Abstract：
OBJECTIVE: Post-transcriptional modifications and their specific mechanisms are the focus of research on the regulation of myocardial damage. Stress granules (SGs) can inhibit the inflammatory response by inhibiting the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. This study investigated whether alkylation repair homologue protein 5 (ALKBH5) could affect myocardial inflammation and apoptosis during diabetic myocardial ischaemia-reperfusion injury (IRI) through the cGAS-STING pathway via SGs.
METHODS: A diabetes ischaemia-reperfusion rat model and a high glucose hypoxia/reoxygenation cell model were established. Adeno-associated virus (AAV) and lentivirus (LV) were used to overexpress ALKBH5, while the SG agonist arsenite (Ars) and the SG inhibitor anisomycin were used as interventions. Then, the levels of apoptosis and related indicators in the cell and rat models were measured.
RESULTS: In the in vivo experiment, compared with the normal sham group, the degree of myocardial tissue damage, creatine kinase-MB and cardiac troponin I in serum, and myocardial apoptosis, the infarcted area of myocardium, and the level of B-cell lymphoma 2 associated X protein, cGAS-STING pathway and inflammatory factors in the diabetes ischaemia-reperfusion group were significantly increased. However, the expression of SGs and the levels of ALKBH5, rat sarcoma-GTPase-activating protein-binding protein 1, T-cell intracellular antigen-1 and Bcl2 were significantly decreased. After AAV-ALKBH5 intervention, the degree of myocardial tissue damage, degree of myocardial apoptosis, and extent of myocardial infarction in myocardial tissue were significantly decreased. In the in vitro experiment, compared with those in the normal control group, the levels of lactate dehydrogenase, inflammation and apoptosis were significantly greater, and cell viability and the levels of ALKBH5 and SGs were decreased in the high glucose and hypoxia/reoxygenation groups. In the high glucose hypoxia/reoxygenation cell model, the degree of cell damage, inflammation, and apoptosis was greater than those in the high glucose and hypoxia/reoxygenation models, and the levels of ALKBH5 and SGs were further decreased. LV-ALKBH5 and Ars alleviated the degree of cell damage and inhibited inflammation and cell apoptosis. The inhibition of SGs could partly reverse the protective effect of LV-ALKBH5. The cGAS agonist G140 antagonized the inhibitory effects of the SG agonist Ars on cardiomyocyte apoptosis, inflammation and the cGAS-STING pathway.
CONCLUSIONS: Both ALKBH5 and SGs inhibited myocardial inflammation and apoptosis during diabetic myocardial ischaemia-reperfusion. Mechanistically, ALKBH5 might inhibit the apoptosis of cardiomyocytes by promoting the expression of SGs through the cGAS-STING pathway.
METHODS: A diabetes ischaemia-reperfusion rat model and a high glucose hypoxia/reoxygenation cell model were established. Adeno-associated virus (AAV) and lentivirus (LV) were used to overexpress ALKBH5, while the SG agonist arsenite (Ars) and the SG inhibitor anisomycin were used as interventions. Then, the levels of apoptosis and related indicators in the cell and rat models were measured.
RESULTS: In the in vivo experiment, compared with the normal sham group, the degree of myocardial tissue damage, creatine kinase-MB and cardiac troponin I in serum, and myocardial apoptosis, the infarcted area of myocardium, and the level of B-cell lymphoma 2 associated X protein, cGAS-STING pathway and inflammatory factors in the diabetes ischaemia-reperfusion group were significantly increased. However, the expression of SGs and the levels of ALKBH5, rat sarcoma-GTPase-activating protein-binding protein 1, T-cell intracellular antigen-1 and Bcl2 were significantly decreased. After AAV-ALKBH5 intervention, the degree of myocardial tissue damage, degree of myocardial apoptosis, and extent of myocardial infarction in myocardial tissue were significantly decreased. In the in vitro experiment, compared with those in the normal control group, the levels of lactate dehydrogenase, inflammation and apoptosis were significantly greater, and cell viability and the levels of ALKBH5 and SGs were decreased in the high glucose and hypoxia/reoxygenation groups. In the high glucose hypoxia/reoxygenation cell model, the degree of cell damage, inflammation, and apoptosis was greater than those in the high glucose and hypoxia/reoxygenation models, and the levels of ALKBH5 and SGs were further decreased. LV-ALKBH5 and Ars alleviated the degree of cell damage and inhibited inflammation and cell apoptosis. The inhibition of SGs could partly reverse the protective effect of LV-ALKBH5. The cGAS agonist G140 antagonized the inhibitory effects of the SG agonist Ars on cardiomyocyte apoptosis, inflammation and the cGAS-STING pathway.
