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肺动脉高压(PAH)是一种慢性进行性疾病,是一种被定义为在海平面,静息状态下通过右心导管测定的平均肺动脉压超过25 mmHg的疾病,世界上大约有1%的人口都受到了PAH的影响[1-2]。缺氧性肺动脉高压(hypoxic pulmonary hypertension, HPH)是其中第三类,它主要是由慢性缺氧所导致的例如慢性阻塞性肺疾病等。HPH的主要特征表现为肺血管的阻力增加,最终导致右心衰竭甚至死亡,是一种预后极差、对医疗负担极重的疾病[3-5]。远端小动脉是HPH的主要靶点,它的主要病理特征是肺血管的重构,血管壁表现出促增殖/抗凋亡和高代谢状态,毛细血管前小动脉肌化,并随着时间的推移导致血管闭塞。其中血管内皮损伤及平滑肌细胞增殖在肺血管的重构中发挥着中重要作用[6-7]。目前虽然已经开发了多种治疗PAH的药物,但重度肺动脉高压病人的长期预后仍然很差[8-9]。因此,我们亟需更多的研究靶点以求改善HPH预后并减轻医疗负担。
线粒体乙醛脱氢酶(ALDH2)隶属于乙醛脱氢酶家族,是一种位于线粒体基质中的变构四聚体酶,最早在肝脏中被发现,后续研究[10-12]发现在心脏、大脑、肠道等多种器官中也大量表达。在多项研究中发现ALDH2通过清除脂质过氧化产生的有毒醛,特别是4-HNE,在心肌损伤和中风中发挥着保护作用。有研究证实ALDH2激活可减轻野百合碱诱导的PAH[13-14],同时,XU等[15]研究发现ALDH2可能通过调控4-HNE的水平减轻HPH的发生。
自噬是将细胞质物质传递到动物细胞中的溶酶体或植物和酵母细胞中的液泡的所有途径的总称。主要可分为三类: 巨自噬、微自噬以及分子伴侣介导的自噬。自噬分为三个过程: 自噬起始、自噬膜延伸和自噬溶酶体形成[16-18]。Beclin1是自噬过程中的关键蛋白之一,在自噬过程中,Beclin1在调节自噬小体膜的形成和物质转运过程中起着重要作用。Beclin1主要通过Atg14L-Vps15-Vps34信号轴调控巨自噬的起始[19]。微管相关蛋白1A/1B轻链3B(MAP1 LC3)是微管相关蛋白,它们介导微管和细胞骨架成分之间的物理相互作用,通过泛素样修饰参与自噬体液泡(自噬体)的形成。并且对于自噬体成熟的后期阶段至关重要,同时它将线粒体消除降低到基础水平以满足细胞能量需求并防止过量活性氧自由基(ROS)产生从而参与线粒体自噬以调节线粒体的融合与分裂[20-21]。SQSTM1(p62)是巨自噬所需的自噬受体。作为多泛素化自噬小体和溶酶体之间的桥梁。直接与降解的物质和MAP1 LC3家族的自噬修饰剂相互作用,在自噬溶酶体的形成中发挥了关键作用[22-23]。最近有研究表明,缺氧能够通过促进自噬的发生进而激动肺动脉平滑肌细胞(PASMC)的增生与迁移,并且抑制自噬能够有效的减缓野百合碱诱导的PAH的发生发展。
本研究拟用通过慢性缺氧构建HPH动物模型,同时使用Alda-1以及Daidzin分别激动、抑制线粒体ALDH2,观察肺组织病理改变,同时建立缺氧性肺动脉高压细胞模型,观察PASMC细胞中自噬水平的变化,同时用CCK8检测细胞的增殖水平、流式细胞术测定细胞凋亡,从而探究ALDH2、自噬与HPH的关系,为临床通过ALDH2抑制自噬从而改善甚至治疗HPH提供新的方法及思路。
线粒体ALDH2通过调控自噬对缺氧性肺动脉高压的保护机制研究
Protective mechanism of mitochondrial ALDH2 on hypoxic pulmonary hypertension by regulating autophagy
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摘要:
目的探讨激活线粒体乙醛脱氢酶2(ALDH2)可以降低因缺氧所引起的平滑肌细胞自噬, 并降低肺动脉平滑肌细胞(PASMCs)的增殖, 分析ALDH2对缺氧性肺动脉高压平滑肌增殖的调控关系。 方法将SPF级雄性SD大鼠分4组: 正常对照组(N组)、常氧+ALDH2特异性激动剂Alda-1组(N+Alda-1组)、缺氧组(H组)、缺氧+Alda-1组(H+Alda-1组), 将PASMCs分为6组: 常氧组(N组)、常氧+Alda-1组(NA组)、缺氧组(H组)、缺氧+Alda-1组(HA组)、缺氧+ALDH2抑制剂Daidzin组(HD组)、缺氧+Alda-1+Daidzin组(HAD组)。HE染色观察各组动物肺组织中肺动脉病理变化; 免疫荧光鉴定肺动脉平滑肌细胞; CCK-8测定各组细胞增殖能力; Annexin V-FITC/PI双染测定细胞凋亡水平; Western blotting检测各组ALDH2、p62、Beclin1、LC3B等蛋白的表达。 结果与N组相比, H组肺小动脉管壁增厚, 管腔变窄; 与HC组相比, H+Alda-1组的肺小动脉管壁厚度减轻, 管腔狭窄程度有所改善; 与N组相比, H组的ALDH2表达降低(P < 0.05), 自噬相关蛋白p62、Beclin1、LC3B均明显升高(P < 0.01), 细胞增殖明显增加(P < 0.01);与H组相比, HA组ALDH2表达明显升高(P < 0.01), 自噬相关蛋白p62、Beclin1、LC3B均明显降低(P < 0.01), 细胞增殖受到明显抑制(P < 0.01)。 结论线粒体ALDH2对缺氧诱导的PASMCs的增殖有着显著的抑制作用, 其机制可能与ALDH2对细胞的自噬调节有关。 