、p62、Beclin-1蛋白相對(duì)表達(dá)量比較缺氧組和TH干預(yù)組LC3B、Beclin-1高于對(duì)照組(P均<0.05)，缺氧組p62降低(P<0.05)。TH干預(yù)組LC3B低于缺氧組，p62增高(P均<0.05)。缺氧組與TH干預(yù)組Beclin-1無統(tǒng)計(jì)學(xué)差異。見表1。

表1　各組LC3B、p62和Beclin-1蛋白相對(duì)表達(dá)量比較±s)

注：與對(duì)照組比較，*P<0.05；與缺氧組比較，#P<0.05。

2.2雷帕霉素處理后各組LC3B、p62、p-mTOR、p-S6蛋白相對(duì)表達(dá)量比較TH干預(yù)組與缺氧組比較LC3B降低，而p62、p-mTOR和p-S6均升高(P均<0.05)；雷帕霉素干預(yù)組與缺氧組比較LC3B升高，p62降低(P均<0.05)，而p-mTOR和p-S6差異無統(tǒng)計(jì)學(xué)意義；雷帕霉素+TH干預(yù)組與TH干預(yù)組比較LC3B升高，p62、p-mTOR和p-S6均降低，結(jié)果均有統(tǒng)計(jì)學(xué)差異(P均<0.05)；雷帕霉素+TH干預(yù)組與雷帕霉素干預(yù)組比較LC3B降低，p62升高(P均<0.05)，而p-mTOR和p-S6差異無統(tǒng)計(jì)學(xué)意義。見表2。

2.3各組細(xì)胞雙熒光自噬流檢測(cè)結(jié)果缺氧組和TH干預(yù)組的黃點(diǎn)和紅點(diǎn)多于對(duì)照組(P均<0.05)，TH干預(yù)組的黃點(diǎn)與紅點(diǎn)少于缺氧組(P均<0.05)。見表3。

3　討論

細(xì)胞自噬是指在細(xì)胞內(nèi)通過形成雙層脂質(zhì)膜包裹細(xì)胞內(nèi)細(xì)胞器或細(xì)胞內(nèi)物質(zhì)(自噬小體)，隨之與溶酶體結(jié)合并降解的過程[5]。LC3B是自噬體膜上的標(biāo)記蛋白,細(xì)胞內(nèi)LC3B蛋白的變化通常與自噬活性聯(lián)系密切，被廣泛用于自噬活性的研究[6]。另一個(gè)被廣泛應(yīng)用的自噬標(biāo)記蛋白p62，其降解程度能有效反映自噬活性[7]。Beclin-1是細(xì)胞自噬的一個(gè)關(guān)鍵調(diào)控因子，是自噬體形成不可或缺的條件[8]。研究顯示，TH是一種多重機(jī)制的治療方式，它對(duì)細(xì)胞的保護(hù)機(jī)制多與應(yīng)用TH的開始時(shí)間有關(guān)。缺血期應(yīng)用TH要優(yōu)于再灌注期，不僅可以減少缺血期產(chǎn)生的病理產(chǎn)物ROS等，還可能影響再灌注期間炎癥信號(hào)通路等[9]。盡早應(yīng)用TH可使機(jī)體獲益更多。TH能有效抑制缺血期H9c2細(xì)胞的壞死與凋亡[10]，缺血前或再灌注治療前應(yīng)用TH均能減少H9c2細(xì)胞凋亡。然而TH調(diào)控自噬及其相關(guān)機(jī)制少見報(bào)道。

表2　各組LC3B、p62、p-mTOR、p-S6蛋白相對(duì)表達(dá)量比較

注：與缺氧組比較，*P<0.05；與TH干預(yù)組比較，#P<0.05；與雷帕霉素干預(yù)組比較，△P<0.05。

表3　各組熒光點(diǎn)數(shù)比較(個(gè)，±s)

