肺動(dòng)脈平滑肌細(xì)胞凋亡在大鼠低氧性肺動(dòng)脈重構(gòu)自然逆轉(zhuǎn)中的作用及分子機(jī)制*

陳 鍵， 王艷霞， 牛 雯， 李志超

(第四軍醫(yī)大學(xué)病理學(xué)與病理生理學(xué)教研室， 陜西 西安 710032)

肺動(dòng)脈平滑肌細(xì)胞凋亡在大鼠低氧性肺動(dòng)脈重構(gòu)自然逆轉(zhuǎn)中的作用及分子機(jī)制*

陳 鍵， 王艷霞， 牛 雯， 李志超△

(第四軍醫(yī)大學(xué)病理學(xué)與病理生理學(xué)教研室， 陜西 西安 710032)

目的： 研究肺動(dòng)脈平滑肌細(xì)胞(pulmonary arterial smooth muscle cells, PASMCs)凋亡在低氧性肺動(dòng)脈重構(gòu)自然逆轉(zhuǎn)中的作用，并探討其可能機(jī)制。方法: 24只SD大鼠隨機(jī)均分為常氧4周組、低氧4周組、低氧4周后復(fù)氧1周組及復(fù)氧6周組。分別檢測(cè)右室收縮壓(right ventricular systolic pressure, RVSP)、肺動(dòng)脈中膜厚度(medial thickness, MT)和中膜面積(medial area, MA)，以及肺動(dòng)脈中膜自噬、凋亡等在低氧-復(fù)氧中的變化。大鼠原代PASMCs分為常氧48 h組、低氧48 h組、低氧48 h后復(fù)氧24 h組及常氧72 h組，觀察PASMCs凋亡和自噬在低氧-復(fù)氧中的變化。再將PASMCs分為常氧72 h組、低氧48 h后復(fù)氧24 h組及低氧48 h+氯喹(自噬抑制劑)干預(yù)后復(fù)氧24 h組，觀察PASMCs低氧階段的自噬對(duì)其復(fù)氧階段凋亡的影響。結(jié)果: (1)低氧使大鼠RVSP、右室肥厚指數(shù)、MT及MA顯著升高(P<0.05)；復(fù)氧后上述指標(biāo)逐漸降低。(2)低氧使肺動(dòng)脈中膜LC3表達(dá)升高，P62表達(dá)降低，復(fù)氧后上述分子的表達(dá)逐步恢復(fù)正常。低氧顯著降低了中膜cleaved caspase-3 的表達(dá)，復(fù)氧1周其表達(dá)顯著高于低氧組。(3)低氧期原代PASMCs cleaved caspase-3/PARP 的表達(dá)顯著低于常氧組，復(fù)氧后其表達(dá)明顯升高(P<0.05)；PASMCs LC3和P62的表達(dá)在低氧期顯著降低(P<0.05)。(4)抑制了PASMCs低氧階段的自噬后，其復(fù)氧階段 cleaved caspase-3/PARP表達(dá)顯著降低(P<0.05)。結(jié)論: PASMCs的凋亡參與了低氧性肺動(dòng)脈重構(gòu)的自然逆轉(zhuǎn)；復(fù)氧期PASMCs凋亡的發(fā)生可能與其低氧期的自噬有關(guān)。

