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TRPV4通道介导慢性低氧肺高压大鼠肺微血管内皮通透性增加

焦海霞^1,2, 袁胜霞^2,3, 黄燕珍², 苏巧文^2,4, 何瑞岚^1,2, 吴枝娟^1,2, 林默君^1,2,*

¹福建医科大学基础医学院，福州 350112；²福建医科大学心血管病离子通道信号调控福建省高校重点实验室，福州 350112；³中国人民解放军联勤保障部队第九〇〇医院，福州 350102；⁴福建医科大学附属泉州第一医院病理科，泉州 362000

摘要

本文旨在研究瞬时受体电位香草素受体4 (transient receptor potential vanilloid 4, TRPV4)通道对慢性低氧肺高压(chronic hypoxia-induced pulmonary hypertension, CHPH)大鼠肺微血管内皮细胞(pulmonary microvascular endothelial cells, PMVECs)通透性的影响，以阐明CHPH发生时血管内皮功能障碍的可能机制。Sprague-Dawley大鼠慢性低氧处理21天建立CHPH模型，组织块贴壁法原代培养PMVECs，采用FITC-葡聚糖法检测细胞通透系数，透射电镜观察紧密连接(tight junction, TJ)结构，实时荧光定量PCR和蛋白印迹法检测TRPV4和TJ相关蛋白Occludin、Claudin-5、ZO-1表达变化，TRPV4 特异性激动剂GSK1016790A (GSK, 10 nmol/L)和抑制剂HC-067047 (HC, 1 μmol/L, 0.5 μmol/L)干预后观察PMVECs内Ca2+浓度变化及其对PMVECs通透性的影响。结果显示：慢性低氧处理21天后成功建立CHPH大鼠模型，CHPH大鼠肺血管内皮间TJ结构破坏，PMVECs屏障功能降低，细胞间通透性增高，TJ相关蛋白表达显著减少，TRPV4表达增高(P < 0.01)。特异性激活TRPV4后CHPH大鼠PMVECs内Ca2+浓度显著增高，TRPV4 特异性抑制剂HC对CHPH大鼠细胞Ca2+增高的抑制效应显著高于正常PMVECs，并可以逆转CHPH大鼠PMVECs的通透性增高，增加三种TJ相关蛋白表达(P < 0.01或P < 0.05)。上述结果提示，TRPV4通道通过增加胞内Ca2+浓度，引起CHPH大鼠PMVECs上TJ结构破坏、TJ相关蛋白表达下降，诱发内皮功能障碍。

关键词： 肺高压; 肺微血管内皮细胞; 紧密连接; 钙离子; TRPV4

分类号：R331.3;R332

TRPV4 channel mediates the increase of pulmonary microvascular endothelial permeability in rats with chronic hypoxic pulmonary hypertension

JIAO Hai-Xia^1,2, YUAN Sheng-Xia^2,3, HUANG Yan-Zhen², SU Qiao-Wen^2,4, HE Rui-Lan^1,2, WU Zhi-Juan^1,2, LIN Mo-Jun^1,2,*

¹School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350122, China；²The Key Laboratory of Fujian Province Universities on Ion Channel and Signal Transduction in Cardiovascular Diseases, Fujian Medical University, Fuzhou 350122, China；³The 900th Hospital of Joint Logistic Support Force, PLA, Fuzhou 350102, China；⁴Department of Pathology, Affiliated Quanzhou First Hospital of Fujian Medical University, Quanzhou 362000, China

Abstract

The purpose of the present study was to investigate the effect of transient receptor potential vanilloid 4 (TRPV4) channel on the permeability of pulmonary microvascular endothelial cells (PMVECs) in rats with chronic hypoxia-induced pulmonary hypertension (CHPH), so as to clarify the mechanism of vascular endothelial dysfunction during the occurrence of pulmonary hypertension (PH). CHPH rat model was established by exposure to chronic hypoxia (CH) for 21 days. Primary PMVECs were cultured by adherent tissue blocks at the edge of the lung. The permeability coefficient of primary cultured PMVECs was detected by fluorescein isothiocyanate (FITC)-dextran. The structure of tight junction (TJ) was observed by transmission electron microscope. The expression of TRPV4 and TJ-related proteins, such as, Occludin, Claudin-5, ZO-1 were examined by real-time fluorescence quantitative PCR and Western blotting. The intracellular calcium concentration ([Ca2+]i) in PMVECs and its effect on PMVECs permeability were observed after the intervention of TRPV4 specific agonist GSK1016790A (GSK, 10 nmol/L) and specific inhibitor HC-067047 (HC, 1 μmol/L, 0.5 μmol/L). The results showed that the CHPH model was successfully established in rats treated with CH for 21 days. In CHPH rats, the structure of TJ was destroyed, the function of PMVECs barrier was decreased, the intercellular permeability was increased, the expression of TJ-related proteins were significantly decreased and the expression of TRPV4 was significantly increased (P < 0.01). The amplitude of [Ca2+]i in PMVECs of CHPH rats was significantly increased after activation of TRPV4. The inhibition ratio of HC on [Ca2+]i in PMVECs of CHPH rats was significantly higher than that in normal PMVECs. TRPV4 specific inhibitor HC reversed the increase of PMVECs permeability and increased the expression of three TJ-related proteins in CHPH rats (P < 0.01, P < 0.05). These results suggest that TRPV4 channel can induce endothelial dysfunction by increasing the [Ca2+]i, resulting in the destruction of TJ structure and the decrease of TJ-related proteins expression on PMVECs in CHPH rats.

Key words: pulmonary hypertension; pulmonary microvascular endothelial cells; tight junction; Ca2+; TRPV4

收稿日期：2020-11-29　　录用日期：2021-03-22

通讯作者：林默君　　E-mail: mjlin@mail.fjmu.edu.cn

引用本文：

焦海霞, 袁胜霞, 黄燕珍, 苏巧文, 何瑞岚, 吴枝娟, 林默君. TRPV4通道介导慢性低氧肺高压大鼠肺微血管内皮通透性增加[J]. 生理学报 2021; 73 (6): 867-877.

JIAO Hai-Xia, YUAN Sheng-Xia, HUANG Yan-Zhen, SU Qiao-Wen, HE Rui-Lan, WU Zhi-Juan, LIN Mo-Jun. TRPV4 channel mediates the increase of pulmonary microvascular endothelial permeability in rats with chronic hypoxic pulmonary hypertension. Acta Physiol Sin 2021; 73 (6): 867-877 (in Chinese with English abstract).