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伞形酮通过抑制RhoA/ROCK信号通路和自噬改善慢性低氧性肺动脉高压

商萍¹, 孙帅波², 刘宝华^1,*

¹温州医科大学附属第二医院康复科，温州 325000 ；²温州医科大学附属第二医院骨外科，温州 325000

摘要

本研究考察低氧对肺动脉平滑肌细胞(pulmonary artery smooth muscle cells, PASMCs)中RhoA/Rho激酶(Rho-kinase, ROCK)信号通路和自噬的影响，探讨伞形酮改善低氧性肺动脉高血压的作用。体外培养PASMCs，并将其分为4组：对照组、低氧组、低氧+伞形酮组、常氧+伞形酮组，免疫荧光染色评估平滑肌肌动蛋白α和LC3，Western blot检测RhoA、ROCK2、p-MYPT1、LC3-II、LC3-I、Beclin-1、p62、C-Caspase 3、Bax和Bcl-2蛋白表达水平。在体研究中，将Sprague-Dawley (SD)大鼠分为3组：对照组、低氧组、低氧+伞形酮组，评估右心室重量和左心室加室内隔重量之比[RV/(LV+S)]，HE染色评估肺动脉形态特征。结果显示，与对照组相比，低氧组PASMCs中LC3-II/LC3-I比例和Beclin-1表达显著增加，p62表达显著降低；RhoA、ROCK2和p-MYPT1蛋白表达显著增加(P < 0.05)。而相比低氧组，低氧+伞形酮组PASMCs中的LC3-II/LC3-I比例和Beclin-1表达降低，p62表达显著增加；RhoA、ROCK2和p-MYPT1蛋白表达显著降低(P < 0.05)。低氧大鼠的肺动脉壁增厚，RV/(LV+S)比值增高，而低氧+伞形酮组有明显改善(P < 0.05)。这些结果表明伞形酮通过抑制PASMCs的RhoA/ROCK信号通路和自噬改善低氧性肺动脉高血压。

关键词： 肺动脉高血压; 伞形酮; 自噬; RhoA

Umbelliferone improves chronic hypoxia-induced pulmonary hypertension by inhibiting the RhoA/ROCK signaling pathway and autophagy

SHANG Ping¹, SUN Shuai-Bo², LIU Bao-Hua^1,*

¹ Department of Rehabilitation, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China；²Department of Orthopedics, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China

Abstract

This study aimed to investigate the effects of hypoxia on RhoA/Rho-kinase (ROCK) signaling pathway and autophagy in pulmonary artery smooth muscle cells (PASMCs), and to explore the underlying mechanism of Umbelliferone (Umb) in ameliorating chronic hypoxic pulmonary hypertension. PASMCs were cultured from Sprague-Dawley (SD) rats and randomly divided into control group, hypoxia group, hypoxia + Umb intervention group and normoxia + Umb intervention group. Alpha smooth muscle actin (α-SMA) and LC3 were assessed by immunofluorescence staining. Protein expression of RhoA, ROCK2, p-MYPT1, LC3-II, Beclin-1, p62, C-Caspase 3, Bax and Bcl-2 was analyzed by Western blotting. In in vivo study, SD rats were divided into control group, hypoxia group and hypoxia + Umb intervention group. Weight ratio of the right ventricle (RV)/left ventricle plus septum (LV+S) was detected, and pulmonary arterial morphological features were examined by HE staining. The results indicated that compared with the control group, the LC3-II/LC3-I ratio and expression of Beclin-1 were significantly increased, while p62 expression was significantly decreased, and the expressions of RhoA, ROCK2 and p-MYPT1 were significantly increased in PASMCs of hypoxia group (P < 0.05). The changes of LC3-II/LC3-I ratio, the expressions of Beclin-1, p62, RhoA, ROCK2 and p-MYPT1 in PASMCs were reversed by Umb treatment (P < 0.05). Consistently, the pulmonary arterial wall was thickened and the RV/(LV+S) ratio was increased in hypoxic rats, which were significantly improved by Umb treatment (P < 0.05). These results suggest that Umb can improve hypoxia-induced pulmonary hypertension by inhibiting the RhoA/ROCK signaling pathway and autophagy in PASMCs. 

Key words: pulmonary arterial hypertension; Umbelliferone; autophagy; RhoA

收稿日期：　　录用日期：

通讯作者：刘宝华　　E-mail: bhbh369@sina.com

DOI: 10.13294/j.aps.2022.0056

引用本文：

商萍, 孙帅波, 刘宝华. 伞形酮通过抑制RhoA/ROCK信号通路和自噬改善慢性低氧性肺动脉高压[J]. 生理学报 2022; 74 (4): 555-562.

SHANG Ping, SUN Shuai-Bo, LIU Bao-Hua. Umbelliferone improves chronic hypoxia-induced pulmonary hypertension by inhibiting the RhoA/ROCK signaling pathway and autophagy. Acta Physiol Sin 2022; 74 (4): 555-562 (in Chinese with English abstract).