低氧性肺動(dòng)脈高壓； 自噬； 細(xì)胞凋亡

低氧性肺動(dòng)脈高壓(hypoxic pulmonary hypertension, HPH)是以肺小動(dòng)脈收縮及重構(gòu)為主要特征的慢性漸進(jìn)性疾病，具有較高的發(fā)病率及死亡率[1]。大量的研究表明，HPH中的肺動(dòng)脈重構(gòu)在恢復(fù)常氧后可以逐步逆轉(zhuǎn)[2]。早先有研究指出，凋亡在低氧性肺動(dòng)脈重構(gòu)的逆轉(zhuǎn)中發(fā)揮著重要作用[3]，但其發(fā)生的具體機(jī)制尚不清楚。自噬是機(jī)體一種進(jìn)化保守的分解代謝過程，可以降解胞質(zhì)的蛋白多聚體以及細(xì)胞器等[4]。傳統(tǒng)的觀念認(rèn)為自噬作為機(jī)體在惡劣條件下的一種應(yīng)激反應(yīng)，通過清除損傷的蛋白質(zhì)及細(xì)胞器、維持機(jī)體代謝穩(wěn)態(tài)等，促進(jìn)了細(xì)胞存活[5]；也有研究認(rèn)為自噬在一定條件下可以誘導(dǎo)凋亡的發(fā)生最終導(dǎo)致細(xì)胞死亡[6]。在HPH發(fā)生發(fā)展的過程中常伴有肺動(dòng)脈平滑肌細(xì)胞(pulmonary arterial smooth muscle cells, PASMCs)自噬的發(fā)生[7]。是否PASMCs低氧階段的自噬促進(jìn)了其復(fù)氧階段凋亡的發(fā)生，最終加速了低氧性肺動(dòng)脈重構(gòu)的逆轉(zhuǎn)，目前尚無(wú)研究報(bào)道。本研究分別觀察了自噬及凋亡在低氧性肺動(dòng)脈重構(gòu)的發(fā)生及逆轉(zhuǎn)中的變化，并探討了PASMCs低氧階段的自噬對(duì)其復(fù)氧階段凋亡的影響。

材 料 和 方 法

1 材料

1.1 實(shí)驗(yàn)動(dòng)物 成年雄性SD大鼠，體重(200±20) g，共24只，購(gòu)于第四軍醫(yī)大學(xué)實(shí)驗(yàn)動(dòng)物中心。

1.2 藥物及試劑 水合氯醛(上海山浦化工有限公司)；BCA蛋白定量試劑盒(上海碧云天有限公司)；LC3兔單克隆抗體(Millipore)；P62兔單克隆抗體(Sigma);cleaved caspase-3和cleaved PARP兔多克隆抗體(CST);β-actin兔多克隆抗體(ImmunoWay);免疫組化試劑(北京中衫生物技術(shù)有限公司);DAB試劑盒(北京中衫生物技術(shù)有限公司);氯喹(chloroquine, CLQ; 購(gòu)自MCE)；CellMax胎牛血清(賽澳美細(xì)胞技術(shù)有限公司)；DMEM高糖培養(yǎng)液(HyClone)。

1.3 實(shí)驗(yàn)儀器 全自動(dòng)調(diào)節(jié)低壓低氧艙(第四軍醫(yī)大學(xué)病理學(xué)與病理生理學(xué)教研室自主研發(fā))；壓力檢測(cè)系統(tǒng)(AD)；常氧、低氧細(xì)胞培養(yǎng)箱(Thermo)；酶聯(lián)免疫檢測(cè)儀(BioTek)；SDS-PAGE凝膠電泳儀及電轉(zhuǎn)儀(Bio-Rad)。

2 實(shí)驗(yàn)方法及步驟

2.1 實(shí)驗(yàn)動(dòng)物分組及處理 24只SD大鼠隨機(jī)分為：常氧4周(normoxia for 4 weeks, N)組、低氧4周(hypoxia for 4 weeks, H)組、低氧4周后復(fù)氧1周(reoxygenation for 1 week after hypoxia for 4 weeks, R1)組以及低氧4周后復(fù)氧6周(reoxygenation for 6 weeks after hypoxia for 4 weeks, R6)組，每組6只。低氧組大鼠置于低壓低氧艙內(nèi)(模擬5 500米高空環(huán)境，10%O2)，每天8 h，連續(xù)4周。復(fù)氧組大鼠先低氧處理4周，然后在常氧條件下分別恢復(fù)1周和6周。常氧組大鼠在常氧條件下飼養(yǎng)。所有大鼠飼養(yǎng)于室溫(18～22 ℃)、濕度50%～70%、12 h光照與12 h 黑暗交替的環(huán)境中，可自由飲水與攝食。

