HMGB1介导的血脑屏障损伤在机械通气致“肺-脑”损伤中的作用及机制研究

Mechanical ventilation (MV) is essential for patients with acute respiratory failure. However,ventilator induced not only lung injury but also brain dysfunction. The mechanism of mechanical ventilation induced “lung-brain” injury has not been identified. Blood-brain barrier is an important structure to maintain a stable environment of brain. High mobility group protein 1 (HMGB 1) is a late inflammatory factor.Recent studies have found that HMGB 1 is related to not only mechanical ventilation-induced lung injury but also Rho/ROCK signaling pathway involved in blood-brain barrier damage. Our preliminary experiment showed that HMGB 1 expression of lung and brain was up-regulated in the model of ventilator induced lung injury in rat. Based on our preliminary experiment, we propose hypothesis that HMGB1 by activating RAGE/Rho/ROCK signaling pathway mediated blood-brain barrier dysfunction, which led to the occurrence of mechanical ventilation induced brain injury. Our project intends to use pathology, molecular biology, RNA gene interference detection technology to discuss the effect and molecular mechanism of HMGB1/RAGE/RhoA/ROCK-mediated blood-brain barrier in mechanical ventilation-induced lung and brain injury. Fuethermore, we provide new ideas for clinical prevention of mechanical ventilation-induced lung and brain injury.

机械通气是一把双刃剑，不仅引起机械通气肺损伤，还可以诱发脑损伤，严重影响患者预后。但机械通气所致“肺-脑”损伤发生机制仍未阐明。血脑屏障是维护脑部内环境稳定的重要结构。高迁移率族蛋白1(HMGB1)作为一种晚期炎性因子，近年证实不仅在机械通气肺损伤中扮演着重要的角色，而且还可能是诱发血-脑屏障损伤的重要介质，并与介导内皮细胞损伤的Rho/ROCK通路激活密切相关。尤其近期我们发现,机械通气肺损伤大鼠的肺、脑组织中HMGB1表达均明显增高。结合前期研究结果，我们推测并提出科学假说：HMGB1通过激活RAGE/Rho/ROCK信号通路介导血脑屏障功能损伤，进而导致机械通气所致脑损伤的发生。本项目拟采用病理学、分子生物学、RNA干扰等技术，重点研究HMGB1/RAGE/RhoA/ROCK介导的血脑屏障损伤在机械通气所致“肺-脑”损伤中的作用和分子机制，为临床机械通气肺、脑损伤的防治提供新思路。

研究背景和目的：血脑屏障是维护脑部内环境稳定的重要结构。高迁移率族蛋白1(HMGB1)作为一种晚期炎性因子，近年证实不仅在机械通气肺损伤中扮演着重要的角色，而且还可能是诱发血-脑屏障损伤的重要介质。本研究旨在探讨HMGB1介导的血脑屏障损伤在机械通气致“肺-脑”损伤中的作用及机制研究。.实验方法：通过制备大鼠机械通气肺损伤模型，分别抑制和诱导HMGB1，从正反两方面观察大鼠肺-脑损伤情况，检测肺组织、脑组织病理学损伤情况、检测肺组织HMGB1蛋白表达情况，检测脑组织HMGB1、RAGE蛋白表达情况。细胞实验通过siRNA干扰RAGE、RhoA、ROCK蛋白表达，观察内皮细胞间紧密连接情况。.实验结果：WB和PCR结果显示，在机械通气所致肺-脑损伤模型中，肺组织HMGB1蛋白表达明显增加、脑组织中HMGB1、RAGE、RhoA、ROCK等蛋白表达明显增加、而紧密连接蛋白Claudin-5、Occludin及Zo-1的表达明显下调。大鼠肺损伤评分、W/D、TNF-α、IL-1β及IL-6浓度、脑含水量、脑组织EB含量明显较高。使用抑制剂抑制HMGB1活性后，脑组织中HMGB1、RAGE、RhoA、ROCK等蛋白表达明显下调、紧密连接蛋白Claudin-5、Occludin及Zo-1的表达明显上升。大鼠肺损伤评分、W/D、TNF-α、IL-1β及IL-6浓度、脑含水量、脑组织EB含量均有明显改善。离体细胞实验研究结果显示，20%牵张强度牵张肺上皮细胞后的培养液培养脑血管内皮细胞后收集细胞后，WB和免疫荧光结果显示HMGB1、RAGE、RhoA、ROCK等蛋白表达明显升高，而在牵张肺泡上皮细胞之前使用HMGB1或HMGB1-siRNA干扰其表达后，上述蛋白表达量均显著下调。最后，我们通过正常大鼠气管内滴注AAV-HMGB1过表达HMGB1进行模拟肺-脑损伤，结果发现正常大鼠过表达HMGB1后出现肺组织和脑组织损伤、WB显示大鼠脑组织HMGB1、RAGE、RhoA、ROCK蛋白表达显著升高。.研究结论：机械通气肺损伤时肺、脑组织中HMGB1表达增加，表达增加的HMGB1诱导下游RAGE/RhoA/ROCK信号通路的激活，最终导致血脑屏障受损、脑组织水肿、发生脑损伤。本研究结果可为临床防治机械通气肺-脑损伤提供潜在的靶点。