注：與對(duì)照組比較，*P<0.05；與缺氧組比較，#P<0.05。

近期文獻(xiàn)報(bào)道，在大鼠心肌頓抑模型中，TH能有效恢復(fù)神經(jīng)功能，并通過抑制自噬改善海馬旁回腦細(xì)胞活性[11]。另外，TH可以通過抑制凋亡、降低過度自噬修復(fù)缺血損傷的腦脊髓神經(jīng)功能[12,13]。在缺血再灌注損傷的心肌細(xì)胞中，TH改善細(xì)胞活性并下調(diào)自噬水平[14]。本研究表明，TH可降低缺氧期間H9c2心肌細(xì)胞自噬水平(降低LC3B、升高p62)，在缺氧損傷環(huán)境下表現(xiàn)出抑制細(xì)胞自噬活性。Beclin-1在自噬誘導(dǎo)的起始階段起重要作用[15]，但缺氧組與TH干預(yù)組相比，Beclin-1表達(dá)并無統(tǒng)計(jì)學(xué)差異。表明TH對(duì)自噬的影響并不依賴Beclin-1的表達(dá)，與以往報(bào)道[14]相符。

為了探討TH對(duì)自噬活性的調(diào)節(jié)機(jī)制，我們進(jìn)一步對(duì)mTOR信號(hào)通路進(jìn)行研究。mTOR信號(hào)通路的激活對(duì)細(xì)胞自噬活性具有負(fù)性調(diào)節(jié)作用[16]。作為mTOR信號(hào)通路的關(guān)鍵成員，mTOR和S6蛋白磷酸化與自噬的調(diào)控密切相關(guān)[17]。p-mTOR及p-S6表達(dá)可反映mTOR通路激活程度[18]，表達(dá)升高提示mTOR信號(hào)通路高度激活，并會(huì)對(duì)自噬活性造成顯著抑制[18]。而雷帕霉素作為自噬活性的經(jīng)典激活劑，可抑制mTOR信號(hào)通路，從而激活自噬。本研究顯示，TH能通過促進(jìn)mTOR和S6蛋白磷酸化來激活mTOR通路(使p-mTOR、p-S6表達(dá)升高)，并抑制缺氧期間H9c2的自噬水平(使LC3B降低、p62升高)。然而當(dāng)雷帕霉素作用于TH干預(yù)下的缺氧期H9c2細(xì)胞時(shí)，TH對(duì)自噬的抑制作用完全消失，取而代之的是自噬活性的強(qiáng)烈激活，并且雷帕霉素也逆轉(zhuǎn)了TH對(duì)p-mTOR和p-S6的促表達(dá)作用。簡(jiǎn)而言之，雷帕霉素的介入抑制了mTOR信號(hào)通路(p-mTOR、p-S6表達(dá)降低)，同時(shí)也強(qiáng)烈激活了原本受到TH抑制的自噬活性，說明TH或可通過激活mTOR信號(hào)通路抑制自噬活性。

自噬流是指自噬的完整過程，自噬小體的雙層膜結(jié)構(gòu)包裹部分胞質(zhì)和細(xì)胞內(nèi)需降解的細(xì)胞器、蛋白質(zhì)等形成自噬體，最后與溶酶體融合形成自噬溶酶體，降解其所包裹的內(nèi)容物，以實(shí)現(xiàn)細(xì)胞穩(wěn)態(tài)和細(xì)胞器的更新[19]。因此，較之以LC3B和p62蛋白表達(dá)來反映細(xì)胞自噬活性，自噬流的檢測(cè)更加直觀，可以更方便地對(duì)自噬形成過程的各個(gè)階段進(jìn)行分析解讀。本研究顯示，與對(duì)照組相比，缺氧組與TH處理組的黃色熒光點(diǎn)數(shù)、紅色熒光點(diǎn)數(shù)增多，提示缺氧促進(jìn)了H9c2心肌細(xì)胞自噬小體和自噬溶酶體的升高，而TH則部分減少了缺氧后H9c2心肌細(xì)胞自噬小體和自噬溶酶體，與缺氧組相比，TH處理組的自噬體、自噬溶酶體減少。說明TH能夠通過抑制缺氧期H9c2心肌細(xì)胞自噬小體的形成，從而抑制自噬流水平。

綜上所述，TH可通過mTOR相關(guān)信號(hào)通路對(duì)細(xì)胞自噬流進(jìn)行調(diào)控，抑制缺氧期H9c2心肌細(xì)胞的自噬流。此研究揭示了TH在心血管系統(tǒng)中又一新的機(jī)制，為缺血性心臟病的治療提供了理論依據(jù)。