2.2 血流動(dòng)力學(xué)檢測(cè)及標(biāo)本收集 大鼠稱重后，10%水合氯醛(3.5 mL/kg)腹腔注射麻醉。經(jīng)右頸外靜脈插管至右心室，壓力檢測(cè)系統(tǒng)記錄右心室收縮壓(right ventricular systolic pressure, RVSP)，可近似視為肺動(dòng)脈壓[8]。測(cè)壓結(jié)束放血處死大鼠，分離出心臟和肺臟。每只大鼠分別取右肺下葉0.3 cm×0.3 cm×0.2 cm大小肺組織，置于4%多聚甲醛中固定，用于后續(xù)病理學(xué)檢測(cè)。剩余肺組織保存于液氮中備用。取出心臟，去除心房和大血管，將右心室壁(right ventricle, RV)與左心室及室間隔(left ventricle plus interventricular septum, LV+S)分離。拭去表面的水并稱重，計(jì)算RV/(LV+S)。

2.3 血管形態(tài)學(xué)檢測(cè) 固定的大鼠肺組織經(jīng)石蠟包埋、切片及HE染色，光鏡下觀察各處理組外周肺動(dòng)脈(直徑30～100 μm)形態(tài)學(xué)改變并拍照。使用圖像處理軟件(Image-Pro Plus)，分別計(jì)算肺動(dòng)脈中膜厚度(medial thickness, MT)百分比[MT%=100×MT/(vessel semi-diameter)]以及中膜面積(medial area, MA)百分比[MA%=100×(cross-sectional medial layer area)/(total cross-sectional vessel area)]。

2.4 免疫組化染色 肺組織石蠟切片經(jīng)梯度二甲苯及乙醇溶液脫蠟后，過氧化氫滅活內(nèi)源性過氧化物酶、檸檬酸緩沖液進(jìn)行抗原修復(fù)等，最后LC3(1∶100)、P62 (1∶100)及cleaved caspase-3(1∶50)抗體溶液孵育4 ℃過夜。次日，滴加辣根過氧化物酶結(jié)合的抗兔抗體、DAB法顯色、蘇木精復(fù)染、脫水、透明、封片后鏡下觀察并拍照。

2.5 大鼠PASMCs的原代培養(yǎng) 大鼠原代PASMCs通過組織塊法進(jìn)行培養(yǎng)。動(dòng)物麻醉后(方法同上)，放血處死，超凈臺(tái)內(nèi)分離出肺動(dòng)脈。在無(wú)菌的PBS中除去肺動(dòng)脈外膜及內(nèi)膜，將剩余中膜剪至1 mm×1 mm×1 mm大小的組織塊，移入培養(yǎng)瓶底面并均勻分布。加入含20%胎牛血清的培養(yǎng)基，將培養(yǎng)瓶倒置于37 ℃、21% O2的細(xì)胞培養(yǎng)箱內(nèi)，3 h后翻瓶。3 d后可見PASMCs從組織塊爬出，待細(xì)胞長(zhǎng)到80%融合后進(jìn)行傳代，使用生長(zhǎng)良好的3～5代細(xì)胞進(jìn)行后續(xù)實(shí)驗(yàn)。

2.6 PASMCs的分組及處理 首先，將PASMCs分為常氧48 h (normoxia for 48 h, N48)組、低氧48 h (hypoxia for 48 h, H48)組、低氧48 h后復(fù)氧24 h (reoxygenation for 24 h after hypoxia for 48 h, H48R24)組以及常氧72 h(normoxia for 72 h, N72)組。常氧組PASMCs置于37 ℃、5%CO2、21%O2條件下分別培養(yǎng)48 h和72 h；低氧組在37 ℃、5%CO2、5%O2條件下培養(yǎng)48 h；復(fù)氧組先在低氧條件下培養(yǎng)48 h后轉(zhuǎn)至常氧繼續(xù)培養(yǎng)24 h。再者，將PASMCs分為常氧48 h組、低氧48 h組、低氧48 h+CLQ(hypoxia+CLQ for 48 h, H48+CLQ)組、常氧72 h(N72)組、低氧48 h后復(fù)氧24 h(H48R24)組和低氧+CLQ干預(yù)48 h后復(fù)氧24 h(reoxygenation for 24 h after hypoxia+CLQ for 48 h, HCLQR24)組。低氧及復(fù)氧組處理方法同上。CLQ干預(yù)組在低氧期間向PASMCs加入CLQ(20 μmol/L)共培養(yǎng)48 h后細(xì)胞換液并轉(zhuǎn)至常氧條件繼續(xù)培養(yǎng)24 h。

