线粒体过度融合引起RVLM小胶质细胞激活参与应激性高血压发病的机制以及Sigma-1受体激活的对抗作用

"Neuroinflammation-sympathetic activation" coupling is the characteristic of stress-induced hypertension (SIH), and plays a key role in the maintenance of neurogenic hypertension and genesis of end-organ damage. Microglia activation is a sign of neuro-inflammation in the autonomic centers of the brain, however the trigger mechanism of microglia activation is unclear. Our preliminary study found that mitochondrial hyperfusion was commonly present in the microglia of the rostral ventrolateral medulla in SIH rats, while sigma-1 (σ1) receptor, the endoplasmic reticulum molecular chaperone located on the mitochondrion-associated endoplasmic reticulum membrane, inhibited the activation of microglia, which indicates a correlation between the abnormality of mitochondria and microglia activation. We speculate that σ1 receptor may inhibit microglia activation via regulation of mitochondrial hyperfusion, and consequently reduce neurological inflammation and lower sympathetic activity. This proposal aims to study the mechanisms of mitochondrial hyperfusion-induced microglia activation and the antagonistic effect of σ1receptor activation on them. Expression vector construction or RNA interference technology are to be exploited to observe the effect of σ1 receptor overexpression or silence on the mitochondrial fusion and activation of microglia in vitro. And the agonist and antagonist of σ1 receptors are to be microinjected into the RVLM of the rats to observe the effect of σ1 receptor on blood pressure. Execution of this proposal may supply new target for the exploration of anti-hypertensive drugs.

“炎症-交感”激活是应激性高血压（SIH）的特征，也是维持神经源性高血压及造成终末器官损伤的重要因素。小胶质细胞（MG）激活是应激致神经炎症的标志，但其激活的触发机制不清。本课题组前期在观察SIH大鼠交感中枢延髓头端腹外侧区线粒体异常与MG激活之间的关系时发现：激活的MG内线粒体过度融合，位于线粒体相关内质网膜上的内质网伴侣分子Sigma-1（σ1）受体表达减少；而激活σ1受体则能减少MG的激活。我们推测σ1受体的动态激活可能通过抑制MG激活减轻神经炎症，进而降低交感活动。拟采用表达载体构建或RNAi等技术观察σ1受体过表达或沉默对体外培养的MG激活和线粒体融合的影响；拟通过定向激活或抑制SIH大鼠脑内σ1受体明确其在体动态调控对交感活动的调节作用。该项目为应激致高血压发病机制提供了新视点，其执行有望把σ1受体确定为抗高血压新药研制的靶点，具有重要意义。

Sigma-1受体(Sig-1R)定位于线粒体相关内质网膜（ＭＡＭs），在海马锥体、黑质多巴胺神经元和胶质细胞选择性高表达。破坏或抑制Sig-1R功能可干扰内质网－线粒体的钙稳态，诱导内质网应激和破坏线粒体功能。本研究的目的是探讨Sig-1R对应激性高血压（SIH）大鼠延髓头端腹外侧区（RVLM）神经炎症的影响及对其血压（BP）和交感神经活动（RSNA）的影响。SIH大鼠模型采用每天两次，每次2h的间歇性电击加噪音刺激15-20天来制备。使用免疫印迹，qRT-PCR和/或免疫荧光染色检测线粒体融合蛋白（MFN1，MFN2和OPA1）以及线粒体分裂蛋白（与动力相关的蛋白1，Drp1）的表达。Prorenin是脑肾素-血管紧张素系统(RAS)的一员，可直接激活MG。SIH大鼠RVLM中prorenin、prorenin受体(PRR)表达增加及NLRP3活化增加。因此离体细胞采用prorenin 刺激。在prorenin刺激的MG中，更多的NLRP3从细胞质转移到线粒体中。Prorenin增加ROS触发M1表型转换和NLRP3激活，而NLRP3阻断剂MCC950降低了Prorenin诱导的M1极化及促炎因子的释放。我们发现Sig-1R在SIH大鼠的RVLM中伴随着促炎表型小胶质细胞增加而下调。显微注射Sig-1R激动剂SKF10047到SIH大鼠的RVLM中来验证Sig-1R对M1表型极化的影响。结果表明，SKF10047降低SIH大鼠RVLM区促炎细胞因子的表达，降低SIH大鼠动脉血压和RSNA。SKF10047处理的应激大鼠小胶质细胞M1/M2表型转换降低。透射电镜显示SKF10047处理的应激大鼠小胶质细胞MAMs升高，与共聚焦显微镜观察到的Mito-Tracker和ER-Tracker共定位增多相一致。透射电子显微镜成像表明SKF10047降低应激相关的线粒体过度融合和伸长。应激线粒体分裂蛋白DRP1的表达降低，Drp1过表达可以通过减少线粒体过度融合减少小胶质细胞炎症因子的释放。结论：Sig-1R可能通过抑制SIH大鼠小胶质细胞的线粒体过度融合而减轻RVLM中的M1免疫表型转换和神经炎症，从而降低SIH大鼠交感活性和血压。阐明Sig-1R受体通过干预该机制产生对抗SIH的效应。干预上述一个或者多个环节，可能为应激性难治性高血压防治提供潜在的新策略。