基于细胞自噬探讨星状神经节阻滞减轻失血性休克后急性肺损伤作用的肠-淋巴机制

The post-hemorrhagic shock mesenteric lymph (PHSML) return is an important bridge and fundamental mechanism of hemorrhagic shock-induced acute lung injury (ALI). Based on the previous experimental result of stellate ganglion block (SGB) lengthening the survival time and improving the intestinal barrier function in rats following hemorrhagic shock, combined with the theoretical basis of excessive autophagy inducing cell injury, we hypothesized that SGB reduced intestinal barrier damage through alleviating ischemia and inflammation-induced autophagy, thereby, decreased the poisonous substances absorption and transportation via PHSML, as a result, alleviated ALI caused by hemorrhagic shock. To test the hypothesis, in the current study, we will investigate the effects of SGB treatment on the intestinal barrier function, the volume and poisonous substances in PHSML, pulmonary permeability, autophagy-related proteins and PI3K, p-Akt, p-mTOR expressions in rats following hemorrhagic shock, and observe the effects of PHSML with SGB or not on the morphology, monolayer permeability, autophagy-related proteins and PI3K, p-Akt, p-mTOR expressions in pulmonary microvascular endothelial cells. Furthermore, we try to observe the effects of agonists, inhibitors and small interference (si) RNA of autophagy on above indices. In conclusion, the present study will clarify the role and intestinal-lymph mechanism of SGB treatment alleviating severe hemorrhagic shock-induced ALI, focussing on autophagy. Furthermore, the current study can not only elucidate the new theory of the ALI induced by severe hemorrhagic shock, but also supply new approaches of prevention and treatment of severe shock and ALI through SGB improving the intestinal barrier function. Thereby, expand the clinical application of SGB in the intensive medicine.

失血性休克后肠淋巴液回流是发生急性肺损伤（ALI）的重要桥梁与基本机制。本项目在前期发现星状神经节阻滞（SGB）延长失血性休克大鼠存活时间、改善肠屏障功能的基础上，结合过度自噬是细胞损伤重要机制的这一理论基础，提出了“SGB通过降低缺血、炎症引起的自噬减轻肠屏障损伤、减少毒性物质经肠淋巴液的吸收与回流，从而减轻失血性休克ALI”的假说。据此，应用清醒大鼠失血性休克模型以及休克肠淋巴液损伤肺微血管内皮细胞模型，以SGB为手段，采用自噬激动剂、抑制剂或siRNA，观察肠屏障功能、肠淋巴液流量与毒性成分、肺通透性、细胞形态与通透性、自噬相关蛋白及PI3K-Akt-mTOR通路的变化，聚焦细胞自噬，系统研究和确立SGB减轻重症休克ALI肠-淋巴机制。在此基础上，实现重症休克ALI发生机制的理论新突破，形成以SGB改善肠屏障功能、防治重症休克与ALI的新策略，藉此拓展SGB在重症医学领域的临床应用。

失血性休克后肠淋巴液（PHSML）回流是发生急性肺损伤（ALI）的重要桥梁与基本机制。本项目应用清醒大鼠失血性休克模型以及PHSML损伤肺微血管内皮细胞（PMVECs）模型，以星状神经节阻滞（SGB）为干预措施，采用自噬激动剂、抑制剂，从动物与细胞两个层面，观察肠屏障功能、肠淋巴液毒性成分、肺通透性、细胞形态与通透性、自噬相关蛋白及PI3K-Akt-mTOR通路的变化，系统研究和确立细胞自噬在SGB减轻重症休克ALI肠-淋巴机制中的作用。动物实验显示，SGB前处理或后处理均可显著提高失血性休克大鼠的存活率，显著降低了肠通透性、改善了肠屏障功能与肠血流、减轻了肠组织损伤与细胞自噬；SGB降低了PHSML内毒素与炎症因子水平；SGB减轻了失血性休克大鼠的肺组织损伤，降低了肺血管通透性增加引起的组织水肿，降低了肺组织细胞自噬相关蛋白表达；抑制细胞自噬（3-MA）可对抗失血性休克对肠、肺损伤的作用，静脉输入PHSML、增强细胞自噬（RAPA）均可抑制SGB的良好作用。在建立大鼠PMVECs原代培养技术的基础上，证实了PHSML降低了PMVECs增殖活性，引起了细胞结构损伤，增加了细胞自噬相关蛋白表达，降低了PI3K-Akt-mTOR信号通路中关键分子mTOR的磷酸化水平；SGB处理的PHSML（PHSML-SGB）处理PMVECs的作用与未处理PHSML相反。这些研究表明，SGB通过抑制细胞自噬减轻了失血性休克后肠屏障功能障碍与结构损伤，进而减轻了PHSML介导的ALI。研究结果不仅丰富了休克ALI的发病学理论，也形成了以SGB改善肠屏障功能、防治重症休克与ALI的新策略，为拓展SGB应用于重症休克的临床防治提供了实验依据和理论基础。