2.7 Western blotting法檢測(cè)蛋白表達(dá) 分別提取組織及細(xì)胞總蛋白，BCA法測(cè)定蛋白濃度。各組樣品取等量蛋白(35 μg)用不同濃度的SDS-PAGE分離蛋白，蛋白經(jīng)濕轉(zhuǎn)法轉(zhuǎn)移至PVDF膜上，5%的脫脂牛奶封閉1 h， LC3 (1∶4 500)、P62 (1∶4 500)、cleaved caspase-3 (1∶800)、cleaved PARP(1∶800)以及β-actin(1∶2 000)抗體溶液孵育，4 ℃過夜。次日，TBST洗膜后加入辣根過氧化物酶標(biāo)記的抗兔Ⅱ抗(1∶2 000)敷育1 h，TBST洗膜，ECL進(jìn)行發(fā)光成像。使用Gel-Pro軟件，對(duì)蛋白條帶進(jìn)行灰度分析。用目的蛋白的灰度值比內(nèi)參照蛋白的灰度值，統(tǒng)計(jì)各蛋白的相對(duì)表達(dá)。

3 統(tǒng)計(jì)學(xué)處理

用SPSS 19.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。數(shù)據(jù)均采用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，多組間比較采用單因素方差分析(one-way ANOVA)，多重比較采用Bonferroni法。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 肺動(dòng)脈血流動(dòng)力學(xué)及結(jié)構(gòu)等指標(biāo)在低氧-復(fù)氧過程中的變化

低氧后RVSP、RV/(LV+S)、外周肺小動(dòng)脈MT及MA與常氧組相比均顯著升高(P<0.05)；恢復(fù)常氧后，上述指標(biāo)逐步降低至正常水平，見圖1。

2 肺動(dòng)脈中膜的自噬和凋亡在低氧-復(fù)氧過程中的變化

低氧后肺動(dòng)脈中膜LC3的表達(dá)與常氧組相比顯著升高，復(fù)氧后其表達(dá)逐步恢復(fù)至常氧水平。與常氧組相比，低氧顯著降低了肺動(dòng)脈中膜 P62的表達(dá)，復(fù)氧后P62的表達(dá)逐步恢復(fù)。低氧后肺動(dòng)脈中膜 cleaved caspase-3的表達(dá)較常氧組顯著降低，復(fù)氧1周后cleaved caspase-3水平較低氧及常氧組顯著升高，見圖2。

3 原代PASMCs的凋亡在低氧-復(fù)氧過程中的變化

低氧48 h組原代PASMCs cleaved caspase-3/PARP的表達(dá)與常氧48 h組相比均顯著降低(P<0.05)；低氧48 h后復(fù)氧24 h，PASMCs cleaved caspase-3/PARP的表達(dá)與常氧72 h及低氧48 h組相比均顯著升高(P<0.05)，見圖3。

4 低氧對(duì)原代PASMCs自噬的影響

低氧48 h組原代PASMCs P62及LC3-II的表達(dá)與常氧48 h組相比均顯著降低(P<0.05)，見圖4。

5 抑制PASMCs低氧階段的自噬后其復(fù)氧階段凋亡的變化

低氧48 h，PASMCs P62及LC3-II的表達(dá)與常氧48 h組相比均顯著降低(P<0.05)；低氧階段用CLQ阻斷自噬后，可見PASMCs P62及LC3-II的表達(dá)與單純低氧組相比顯著升高(P<0.05)。低氧48 h后復(fù)氧24 h，PASMCs cleaved caspase-3/PARP的表達(dá)與常氧72 h組相比顯著升高(P<0.05)；低氧期間用CLQ阻斷了PASMCs的自噬后繼續(xù)復(fù)氧24 h，可見PASMCs cleaved caspase-3/PARP的表達(dá)與低氧48 h后復(fù)氧24 h組相比顯著降低(P<0.05),見圖5。

