心脏干细胞源外泌体介导长链非编码RNA-DMIR在糖尿病心肌缺血再灌注损伤细胞焦亡中的作用机制研究

Diabetes mellitus increases the sensitivity of myocardial cells to ischemic injury and eventually leads to a marked increase in the incidence and mortality of diabetic ischemic heart disease. We found that exosomes from cardiac stem cell (CSC) reduced apoptosis of ischemia-reperfusion (I/R) cardiomyocyte and the long-chain non-coding RNAs (lncRNAs) were differential expressed after exosome treatment by using microarray technology. In addition, CSC exosomes significantly inhibited cell apoptosis following diabetic I/R injury, suggesting that the anti-pyroptosis effect of exosomes may be related to the regulation of lncRNAs in exosomes. Therefore, we hypothesized that CSCs secrete exosomes and the exosomes transmit lncRNA signals into diabetic myocardial I/R cardiomyocytes and regulate mitochondrial division and fusion dynamics, thereby inhibiting cell pyroptosis. To explore the mechanisms of lncRAN-DMIR of CSC-derived exosomes in inhibiting the pyroptosis in diabetic I/R cardiomyocytes, and to provide a new theoretical basis for the prevention and treatment of diabetic myocardial I/R injury, the lncRNA co-expressed in CSC-derived exosomes and diabetic I/R cardiomyocytes(lncRNA-DMIR) will be screened later in conjunction with lncRNA microarrays, bioinformatics analysis and the use of gain-/loss-of-function experiments in the whole animal and cardiomyocytes.

糖尿病使心肌对缺血性损伤敏感性增加，最终导致糖尿病缺血性心脏病发病率与死亡率明显增高。我们前期发现心脏干细胞（CSC）源外泌体可抑制缺血再灌注（I/R）心肌细胞凋亡，应用芯片技术明确此过程中lncRNA差异表达明显。此外，CSC源外泌体可抑制糖尿病心肌I/R损伤后细胞焦亡，提示外泌体抗焦亡作用可能与外泌体内lncRNA调控有关。因此，我们提出假设：CSC分泌外泌体将lncRNA信号传递至糖尿病心肌I/R心肌细胞内，调控线粒体分裂融合动态，从而抑制细胞焦亡。后期拟结合lncRNA芯片、生物信息学分析技术等筛选出CSC源外泌体和糖尿病I/R心肌细胞共表达的lncRNA（lncRNA-DMIR），拟在整体动物和细胞水平利用gain-/loss-of-function试验探索CSC源外泌体lncRAN-DMIR抑制糖尿病I/R心肌细胞焦亡的机制，为临床糖尿病心肌I/R损伤的防治提供新的理论依据。

糖尿病使心肌对缺血性损伤敏感性增加，最终导致糖尿病缺血性心脏病发病率与死亡率明显增高。线粒体分裂与融合平衡和细胞焦亡在心肌缺血再灌注损伤（I/R）中发挥重要作用。研究发现，干细胞源外泌体可通过与心肌细胞间的信息交流参与调控缺血再灌注损伤后细胞焦亡，含有大量非编码RNA的外泌体可能是介导旁分泌效应的重要载体。本课题探讨了骨髓间充质干细胞源外泌体（BMSCs-Exo）是否通过传递长链非编码RNA（LncRNA）至糖尿病心肌I/R损伤后心肌细胞内，调控线粒体分裂融合动态，从而抑制细胞焦亡。本研究使用H9c2心肌细胞建立体外高糖缺氧复氧（H/R）损伤模型，同时在体内建立糖尿病小鼠心肌缺血再灌注（I/R）损伤模型。结果显示，糖尿病心肌I/R损伤可通过激活DRP1使心肌细胞线粒体分裂/融合动态失衡，促进细胞焦亡，而BMSCs-Exo干预可对其进行逆转。BMSCs-Exo不仅减少了糖尿病小鼠I/R损伤后心肌梗死面积，而且降低了高糖H/R处理后H9c2心肌细胞LDH释放。BMSCs-Exo可通过线粒体分裂蛋白动力素相关蛋白1（DRP1）降低线粒体分裂，抑制NLRP3/Caspase-1/GSDMD轴介导的高糖H/R状态下心肌细胞焦亡。随后，我们采用转录组测序分析高糖H/R与BMSCs-Exo处理后高糖H/R H9c2心肌细胞中差异表达的LncRNA与mRNA，我们发现Lnc LOC100911553（即 Lnc-DMIR）在BMSCs-Exo处理后高糖H/R H9c2心肌细胞及BMSCs-Exo中呈高表达。敲低BMSCs中LOC100911553可以逆转BMSCs-Exo对高糖H/R处理的H9c2心肌细胞LDH释放以及NLRP3/Caspase-1/GSDMD信号通路的抑制作用，也可消除BMSCs-Exo对高糖H/R处理后H9c2心肌细胞线粒体的保护作用。我们通过LncRNA下拉分析鉴定了LOC100911553与IGF2BP1的靶向关系，同时探索LOC100911553/IGF2BP1/NLRP3的调控关系，观察到LOC100911553通过隔离IGF2BP1加速了NLRP3 mRNA的降解。综上所述，BMSCs-Exo可通过传递Lnc LOC100911553 隔离IGF2BP1降低NLRP3表达，失活NLRP3/Caspase-1/GSDMD信号通路，调控线粒体分裂/融