STIM1/Orai1调控重症休克血管低反应性的作用机制研究

Vascular hyporeactivity is crucial to persistent low pressure during severe shock in clinic. It has been demonstrated that once ER Ca2+ is depleted, STIM1 functions as an ER Ca2+ sensor, translocates to the close proximity of plasma membranes and activates Orai channels to mediate extracellular Ca2+ influx. Whether STIM1/Orai1 was involved in vascular hyporeactivity following severe shock remained obscure. Our previous experiments have found that L-VDCC mediated extracellular Ca2+ influx was inhibited while the expression of Orai1 was increased; intracellular Ca2+ overload and inhibited Ca2+ response to vasoconstrictors were persistent in smooth muscle cells following shock. We suggested that STIM1/Orai1 might contribute to the regulation of Ca2+ homeostasis and vascular hyporeactivity in severe shock. The present study is based on a model rat with severe hemorrhagic shock and an in vitro model of hypoxia-reoxygenation injury in artery and smooth muscle cells, simultaneous vessel diameter and Ca2+ measurement, patch clamp technique and molecular biology techniques were combined to try to prove our hypothesis, and further to explore the underling mechanisms involved in vascular hyporeactivity. This study has very important significance with regard to further elucidation of the mechanisms involved in the refractory hypotension during severe shock and highlighting the need to develop new treatment targets.

血管低反应性是导致重症休克患者顽固性低血压的主要原因。内质网钙库Ca2+感受器基质交感分子1（STIM1）转位激活细胞膜上Orai1通道介导的外Ca2+内流参与调控细胞内Ca2+稳态，但其与休克血管低反应性之间的关系及其机制目前未见报道。前期研究发现重症休克晚期小动脉平滑肌Orai1的表达增加、电压依赖性钙通道抑制；而细胞内仍存在钙超载现象，血管收缩物质引起的细胞内Ca2+升高不足导致小动脉反应性下降。由此我们推测：STIM1/Orai1参与调控重症休克平滑肌细胞内的钙稳态失调和血管低反应性的发生。本研究拟采用大鼠重症失血性休克模型和体外缺氧复氧模型，联合应用血管直径和胞内钙同步检测技术、膜片钳和分子生物学等技术，从整体-血管-细胞多层次证明我们的假说，并探讨STIM1/Orai1调节休克细胞内钙稳态失调和血管低反应性的分子机制。此研究将为重症休克的防治和有效药物的开发提供新的思路和靶点。

重症休克晚期血管低反应性是导致微循环障碍、顽固型低血压的重要原因。探讨重症休克时血管反应性低下的原因和机制，对寻求及时有效的治疗手段具有重要意义。本项目在前期研究的基础上，将大鼠失血性休克模型和小动脉平滑肌细胞缺氧复氧模型相结合，分别从动物水平和细胞分子不同层面上探讨了重症休克小动脉平滑肌细胞 STIM1/Orai的表达和功能变化与休克血管低反应性之间的相关性。研究发现，休克时STIM1的表达下调和功能异常导致细胞内钙稳态失调进而加重了血管平滑肌细胞的线粒体损伤，促使线粒体通透性转变孔MPTP的重要结构蛋白CyPD的表达上调，MPTP开放时间增加，导致休克大鼠小动脉血管反应性下降，生存时间缩短。过表达STIM1可显著抑制MPTP的开放，改善休克大鼠肠系膜动脉的血管反应性。此外，重症休克过程中伴随的缺氧和复氧导致细胞内活性氧的大量产生，还原型谷胱甘肽和氧化型谷胱甘肽的比例失衡，导致细胞内蛋白谷胱甘肽化修饰增加。由于平滑肌细胞的STIM1/ORAI电流较小，我们采用四环素诱导的稳定过表达STIM1/ORAI的Trex细胞系来观察缺氧复氧或氧化应激（H2O2）对细胞电流以及STIM1向细胞膜上转位的影响。研究发现，在没有TG诱导的情况下，缺氧复氧可导致细胞内肌浆网上的STIM1蛋白向细胞膜上聚集，而H2O2虽然可以显著抑制STIM1/ORAI的电流，但不影响STIM1向细胞膜上的转位。该项目的实施首次发现重症休克时肠系膜动脉平滑肌细胞STIM1表达下调与线粒体的功能障碍相关，提示以STIM1为靶点的药物筛选有望改善休克血管的反应性。该研究将为重症休克顽固性低血压发生机制的深入研究以及推动新的有效靶点药物的开发提供更充分的科学依据，并为重症休克的治疗提供新思路。