Figure 1.The changes of hemodynamic and structural parameters in pulmonary arteries following hypoxia-reoxygenation.N: normoxia for 4 weeks; H: hypoxia for 4 weeks; R1: reoxygenation for 1 week after hypoxia for 4 weeks; R6: reoxygenation for 6 weeks after hypoxia for 4 weeks. Mean±SD.n=3～6.*P<0.05,**P<0.01vsN;#P<0.05,##P<0.01vsH;&&P<0.01vsR1.

圖1 肺動(dòng)脈血流動(dòng)力學(xué)及結(jié)構(gòu)等指標(biāo)在低氧-復(fù)氧過程中的變化

Figure 2.The changes of autophagy and apoptosis in the pulmonary arterial medial layer following hypoxia-reoxygenation(×400). Representative images of immunostaining for LC3, P62, or cleaved caspase-3 in the pulmonary arterial medial layer. N: normoxia for 4 weeks; H: hypoxia for 4 weeks; R1: reoxygenation for 1 week after hypoxia for 4 weeks; R6: reoxygenation for 6 weeks after hypoxia for 4 weeks.

圖2 肺動(dòng)脈中膜的自噬和凋亡在低氧-復(fù)氧過程中的變化

Figure 3.The changes of primary PASMC apoptosis following hypoxia-reoxygenation. Western blotting analysis of the cleaved caspase-3 and cleaved PARP expression. Densitometry analysis of protein abundance was conducted by normalizing to that of β-actin. N48: normoxia for 48 h; H48: hypoxia for 48 h; H48R24: reoxygenation for 24 h after hypoxia for 48 h; N72: normoxia for 72 h. Mean±SD.n=3.*P<0.05vsN48;#P<0.05vsN72;&P<0.05,&&P<0.01vsH48.

圖3 原代PASMCs的凋亡在低氧-復(fù)氧過程中的變化

Figure 4.The effect of hypoxia on primary PASMC autophagy. Western blotting analysis of the LC3-II and P62 expression. Densitometry analysis of protein abundance was conducted by normalizing to that of β-actin. N48: normoxia for 48 h; H48: hypoxia for 48 h. Mean±SD.n=3.*P<0.05,**P<0.01vsN48.

圖4 低氧對(duì)原代PASMCs自噬的影響

討 論

HPH是一種原發(fā)或繼發(fā)性的肺血管阻力進(jìn)行性升高的疾病，如未及時(shí)有效地接受治療常常導(dǎo)致病人發(fā)生右心衰竭直至死亡[9]。目前治療HPH藥物的種類逐漸增多，但大多都不能有效地抑制或逆轉(zhuǎn)肺動(dòng)脈重構(gòu)。大量的研究證實(shí)，以往認(rèn)為是不可逆性改變的低氧性肺動(dòng)脈重構(gòu)，在恢復(fù)常氧后可以逐步逆轉(zhuǎn)[2, 10]，但其機(jī)制尚不清楚。曾有研究表明，凋亡在低氧性肺血管重構(gòu)的逆轉(zhuǎn)中發(fā)揮著重要作用，然而此研究并未就凋亡發(fā)生的細(xì)胞類型及機(jī)制作詳細(xì)的探討[3]。本研究證實(shí)，低氧性肺動(dòng)脈重構(gòu)在恢復(fù)常氧后逐步逆轉(zhuǎn)至正常水平，進(jìn)一步探明PASMCs特異性的凋亡參與了低氧性肺動(dòng)脈重構(gòu)復(fù)氧逆轉(zhuǎn)的過程。

