STIM1/Orai1介导的钙池操纵性钙内流在氢气治疗脓毒症肺损伤中的机制研究

Lung is the most easily damaged organ by sepsis. Currently there is still no comprehensively effective therapeutics on sepsis-induced acute lung injury. Sepsis-induced depletion of calcium store in the endoplasmic reticulum can continuously activate store-operated calcium entry (SOCE) mediated by stromal interaction molecule 1 (STIM1) and Orai1. Meanwhile, expression of plasma membrane STIM1 can be abnormally elevated. These changes cause a series of cell dysfunction including failure of intracellular calcium homeostasis, abnormal expression of cell-cell tight junction proteins and even apoptosis. Our preliminary results indicate that hydrogen treatment can significantly alleviate lung injury in sepsis, protect pulmonary microvascular endothelial cells from hyperpermeability, and decrease the expression of plasma membrane STIM1, but the underlying mechanism that how hydrogen maintains calcium homeostasis of endothelial cells in septic models remains unclear. In this study, we propose to investigate the molecular mechanism of STIM1-Orai1 regulation by hydrogen treatment, explore the effect of hydrogen treatment on calcium homeostasis in pulmonary microvascular endothelial cells, and further elucidate the dependency of hydrogen's effect on calcium and STIM1 during treatments on sepsis. The results of this study can help us understand the mechanism of hydrogen treatment on sepsis, illustrate the significance of hydrogen treatment on calcium homeostatic failure-associated diseases, and provide solid theoretical basis for the clinical application of hydrogen in the future.

肺脏是脓毒症最易损伤的靶器官，目前仍缺乏针对脓毒症急性肺损伤的全面而有效的治疗方法。脓毒症时细胞内质网钙库耗竭使STIM1/Orai1介导的钙池操纵性钙内流持续进行，且细胞膜STIM1表达异常升高，导致了包括细胞内钙稳态被破坏、内皮细胞紧密连接蛋白表达异常乃至凋亡等一系列细胞功能损伤。我们的前期研究发现，氢气可显著减轻脓毒症小鼠的肺损伤，维持肺微血管内皮细胞通透性，并降低脓毒症时细胞膜STIM1的表达，但氢气治疗对保护脓毒症模型钙稳态的具体分子机制尚不明确。本项目中，我们将研究氢气对钙池操纵性钙内流的关键蛋白STIM1和Orai1的调控机制，探索脓毒症时氢气在治疗肺损伤过程中对肺微血管内皮细胞钙稳态的影响，并阐明氢气在治疗脓毒症过程中对钙离子和STIM1的依赖性，以了解氢气治疗脓毒症的分子机制，明确氢气在缓解钙稳态失调所致疾病中的价值和意义，并为氢气未来的在临床上的应用提供充分的理论依据。

急性肺损伤（ALI）是脓毒症患者主要并发症之一，肺微血管内皮细胞（PMVEC）损伤将直接导致ALI的发生，钙稳态失调是脓毒症导致肺微血管内皮细胞功能损伤的关键机制之一。本课题组前期发现氢气明显减轻脓毒症小鼠肺损伤，维持肺微血管内皮细胞通透性，并降低脓毒症时细胞膜STIM1的表达，但氢气治疗对保护脓毒症模型钙稳态的具体分子机制仍不清。本研究主要探讨STIM1/Orai1介导的钙池操纵性钙内流在氢气治疗脓毒症肺损伤中的作用，采用Western blot、RT-PCR、免疫荧光和钙离子染色等分子生物学技术，在脓毒症小鼠肺损伤模型及PMVEC培养模型的基础上，从在体、离体和分子水平等不同层次证明：STIM1/Orai1在脓毒症肺损伤中的作用；氢气通过下调PMVEC中STIM1及Orai1的表达及相互作用来改善脓毒症肺损伤，以了解氢气治疗脓毒症的分子机制，上述研究结果将为氢气治疗脓毒症的临床应用奠定理论基础，为研发治疗脓毒症的药物靶点提供依据。