FAK介导的α2AR活化对肾缺血再灌注致PMVECs损伤的保护作用和机制研究

Acute lung injury(ALI) induced by Acute kidney injury(AKI) is one of clinical manifestations of perioperative multiple organs dysfunction or failure. The mortality of combined AKI and ALI could be even 80%. Therefore, it is of great significance to search for related pulmonary protective methods and mechanisms. Pulmonary microvascular endothelia cells(PMVECs) injury is central to the pathogenesis of AKI-induced ALI. The endothelial-targeted interventions could be importantly potential value to prevent ALI. Our previous researches proved that activation of α2 adrenoceptor(α2 AR) could attenuate ALI induced by AKI. However, the mechanisms are still unclear. Focal adhesion kinase(FAK)as a no-receptor tyrosine protein kinase, being one intersection of cytoplasmic signal crosstalk, may mediated the permeability, apoptosis and inflammatory response in PMVECs. Latest research demonstrated that stimulation of α2 AR would activate FAK in nervous system. Herein, the aim of the current study is to authenticate the mechanism of pulmonary protection induced by α2 AR activation by using real-time microscopy, Western blot, Real-time PCR, ELISA and so on, which could be that FAK mediated α2 AR activation maintain endothelia barrier integrity and functions as well as mitigate apoptosis and inflammatory response in PMVECs. The novel strategy for relieving AKI-induced ALI may be anticipated.

急性肾损伤（AKI）所致急性肺损伤（ALI）是围术期多器官功能障碍或衰竭表现之一，AKI合并ALI时死亡率高达80%，探索相应肺保护手段和机制意义重大。AKI致ALI机制中，肺微血管内皮细胞（PMVECs）受损是核心环节，以其为靶目标的防治研究具有重要潜在价值。我们前期研究证实α2肾上腺素受体（α2AR）激动可减轻AKI所致ALI，但机制不清。黏着斑激酶（FAK）作为一种胞内多信号转导通路交汇点的非受体型酪氨酸蛋白激酶，对PMVECs间的通透性以及凋亡炎症反应具有潜在调节作用。最新神经系统研究显示α2AR活化能激活FAK。本研究拟通过Real time Microscopy、Western blot等技术验证α2AR激动的肺保护机制是：PMVECs上α2AR活化，激活胞内FAK，维持PMVECs屏障完整性和功能，同时降低PMVECs炎症和凋亡反应。以期为AKI致ALI的防治提供一种新策略。

肾缺血再灌注（rI/R）所致急性肾损伤（AKI）是围术期常见并发症，且常伴肾外多器官的损伤和功能障碍，此为其高死亡率的重要原因。肺脏是极易受累器官之一，AKI合并ALI时死亡率甚至高达80%。深入探索AKI病理背景下的肺保护手段和机制，具有重要临床价值。我们前期研究提示α2肾上腺素受体（α2AR）活化后，对多种器官缺血再灌注具有保护作用。右美托咪定（Dexmedetomidine, Dex）是一种高选择性α2AR激动剂。为进一步探索α2AR 活化后的肺保护机制，本项目运用Dex作为α2AR激动剂，进行体内外实验，用FITC-荧光白蛋白及Transwell检测肺微血管内皮细胞（PMVECs）通透性，免疫荧光和Western Blot检测蛋白表达，TUNEL法检测细胞凋亡，BL-420F多功能生物机能实验系统采集神经放电频率，ELISA检测血清炎性因子水平，HE染色评估器官病理。结果显示：① Dex减轻C57BL/6J小鼠rI/R所致的肺损伤和肺微血管的高通透性，该作用可被α2AR抑制剂阿替美唑（Atip）或黏着斑激酶（FAK）的抑制剂消除；②离体情况下，Dex可减轻C57BL/6J小鼠rI/R血清所致的单层PMVECs高通透性；③Dex时间和剂量依赖性引起PMVEC中FAK（Tyr397）磷酸化，活化的FAK可减轻肾rI/R血清所致的PMVEC高通透性；④Dex通过激活FAK而维持细胞骨架和细胞连接的完整性；⑤Dex增加肾rI/R小鼠颈迷走神经的放电频率，这一作用可被Atip或脾切除术所拮抗；⑥Dex和迷走神经刺激术（VNS）使得血清中的乙酰胆碱（ACh）增加、儿茶酚胺（CA）减少；⑦Dex和VNS减少血清中促炎因子水平；⑧Dex和VNS减轻小鼠肾I/R中的肾损伤和肾细胞凋亡。上述研究证实α2AR活化对rI/R所致ALI的保护机制是：激活FAK信号通路调节PMVEC细胞骨架和细胞连接维护内皮完整性。此外，Dex具有的抗炎作用依赖于迷走神经的完整性，其对肾rI/R致肾损伤的保护作用可能是通过胆碱能抗炎通路（cholinergic anti-inflammatory pathway，CAP）实现。该实验为今后围术期应用α2AR激动剂防治AKI及其所致的ALI提供理论支持。