關(guān)于PASMCs恢復(fù)常氧之后凋亡增加的機(jī)制，目前尚無(wú)報(bào)道。從惡劣的低氧條件轉(zhuǎn)至相對(duì)優(yōu)越的常氧條件，細(xì)胞的凋亡反而較前增多，我們推測(cè)PASMCs發(fā)生復(fù)氧凋亡最根本的動(dòng)因可能存在于低氧階段。自噬作為機(jī)體的一種適應(yīng)性分解代謝反應(yīng)，可以清除胞內(nèi)損傷的蛋白質(zhì)及細(xì)胞器等。在大多數(shù)應(yīng)激條件下，自噬序貫性的分解代謝過程通?？梢源龠M(jìn)細(xì)胞存活[11]，然而也有研究報(bào)道細(xì)胞自噬在一定程度上可以誘導(dǎo)凋亡的發(fā)生[12]。那么低氧期PASMCs是否發(fā)生了自噬，我們的結(jié)果顯示低氧期大體肺動(dòng)脈中膜P62表達(dá)降低，LC3 的表達(dá)升高，提示PASMCs自噬增加。但低氧階段，原代培養(yǎng)的PASMCs P62及LC3-II的表達(dá)均顯著降低。在自噬發(fā)生的過程中，LC3-II的主要作用是形成自噬體雙層膜，P62可特異性地識(shí)別和選擇待降解的目標(biāo)分子并將其運(yùn)至自噬體[13]。最終此兩者在自噬體與溶酶體融合后，而被降解掉。因此，只有當(dāng)細(xì)胞發(fā)生過度自噬時(shí)，大量的自噬體被溶酶體降解掉后，細(xì)胞中P62和LC3-II表達(dá)才會(huì)同時(shí)減少[14]。我們后續(xù)在低氧條件下應(yīng)用了自噬抑制劑CLQ(升高溶酶體內(nèi)的pH，抑制自噬體與溶酶體融合)后，可見PASMCs P62及LC3-II的表達(dá)均顯著增多。由此我們可以得出，低氧期原代培養(yǎng)的PASMCs發(fā)生了過度自噬。低氧期，大體肺動(dòng)脈中PASMCs的自噬只是適度增加，而離體培養(yǎng)的PASMCs卻發(fā)生了過度自噬，我們推測(cè)此差異可能與在體情況下神經(jīng)體液等因素的調(diào)節(jié)有關(guān)。

Figure 5.The effect of PASMC autophagy under hypoxia on its apoptosis during reoxygenation. To ensure the effect of chloroquine (CLQ) on the inhibition of PASMC autophagy, Western blotting analysis of the P62 and LC3-II expression of different groups was performed (A). To further examine the effect of PASMC autophagy under hypoxia on its apoptosis during reoxygenation, the expression of cleaved caspase-3/PARP was also examined (B). N48: normoxia for 48 h; H48: hypoxia for 48 h; H48+CLQ: hypoxia+CLQ for 48 h; H48R24: reoxygenation for 24 h after hypoxia for 48 h; N72: normoxia for 72 h; HCLQR24: reoxygenation for 24 h after hypoxia+CLQ for 48 h. Mean±SD.n=3.*P<0.05,**P<0.01vsN48 or N72;&&P<0.01vsH48;#P<0.05,##P<0.01vsH48R24.

圖5 PASMCs低氧階段的自噬對(duì)其復(fù)氧階段凋亡的影響

通常低氧階段會(huì)有大量的線粒體活性氧簇(mitochondrial reactive oxygen species, mROS)產(chǎn)生，而mROS會(huì)引起細(xì)胞器尤其是線粒體損傷[15]，損傷的線粒體常常會(huì)啟動(dòng)線粒體自噬[16]。所以低氧結(jié)束時(shí)，細(xì)胞中線粒體的數(shù)量處于相對(duì)較低的水平，加之恢復(fù)常氧后PASMCs處在一個(gè)相對(duì)高氧的環(huán)境，因此PASMCs在復(fù)氧早期會(huì)產(chǎn)生過量的ROS[17]。而過量ROS常會(huì)引起胞漿內(nèi)蛋白及細(xì)胞器等發(fā)生氧化損傷，最終導(dǎo)致細(xì)胞凋亡[18]。據(jù)此我們推測(cè)可能是低氧階段增加的自噬，誘導(dǎo)了復(fù)氧階段PASMCs凋亡的發(fā)生。我們的結(jié)果顯示抑制了PASMCs低氧階段的自噬后，其復(fù)氧階段凋亡的水平顯著降低，該結(jié)果說明PASMCs低氧階段的自噬促進(jìn)了其復(fù)氧階段凋亡的發(fā)生。

