中性粒细胞TRPM2通道在脓毒症细菌清除中的作用及机制

Increasing evidence supports that immunosuppression exits in septic patients and plays a central role in the pathogenesis of sepsis. The immune cells of both innate and adaptive system are severely suppressed in septic patients, resulting in reduced capability of immune cells to eradicate invading pathogens. Transient Receptor Potential Melastatin 2 (TRPM2), a nonselective Ca2+-permeable channel, is expressed abundantly in macrophages and neutrophils. Our recent study demonstrated a protective role of TRPM2 in controlling bacterial clearance during sepsis possibly by promoting bacterial killing capability of macrophages. However, the role of neutrophil TRPM2 in bacterial clearance during sepsis and its underlying mechanism remain unclear. Using neutrophils from Trpm2-knockout mice, we will firstly investigate the effect of TRPM2 on the bacterial killing capability and degranulation of neutrophils by bacterial killing experiment and flow cytometry. Then, we will further to clarify the detailed molecular mechanism of the effect of TRPM2 on the bacterial killing capability and degranulation of neutrophils by bacterial killing experiment, electrophysiology, intracellular calcium concentration detection, MAPKs inhibitors, flow cytometry, and immunoblot. We will finally confirm the protective role of TRPM2 in promoting bacterial clearance during murine sepsis by regulating the degranulation of neutrophils and its underlying mechanism. Our study will uncover a novel mechanism of the protective role of TRPM2 in sepsis, which provides a new target for the prevention and treatment of sepsis.

免疫细胞功能抑制导致脓毒症患者体内细菌清除能力严重下降在脓毒症的发病机制中发挥重要作用。 瞬时受体电位通道M2 (TRPM2) 是高表达于巨噬细胞和PMN的非特异性Ca2+通道。我们前期研究发现TRPM2通道通过促进巨噬细胞杀菌功能保护脓毒症。然而，中性粒细胞 (PMN)TRPM2通道在脓毒症细菌清除中的作用及机制尚不清楚。本研究使用TRPM2-KO小鼠PMN，通过离体杀菌实验和流式细胞检测TRPM2对PMN脱颗粒和杀菌能力的影响；通过杀菌实验、电生理、钙离子测定、流式细胞检测和Western blot等方法深入研究TRPM2对PMN脱颗粒和杀菌作用影响的具体分子机制；使用TRPM2-KO小鼠，通过小鼠脓毒症模型，验证TRPM2通过调控PMN脱颗粒作用促进脓毒症小鼠体内细菌清除保护脓毒症的作用及其机制。本研究将进一步揭示TRPM2保护脓毒症的新机制，为脓毒症的治疗提供新的靶点奠定理论基础。

中性粒细胞（PMN））释放的弹性酶对消除革兰氏阴性菌至关重要。细胞外钙离子内流对PMN弹性蛋白酶释放和细菌清除能力中起着关键作用。瞬时受体电位通道M2（TRPM2）是一种ca2+-通透性阳离子通道，在PMN高度表达。在此，我们探讨trpm2在PMN细菌清除中的作用和可能的机制。基因敲除TRPM2（TRPM2-KO）），减少了小鼠骨髓来源PMN弹性蛋白酶的释放，且同时降低了其清除大肠杆菌的能力。与野生(WT）小鼠相比，TRPM2-KO脓毒症小鼠腹腔灌洗液中弹性蛋白酶脓度明显降低且细菌清除力下降，生存率下降。甲酰肽（fMLP)）刺激TRPM2-KO PMN，其弹性蛋白酶释放明显低于WT PMN。P38 MAPK抑制剂能明显降低fMLP刺激WT PMN释放弹性蛋白酶的能力，同时在fMLP刺激下TRPM2-KO小鼠PMN的P38 MAPK磷酸化水平明显低于WT小鼠PMN。抑制细胞外钙离子内流明显降低WT小鼠PMN的p38 MAPK磷酸化和弹性蛋白酶释放。TRPM2-KO小鼠PMN在fMLP刺激下其细胞内钙离子脓度明显低于WT小鼠PMN。我们的结果发现，TRPM2可能通过调控弹性蛋白酶在PMN杀菌中起到重要作用。TRPM2介导的钙离子内流可能部分通过调控P38MAPK磷酸化控制PMN弹性蛋白酶的释放。