CONCLUSIONS: Both ALKBH5 and SGs inhibited myocardial inflammation and apoptosis during diabetic myocardial ischaemia-reperfusion. Mechanistically, ALKBH5 might inhibit the apoptosis of cardiomyocytes by promoting the expression of SGs through the cGAS-STING pathway.
摘要：
目的：转录后修饰及其具体机制是心肌损伤调控的研究重点。应激颗粒(SGs)可通过抑制环GMP-AMP合酶(cGAS)-干扰素基因刺激因子(STING)通路抑制炎症反应。这项研究调查了烷基化修复同源蛋白5（ALKBH5）是否可以通过SGs通过cGAS-STING途径影响糖尿病心肌缺血再灌注损伤（IRI）期间的心肌炎症和凋亡。
方法：建立糖尿病缺血再灌注大鼠模型和高糖缺氧/复氧细胞模型。使用腺相关病毒（AAV）和慢病毒（LV）过表达ALKBH5，而SG激动剂亚砷酸盐（Ars）和SG抑制剂茴香霉素用作干预措施。然后，检测细胞和大鼠模型中细胞凋亡及相关指标水平。
结果：在体内实验中，与正常假手术组相比，心肌组织损伤的程度，血清肌酸激酶-MB和心肌肌钙蛋白I，和心肌细胞凋亡，心肌的梗塞区域,和B细胞淋巴瘤2相关X蛋白的水平，糖尿病缺血再灌注组cGAS-STING通路和炎症因子显著升高。然而,SGs的表达和ALKBH5，大鼠肉瘤-GTP酶激活蛋白结合蛋白1，T细胞胞内抗原1和Bcl2的水平显着降低。经过AAV-ALKBH5干预，心肌组织损伤的程度，心肌细胞凋亡程度，心肌组织中心肌梗死程度明显下降。在体外实验中,与正常对照组相比，乳酸脱氢酶的水平，炎症和细胞凋亡显著增加，高糖和缺氧/复氧组的细胞活力和ALKBH5和SGs水平降低。在高糖缺氧/复氧细胞模型中，细胞损伤的程度,炎症,细胞凋亡高于高糖和缺氧/复氧模型，ALKBH5和SGs水平进一步下降。LV-ALKBH5和Ars可减轻细胞损伤程度，抑制炎症反应和细胞凋亡。SGs的抑制可以部分逆转LV-ALKBH5的保护作用。cGAS激动剂G140拮抗SG激动剂Ars对心肌细胞凋亡的抑制作用,炎症和cGAS-STING途径。
结论：ALKBH5和SGs均抑制糖尿病心肌缺血再灌注过程中的心肌炎症和细胞凋亡。机械上,ALKBH5可能通过cGAS-STING通路促进SGs的表达而抑制心肌细胞凋亡。
方法：建立糖尿病缺血再灌注大鼠模型和高糖缺氧/复氧细胞模型。使用腺相关病毒（AAV）和慢病毒（LV）过表达ALKBH5，而SG激动剂亚砷酸盐（Ars）和SG抑制剂茴香霉素用作干预措施。然后，检测细胞和大鼠模型中细胞凋亡及相关指标水平。
结果：在体内实验中，与正常假手术组相比，心肌组织损伤的程度，血清肌酸激酶-MB和心肌肌钙蛋白I，和心肌细胞凋亡，心肌的梗塞区域,和B细胞淋巴瘤2相关X蛋白的水平，糖尿病缺血再灌注组cGAS-STING通路和炎症因子显著升高。然而,SGs的表达和ALKBH5，大鼠肉瘤-GTP酶激活蛋白结合蛋白1，T细胞胞内抗原1和Bcl2的水平显着降低。经过AAV-ALKBH5干预，心肌组织损伤的程度，心肌细胞凋亡程度，心肌组织中心肌梗死程度明显下降。在体外实验中,与正常对照组相比，乳酸脱氢酶的水平，炎症和细胞凋亡显著增加，高糖和缺氧/复氧组的细胞活力和ALKBH5和SGs水平降低。在高糖缺氧/复氧细胞模型中，细胞损伤的程度,炎症,细胞凋亡高于高糖和缺氧/复氧模型，ALKBH5和SGs水平进一步下降。LV-ALKBH5和Ars可减轻细胞损伤程度，抑制炎症反应和细胞凋亡。SGs的抑制可以部分逆转LV-ALKBH5的保护作用。cGAS激动剂G140拮抗SG激动剂Ars对心肌细胞凋亡的抑制作用,炎症和cGAS-STING途径。
结论：ALKBH5和SGs均抑制糖尿病心肌缺血再灌注过程中的心肌炎症和细胞凋亡。机械上,ALKBH5可能通过cGAS-STING通路促进SGs的表达而抑制心肌细胞凋亡。
