MBOAT2诱导血管内皮细胞线粒体自噬障碍及其机制

MBOAT2 induces mitophagy dysfunction in vascular endothelial cells and its mechanism

摘要:

目的]探讨膜结合O-酰基转移酶结构域蛋白2(MBOAT2)对血管内皮细胞线粒体自噬的影响及其作用机制。 [方法]采用Western blot检测人动脉粥样硬化斑块组织中MBOAT2蛋白表达水平；构建MBOAT2过表达的人脐静脉内皮细胞模型,采用Western blot、透射电镜、JC-1染色、MitoSOX Red染色、mRFP-GFP-LC3双荧光及免疫荧光染色等方法,观察MBOAT2过表达对线粒体损伤、线粒体自噬、线粒体DNA(mtDNA)胞质释放及环鸟苷酸-腺苷酸合酶(cGAS)-干扰素基因刺激蛋白(STING)信号通路激活的影响,并采用cGAS特异性抑制剂RU.521进行干预,以验证cGAS-STING信号通路在线粒体损伤及线粒体自噬障碍中的作用。 [结果]与对照组相比,人动脉粥样硬化斑块组织中MBOAT2蛋白表达水平升高70.7%(P＜0.05)；MBOAT2过表达后,人脐静脉内皮细胞中MIC60、ATP5L及ATP5I的表达水平分别下调15.6%、18.9%和25.8%(P＜0.05或P＜0.01),同时伴随线粒体超微结构异常、膜电位下降及线粒体活性氧(mtROS)水平升高；PINK1、Parkin、LC3Ⅱ及p62蛋白表达水平分别升高53.7%、53.8%、54.9%和112.1%(P＜0.05或P＜0.01),黄色自噬小体与红色自噬溶酶体的数量均增加；此外,cGAS蛋白表达水平升高121%,TANK结合激酶1(TBK1)磷酸化水平升高52.5%(P＜0.05或P＜0.01),且可观察到mtDNA向胞质释放。与MBOAT2过表达组相比,经RU.521干预后,cGAS蛋白表达水平降低58.4%,TBK1磷酸化水平降低64.7%；PINK1、Parkin、LC3Ⅱ/LC3Ⅰ比值及p62分别下降44.1%、53.4%、44.7%和65.0%(P＜0.05或P＜0.01)。同时,黄色自噬小体与红色自噬溶酶体的数量均减少,mtROS水平降低,线粒体嵴结构损伤有所减轻。 [结论]MBOAT2通过诱导血管内皮细胞发生线粒体自噬障碍,进而激活mtDNA-cGAS-STING信号通路。

Abstract:

Aim To investigate the effect of membrane-bound O-acyltransferase domain-containing 2 (MBOAT2) on mitophagy in vascular endothelial cells and its mechanisms. Methods Western blot was used to detect MBOAT2 protein expression in human atherosclerotic plaques. A human umbilical vein endothelial cell model with MBOAT2 overexpression was established. Western blot, transmission electron microscopy, JC-1 staining, MitoSOX Red staining, mRFP-GFP-LC3 dual-fluorescence staining and immunofluorescence staining were employed to assess the effects of MBOAT2 overexpression on mitochondrial injury, mitophagy, cytosolic mtDNA release, and activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. In addition, the cGAS specific inhibitor RU.521 was used for intervention to verify the role of the cGAS-STING signaling pathway in mitochondrial injury and mitophagy dysfuntion. Results Compared with the control group, the expression of MBOAT2 protein in human atherosclerotic plaques increased by 70.7% (P＜0.05). After overexpression of MBOAT2, the expression levels of MIC60, ATP5L and ATP5I in human umbilical vein endothelial cells were downregulated by 15.6%, 18.9% and 25.8%, respectively (P＜0.05 or P＜0.01), accompanied by abnormal mitochondrial ultrastructure, decreased membrane potential and increased mitochondrial reactive oxygen species (mtROS); the expression levels of PINK1, Parkin, LC3Ⅱ and p62 proteins increased by 53.7%, 53.8%, 54.9% and 112.1%, respectively (P＜0.05 or P＜0.01), and the number of yellow autophagosomes and red autolysosomes increased; in addition, the expression level of cGAS protein increased by 121%, the phosphorylation level of TANK-binding kinase 1 (TBK1) increased by 52.5% (P＜0.05 or P＜0.01), and the release of mtDNA into the cytoplasm was observed. Compared with the MBOAT2 overexpression group, after RU.521 intervention, the expression level of cGAS protein decreased by 58.4% and the phosphorylation level of TBK1 decreased by 64.7%; PINK1, Parkin, LC3Ⅱ/LC3Ⅰ ratio and p62 decreased by 44.1%, 53.4%, 44.7% and 65.0%, respectively (P＜0.05 or P＜0.01). At the same time, the number of yellow autophagosomes and red autolysosomes decreased, the level of mtROS decreased, and the damage to mitochondrial cristae structure was alleviated. Conclusion MBOAT2 induces mitophagy dysfunction in vascular endothelial cells, thereby activating the mtDNA-cGAS-STING signaling pathway.