綜上所述，本研究提示PASMCs的凋亡參與了低氧性肺動(dòng)脈重構(gòu)復(fù)氧逆轉(zhuǎn)的過程，且PASMCs凋亡的發(fā)生可能與其低氧階段發(fā)生的自噬有關(guān)，這為低氧期應(yīng)用自噬誘導(dǎo)藥物來(lái)治療低氧相關(guān)肺動(dòng)脈高壓提供了新的理論基礎(chǔ)。

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(責(zé)任編輯： 盧 萍， 羅 森)

Effect of PASMC apoptosis on reversal of hypoxic pulmonary arterial remodeling during reoxygenation and its related molecular mechanism

CHEN Jian, WANG Yan-xia, NIU Wen, LI Zhi-chao

(DepartmentofPathologyandPathophysiology,TheFourthMilitaryMedicalUniversity,Xi’an710032,China.E-mail:lizhic@fmmu.edu.cn)

AIM: To explore the effect of pulmonary arterial smooth muscle cell (PASMC) apoptosis on the reversal of hypoxic pulmonary arterial remodeling during reoxygenation and its possible mechanism. METHODS: Male SD rats (n=24) were randomly divided into normoxia for 4 weeks group, hypoxia for 4 weeks group, reoxygenation for 1 week after hypoxia for 4 weeks group and reoxygenation for 6 weeks after hypoxia for 4 weeks group. Right ventricular systolic pressure (RVSP), right ventricular hypertrophy index, pulmonary arterial medial thickness (MT) and medial area (MA) as well as autophagy and apoptosis in the pulmonary arterial medial layer were examined during hypoxia-reoxygenation. The rat primary PASMCs were divided into normoxia for 48 h group, hypoxia for 48 h group, reoxygenation for 24 h after hypoxia for 48 h group and normoxia for 72 h group to explore the changes of PASMC autophagy and apoptosis following hypoxia-reoxygenation. Finally, primary PASMCs were divided into normoxia for 72 h group, reoxygenation for 24 h after hypoxia for 48 h group and reoxygenation for 24 h after hypoxia for 48 h + chloroquine (inhibitor of autophagy) group to investigate the effect of PASMC autophagy during hypoxia on the apoptosis during reoxygenation. RESULTS: After hypoxia for 4 weeks, the RVSP, during right ventricular hypertrophy index, MT and MA increased significantly compared with normoxia group (P<0.05), and gradually decreased during reoxygenation. The expression of LC3 in the pulmonary arterial medial layer increased evidently after hypoxia and gradually reversed during reoxygenation. Moreover, the P62 and cleaved caspase-3 expression decreased after hypoxia compared with normoxia group, and increased markedly following reoxyge-nation. The expression of cleaved caspase-3/PARP in rat primary PASMCs decreased significantly under hypoxia (P<0.05), and increased evidently during reoxygenation. The expression of P62 and LC3-II decreased markedly under hypoxia (P<0.05). After inhibition of PASMC autophagy under hypoxia, the expression of cleaved caspase-3/PARP decreased remarkably during reoxygenation (P<0.05). CONCLUSION: The PASMC apoptosis participates in the reversal of hypoxic pulmonary arterial remodeling, and the PASMC autophagy under hypoxia might facilitate its apoptosis during reoxygenation.

Hypoxic pulmonary hypertension; Autophagy; Apoptosis

1000- 4718(2017)04- 0583- 07

2016- 12- 06

2017- 02- 15

國(guó)家自然科學(xué)基金資助項(xiàng)目(No. 81471816； No. 81270328)

R363.21； R541.3

A

10.3969/j.issn.1000- 4718.2017.04.002

△通訊作者 Tel: 029-84772705; E-mail: lizhic@fmmu.edu.cn