STIM1-nSOCE调控内质网线粒体轴在脑缺血再灌注损伤中的作用及机制研究

With high rate of morbidity, disability and mortality, cerebral ischemic stroke (IS) has been a serious threat to our health and life quality. Despite some reperfusion therapies being extensively applied in clinic, more attentions should be paid to alleviate ischemia-reperfusion injury. Endoplasmic reticulum-mitochondrial axis dysfunction, which cause homeostasis imbalance and then neuronal death, may be the key point of IS injury, but its exact molecular pathology is need to be illuminated. Our previous studies have found that neuronal hypoxia can activate STIM1-mediated neuronal Ca2+ store-operated calcium entry (STIM1-nSOCE), causing mitochondrial homeostasis; inhibition of STIM1-nSOCE can restore oxidative stress-induced ER and mitochondrial dysfunction. Moreover, STIM1-nSOCE is also involved in the regulation of endoplasmic reticulum-mitochondrial calcium signaling, suggesting that STIM1-nSOCE may play an important role in the regulation of endoplasmic reticulum-mitochondrial axis function after ischemia-reperfusion injury. Therefore, this study intends to clarify the role of STIM1-nSOCE in cerebral ischemia-reperfusion injury by using SOCE drug intervention and STIM1-shRNA, CRISPR / Cas9 gene intervention, furtherly clarifying the mechanism of ischemia-reperfusion injury and revealing new intervention targets of ischemia-reperfusion.

缺血性脑卒中(IS）发生率及残死率高，尽管再灌注治疗得到广泛应用，但目前仍缺乏有效的药物干预。内质网-线粒体轴功能障碍可引起内环境稳态失衡，参与IS损伤的关键环节，然其具体分子病理机制尚不清楚。我们的前期研究发现，缺血缺氧可激活STIM1介导的神经元钙库操纵性钙内流（STIM1-nSOCE），引起线粒体稳态失衡；而抑制STIM1-nSOCE可恢复氧化应激所致的内质网功能及线粒体功能障碍。结合文献报道，STIM1-nSOCE参与调控内质网-线粒体间钙信号交流，提示STIM1-nSOCE可能在IS后内质网-线粒体轴功能调控中发挥重要调控作用。因此，本课题拟利用nSOCE药物干预及STIM1-shRNA、CRISPR/Cas9基因干预等技术，明确STIM1-nSOCE调控内质网-线粒体轴在脑缺血再灌注损伤中的作用，为进一步阐明IS致伤机制、揭示新的IS干预靶点及防治策略，提供有价值的理论依据。

缺血性脑卒中(IS）发生率及残死率高，尽管再灌注治疗得到广泛应用，但目前仍缺乏有效的药物干预。内质网-线粒体轴功能障碍可引起内环境稳态失衡，参与IS损伤的关键环节， 然其具体分子病理机制尚不清楚。本项目采用药物干预及STIM1-shRNA、CRISPR/Cas9基因干预等技术探讨 STIM1-nSOCE调控内质网-线粒体轴在神经元氧糖剥夺再灌注损伤中作用及机制。研究发现：（一）氧糖剥夺再灌注损伤（OGD/Rep.）后钙库操纵性钙内流功能异常：其主要构成分子STIM1及Orai1基因及蛋白表达无明显改变，然STIM1斑块化及细胞膜定位明显增高；内质网钙库钙水平下降且介导的SOCE水平也明显下降；（二）抑制STIM1-nSOCE可明显减轻神经元OGD/Rep.损伤：神经元LDH释放减少及凋亡下降；（三）、抑制STIM1-nSOCE可减轻神经元OGD/Rep.引起的内质网-线粒体功能障碍：减轻内质网应激、线粒体氧化应激、线粒体融合/分裂异常、线粒体钙超载及线粒体膜电位去极化；（四）抑制STIM1-nSOCE可通过调控自噬流改善自噬降解受阻，从而促进受损线粒体清除，从而发挥神经保护作用；（五）敲除神经元细胞系HT-22 STIM1表达，Patharray分析表明并未引起明显基因差异性表达，提示其可能通过调控蛋白降解、修饰或通道蛋白开放等发挥作用；（六）抑制STIM1可明显减轻OGD/Rep.损伤引起的线粒体-内质网轴关键调控分子MFN2表达下降，而上调MFN2表达可明显减轻OGD/Rep.损伤引起的线粒体功能障碍。综上，STIM1-nSOCE参与调控ODG/Rep.损伤后内质网-线粒体轴功能调节。抑制STIM1nSOCE主要通过减少MFN2降解维持内质网-线粒体互作、促进受损线粒体自噬清除等机制改善内质网-线粒体功能障碍，从而发挥神经元保护作用。本研究为进一步阐明IS致伤机制、揭示新的IS干预靶点及防治策略，提供一定的理论依据。