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Effect and mechanism of therapeutic hypothermia on autophagy in hypoxia cardiomyocytes

YANGYufei,ZHAOCong,YANGPingzhen,WANGXianbao,DENGYi,CHENAihua

(ZhujiangHospital,SouthernMedicalUniversity,Guangzhou510280,China)

ObjectiveTo investigate the effects of therapeutic hypothermia (TH) on autophagy and autophagic flux of cardiomyocytes in ischemia and to explore the underlying mechanism. MethodsH9c2 rat cardiomyocytes were divided into the control group, hypoxia group, hypoxia + TH group, rapamycin + hypoxia group and rapamycin + hypoxia+ TH group. Cells in the control group were normally cultured at 37 ℃ and received no further treatments. Cells with or without TH treatment in the other four groups were subjected to hypoxia in a hypoxic chamber at 32 ℃ or 37 ℃ respectively. Then, 0.1 μmol/L rapamycin was added 2 h before cells were subjected to hypoxia at 32 ℃ or 37 ℃. Western blotting was performed to detect protein expression of LC3B, p62, Beclin-1, phospho-mTOR (p-mTOR) and phospho-S6 (p-S6). The autophagic flux was detected under adenovirus GFP-mRFP-LC3 confocal laser scanning microscopy.Results①The protein expression of LC3B, p62 and Beclin-1 in the control group, hypoxia group and hypoxia + TH group: compared with the control group, LC3B and Beclin-1 expression was significantly increased in the hypoxia group and hypoxia + TH group (allP<0.05), while p62 expression was significantly decreased in the hypoxia group (P<0.05). Hypoxia + TH group exhibited lower LC3B expression as well as higher p62 expression as compared with that of the hypoxia group (P<0.05). There was no significant difference in Beclin-1 expression between the hypoxia group and hypoxia+TH group. ② The expression of autophagy-related proteins LC3B, p62 and mTOR pathway-related proteins p-mTOR and p-S6 after rapamycin treatment: compared with the hypoxia group, hypoxia + TH group showed decreased expression of LC3B and increased expression of p62, p-mTOR and p-S6 (allP<0.05). Compared with the hypoxia group, rapamycin+hypoxia group showed higher expression of LC3B and lower expression of p62 (allP<0.05), while there were no significant differences in p-mTOR and p-S6. Rapymycin + hypoxia+TH group showed increased LC3B and decreased p62, p-mTOR and p-S6 expression as compared with that of the hypoxia+TH group, and significant difference was found between them (allP<0.05). The rapamycin + hypoxia+TH group showed lower LC3B and higher p62 expression (allP<0.05), but there were no significant differences in p-mTOR and p-S6 expression between rapamycin + hypoxia+TH group and rapamycin +hypoxia group. ③ Detection of autophagic flux: compared with the control group, the green and red puncta were significantly higher in the hypoxia group and hypoxia + TH group (allP<0.05). Hypoxia + TH group showed less green and red puncta than those of the hypoxia group (allP<0.05). ConclusionTH inhibits the autophagic flux of hypoxic cardiomyocytes and the mechanism may be related to the activation of mTOR pathway-related proteins.

therapeutic hypothermia; cardiomyocytes; hypoxia; autophagy; rapamycin

國家自然科學(xué)基金資助項(xiàng)目(81400190，81270218)；廣東省自然科學(xué)基金資助項(xiàng)目(2015A030310478)；廣東省科技計(jì)劃項(xiàng)目(2014A020212191)。

楊羽菲(1989-)，女，在讀碩士研究生，主要研究方向?yàn)樽允稍谛募∪毖獡p傷中的影響及機(jī)制。E-mail: lvyyang0608@163.com

簡(jiǎn)介：陳愛華(1956-)，男，博士，教授，主要研究方向?yàn)樽允稍谛募∪毖腿毖俟嘧p傷中的影響及機(jī)制。E-mail: zj_chenaihua@126.com

10.3969/j.issn.1002-266X.2016.22.001

R541.2

A

1002-266X(2016)22-0001-04

2016-02-25)