METTL3调控PGC-1αm6A甲基化修饰在Reg3β抵抗糖尿病心肌炎症反应中的研究

Diabetes Mellitus (DM) are prone to cardiac dysfunction and increasing risk of adverse cardiovascular events caused by metabolic inflammation. Despite the enthusiasm and effort invested in developing targeted therapeutics for cardiac metaflamamtion, to date, no effective approaches are available to improve the cardiovascular outcomes of diabetic patients. Unpublished data from our group shown that the regenerative protein 3β (Reg3β) can inhibit hyperglycemia-induced apoptosis in H9C2 cardiomyocytes, which may be associated with the activation of JAK2/STAT3 pathway and the decreased expression of inflammatory factor, such as IL-1β and TNF-α. However, we have not explored the mechanism of the anti-inflammatory of Reg3β. Previous studies have confirmed that hyperglycemia inhibit the expression of PGC-1α followed by activation of NF-κB-mediated inflammatory response. Our pre-test results found that Reg3β increased the level of PGC-1α mRNA and inhibited the expression of m6A RNA methyltransferase METTL3. Bioinformatics analysis found that the conditional knockdown of METTL3 reduced the m6A methylation enrichment of PGC-1α in cerebellum. Based on these facts, we hypothesizes that Reg3β can inhibit PGC-1α m6A methylation mediated by METTL3 to attenuate myocardial metaflammation in DM via JAK2/STAT3 pathway by using neonatal rat cardiomyocyte and Reg3β-/- mice. This study not only helps to understand the molecular mechanisms of m6A methylation, but also strengthens the knowledge of fundamental research about cardiovascular disease in diabetes mellitus. This thesis will contribute to developing new strategies for the prevention and treatment of cardiovascular disease in DM, which is valuable and significant for academic research and public health.

糖尿病极易因心肌代谢性炎症损伤而增加不良心血管事件风险，但目前仍缺乏有效的治疗手段，亟需针对性开展干预靶点研究。申请者待发表数据显示，Reg3β抑制高血糖诱导的心肌细胞凋亡，且与JAK2/STAT3通路活化和炎症因子减少有关，但Reg3β的抗炎机制仍不明确。申请者预实验发现，Reg3β增加PGC-1α mRNA水平，抑制m6A RNA甲基化转移酶METTL3表达，敲减METTL3可减少PGC-1α m6A修饰。据此，我们推测Reg3β能活化JAK2/STAT3通路抑制METTL3介导的PGC-1α m6A修饰以抵抗糖尿病心肌炎症损伤。本研究针对糖尿病心肌代谢性炎症损伤的核心发病机制，采用CRISPR/Cas9技术构建Reg3β-/-小鼠，结合m6A甲基化研究前沿，探索Reg3抗炎机制，既有助于深入理解m6A的调控网络，又能为防治糖尿病心血管疾病开拓新思路，具有较高的研究意义和临床价值。

糖尿病心血管并发症是严重影响我国人民身心健康的疾病。本课题拟确认Reg3β的糖尿病心肌保护作用及其关键环节，为防治糖尿病心血管疾病开拓新思路。为此，本项目设计相关内容并取得以下重要成果：1）成功构建Reg3β干扰载体（pG1.2-Reg3β shRNA）和过表达载体（pIRES2-Reg3β cDNA），其中Reg3β干扰载体针对159-178位碱基，抑制率为67.93%。2）与对照小鼠相比，db/db小鼠在16周龄时血糖升高且体重增加，心脏彩超提示左心室舒张功能受损，Masson染色显示心肌纤维化增多。3）免疫组化提示12周龄和16周龄db/db小鼠心脏组织Reg3β表达减少；在H9C2心肌细胞时中，33.3mM葡萄糖能抑制细胞内Reg3β蛋白表达。4）Reg3β干预和pIRES2-Reg3β cDNA可促进JAK2和STAT3的磷酸化，减少Caspase3的活化，抑制H9C2心肌细胞凋亡；JAK2和STAT3的抑制剂可抑制Reg3β的抗凋亡作用。5）人心脏组织中Reg3β和METTL3表达量成负相关，METTL3敲减后的小鼠小脑组织内PGC-1α m6A减少。PCR显示Reg3β减少H9C2细胞METTL3 mRNA并增加PGC-1α mRNA表达量。6）具有心血管保护作用的GLP-1受体激动剂Exendin-4同样可诱导Reg3β表达上调。7）临床研究发现，新冠肺炎患者中，与非糖尿病患者相比，糖尿病患者心肌损伤发生率和死亡风险均增加约3倍。糖尿病合并心肌损伤患者中心粒细胞计数和IL-6水平更高，而淋巴细胞和其他免疫细胞均为较低水平。Logistics回归分析显示，与糖尿病患者心肌损伤发生风险独立相关的免疫指标是CD3+CD4+ T性细胞计数 ≤288 cells/μl (校正后OR, 2.501; 95% CI, 1.282-4.877; p=0.007), IL-6˃25.68mpg/ml (校正后OR, 4.345; 95% CI, 2.192-10.374; p˂0.001)。以上成果提示虽然糖尿病容易出现心肌损伤，但Reg3β具有潜在的糖尿病心肌保护作用。因此，开发以Reg3β为靶点的药物将为糖尿病心血管并发症的治疗提供新的策略，具有较高的临床应用前景和社会意义。项目执行过程中已发表SCI论文4篇，统计源文章1篇，已录用统计源文章1篇，培养研究生3人。