Abstract:ObjectiveTo investigate the effect of activating mitochondrial aldehyde dehydrogenase on the autophagy of smooth muscle cells induced by hypoxia and the proliferation of pulmonary artery smooth muscle cells, and to analyze the regulation of mitochondrial aldehyde dehydrogenase on the proliferation of smooth muscle cells induced by hypoxia pulmonary arterial hypertension. MethodsMale SD rats of SPF grade were divided into four groups: normal control group (N group), normoxia+ALDH2 agonist Alda-1 group (N+Alda-1 group), model group (H group), and hypoxia+Alda-1 group (H+Alda-1 group).PASMCs was divided into six groups: normoxia group (N group), normoxia+Alda-1 group (NA group), hypoxia group (H group), hypoxia+Alda-1 group (HA group), hypoxia+ALDH2 inhibitor Daidzin group (HD group), and hypoxia+Alda-1+Daidzin group (HAD group).HE staining was performed to observe the pathological changes of pulmonary artery in the lung tissue of each group, immunofluorescence was used to identify pulmonary artery smooth muscle cells, CCK-8 was applied to determine the cell viability and proliferation of each group, Annexin V-FITC/PI double staining was employed to detect apoptosis level, and Western blotting was used to analyze the protein expression of ALDH2, p62, Beclin1 and LC3B. ResultsCompared with the N group, the walls of pulmonary arterioles was thickened and the lumen was narrowed in the H group.Compared with the H group, the thickness of pulmonary small artery wall was reduced, and the degree of stenosis was improved in the H+Alda-1 group.Compared with the group N, the expression of ALDH2 decreased (P < 0.05), the expression of autophagy-related proteins p62, Beclin1 and LC3B increased significantly (P < 0.01), and the cell proliferation increased significantly in the group H (P < 0.01).Compared with the group H, the expression of ALDH2 was significantly increased (P < 0.01), the expression of autophagy-related proteins p62, Beclin1 and LC3B obviously decreased (P < 0.01), and the cell proliferation was markedly inhibited in the HA group (P < 0.01). ConclusionsMitochondrial ALDH2 has a significant inhibitory effect on the proliferation of hypoxia-induced PASMCs, and the mechanism may be related to the autophagy regulation of ALDH2 on cells. -
Key words:
- pulmonary hypertension /
- mitochondrial aldehyde dehydrogenase /
- autophagy
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