内质网应激在雌激素提高重症休克淋巴管收缩功能中的作用与机制

Lymphatic contractile dysfunction is an important reason of refractory shock. Estrogen treatment could alleviate the organ injury of severe hemorrhagic shocked animal, but, it is worthy to observe whether estrogen treatment enhancing lymphatic contraction following severe shock. Endoplasmic reticulum stress (ERS) is involved in the development of hemorrhagic shock. However, whether the mechanism by which estrogen regulating lymphatic contraction is related to ERS, it remains unknown. Based on the previous experimental result of 17β estradiol improving mesentery lymphatic contraction in rats following hemorrhagic shock, in the present study, we will investigate that the effects of estrogen treatment on the contraction of mesenteric lymphatics in vivo and in vitro, the contraction of lymphatic smooth muscle cells (LSMCs), the relationship between the contraction and Ca2+ concentration, the apoptosis, the expressions of ERS-related markers, calcium channel-related proteins, and apoptosis-related proteins using the models of hemorrhagic shock in rat and hypoxia/reoxygenation in LSMCs. Meanwhile, we try to observe the effects of agonists, inhibitors and small interference (si) RNA of estrogen receptors and ERS on above indices. In conclusion, the present study will clarify the role and regulatory mechanism of ERS in estrogen treatment enhancing lymphatic contraction following severe shock. Furthermore, the current study can not only elucidate the new theory of the lymphatic contractile dysfunction induced by severe hemorrhagic shock, but also supply new approaches of prevention and treatment of severe shock through estrogen regulating lymphatic contraction, thereby, provide the experimental evidences and theoretical support for clinical application of estrogen.

淋巴管收缩功能障碍是休克难治的关键环节；雌激素治疗可减轻重症失血性休克动物的器官损伤，但能否提高淋巴管收缩性，值得研究；内质网应激参与了失血性休克的发展进程；但雌激素调控淋巴管收缩性的机制是否与内质网应激有关，尚不明确。本项目在前期发现17β雌二醇提高失血性休克大鼠肠系膜淋巴管收缩性的基础上，应用大鼠失血性休克模型以及淋巴管平滑肌细胞（LSMCs）缺氧/复氧模型，采用雌激素及其受体、内质网应激的激动剂、抑制剂或siRNA，观察活体与离体肠系膜淋巴管收缩性、LSMCs收缩性及其与钙离子浓度的关系、内质网应激标志物、钙通道蛋白、凋亡与相关蛋白等变化，系统研究和确立内质网应激在雌激素提高重症休克淋巴管收缩功能中的作用与调控机制。在此基础上，实现重症失血性休克降低淋巴管收缩功能机制的理论新突破，形成以雌激素调控淋巴管收缩功能、防治重症休克的新策略，藉此为拓展雌激素至临床应用提供实验依据和理论基础。

淋巴管收缩功能障碍是加重组织水肿、导致休克难治的关键环节。本项目应用大鼠失血性休克模型以及淋巴管平滑肌细胞（LSMCs）缺氧/复氧、内质网应激（ERS）模型，采用雌激素（E2）及其受体（ERs）、ERS的激动剂/抑制剂，观察活体与离体肠系膜淋巴管收缩性、LSMCs收缩与钙离子浓度的关系、ERS标志蛋白、钙通道蛋白、凋亡与相关蛋白等变化，系统研究和确立ERS在雌激素提高重症休克淋巴管收缩功能中的作用与调控机制。动物实验研究证实，E2降低了失血性休克大鼠肠系膜淋巴管组织ERS标志蛋白GRP78表达，E2及ERS抑制剂4-PBA分别显著延长了休克大鼠的存活时间，雌激素及其受体激动剂、ERS抑制剂均可分别明显改善失血性休克大鼠肠系膜淋巴管的收缩功能，ERs抑制剂、ERS激动剂分别抑制了雌激素和/或ERs激动剂提高失血性休克大鼠肠系膜淋巴管收缩功能的作用。细胞实验证实，E2及其受体激动剂提高了缺氧/复氧或ERS模型LSMCs的活性、收缩性，降低了ERS激动剂TM处理后LSMCs的ERS标志蛋白GRP78、ATF6、IRE-1α表达，且ERs抑制剂降低了E2的有利作用。研究结果表明，ERS参与了重症失血性休克淋巴管收缩功能低下的发生机制，丰富了休克淋巴微循环障碍的理论；雌激素通过抑制ERs依赖的ERS改善了失血性休克肠系膜淋巴管收缩功能，为拓展雌激素应用于重症休克的临床防治提供了实验依据和理论基础。