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白藜芦醇通过调控HIF-1α/NOX4/ROS通路抑制低氧诱导的大鼠肺动脉平滑肌细胞氧化应激与增殖

何丽囡¹, 兰钰茹¹, 贺光明², 郭姝瑾³, 文富强¹, 汪涛^1,*

¹四川大学华西医院/华西临床医学院呼吸病学研究室，成都 610041；²四川省肿瘤医院重症医学科，成都 610041；³电子科技大学中国科学院四川转化医学研究医院，成都 610054

摘要

本文旨在明确白藜芦醇对低氧诱导的肺动脉平滑肌细胞(pulmonary artery smooth muscle cells, PASMCs)氧化应激与增殖的作用及分子机制。体外分离培养原代大鼠PASMCs，采用不同浓度的白藜芦醇(10、20和40 μmol/L)或NADPH氧化酶(NADPH oxidases, NOXs)抑制剂VAS2870 (10 μmol/L)预处理0.5 h，然后将细胞置于常氧(21% O2, 5% CO2)或低氧(2% O2, 5% CO2)中培养24 h。采用CCK-8法和增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)的表达水平检测细胞增殖，用DCFH-DA测定细胞内活性氧(reactive oxygen species, ROS)的生成，用real-time RT-PCR和Western blot检测NOX1、NOX4和低氧诱导因子-1α (hypoxia inducible factor 1α, HIF-1α)的表达水平，通过小干扰RNAs (small interference RNAs, siRNAs)特异性沉默Hif-1α和Nox4后确定相关信号通路。结果显示，白藜芦醇和VAS2870均能显著抑制低氧诱导的大鼠PASMCs细胞增殖和ROS生成，同时白藜芦醇还能有效阻止低氧诱导的HIF-1α蛋白的聚集和NOX4的表达上调，而对NOX1没有明显的影响。沉默Hif-1α或Nox4后，低氧诱导的大鼠PASMCs细胞增殖和ROS累积均显著降低，且能被白藜芦醇进一步抑制。上述结果提示，白藜芦醇可能通过阻断HIF-1α/NOX4/ROS信号通路抑制低氧诱导的大鼠PASMCs氧化应激和增殖。

关键词： 白藜芦醇; 低氧; 低氧诱导因子-1α; NADPH氧化酶4; 活性氧

分类号：R34; Q291

Resveratrol inhibits hypoxia-induced oxidative stress and proliferation in pulmonary artery smooth muscle cells through the HIF-1α/NOX4/ROS signaling pathway

HE Li-Nan¹, LAN Yu-Ru¹, HE Guang-Ming², GUO Shu-Jin³, WEN Fu-Qiang¹, WANG Tao^1,*

¹Laboratory of Pulmonary Diseases, West China Hospital/West China School of Medicine, Sichuan University, Chengdu 610041, China；²Department of Critical Care Medicine, Sichuan Cancer Hospital & Institute, Chengdu 610041, China；³Sichuan Research Hospital of Translational Medicine, Chinese Academy of Sciences, University of Electronic Science and Technology of China, Chengdu 610054, China

Abstract

The purpose of the present study was to determine the effects of resveratrol on hypoxia-induced oxidative stress and proliferation in pulmonary artery smooth muscle cells (PASMCs) and the underlying mechanism. Primary rat PASMCs were isolated and cultured in vitro and pretreated with different concentrations of resveratrol (10, 20, and 40 µmol/L) or the NADPH oxidase (NOX) inhibitor VAS2870 (10 µmol/L) for 0.5 h. The cells were then cultured under normoxia (21% O2, 5% CO2) or hypoxia (2% O2, 5% CO2) for 24 h. The proliferation of cells was measured using the CCK-8 method and the expression of proliferating cell nuclear antigen (PCNA). The production of reactive oxygen species (ROS) was detected by DCFH-DA. The expression of rat NOX1, NOX4 and hypoxia inducible factor 1α (HIF-1α) was detected by real-time RT-PCR and Western blotting assays. The related signaling pathways were determined using the small interference RNAs (siRNAs) specifically targeting Hif-1α and Nox4. The results showed that resveratrol and VAS2870 significantly inhibited hypoxia-induced cell proliferation and ROS production in rat PASMCs. Resveratrol also effectively prevented hypoxia-induced increase of HIF-1α protein levels and NOX4 up-regulation, but had little effect on NOX1. After the knocking down of Hif-1α or Nox4 with siRNAs, hypoxia-induced cell proliferation and ROS accumulation were significantly decreased, and both were further inhibited by resveratrol treatment. These results suggest that resveratrol inhibits hypoxia-induced oxidative stress and cell proliferation in rat PASMCs possibly through blocking the HIF-1α/NOX4/ROS pathway.

Key words: resveratrol; hypoxia; hypoxia inducible factor 1α; NADPH oxidase 4; reactive oxygen species

收稿日期：2019-10-30　　录用日期：2020-05-04

通讯作者：汪涛　　E-mail: tao.wang@scu.edu.cn

DOI: 10.13294/j.aps.2020.0067

引用本文：

何丽囡, 兰钰茹, 贺光明, 郭姝瑾, 文富强, 汪涛. 白藜芦醇通过调控HIF-1α/NOX4/ROS通路抑制低氧诱导的大鼠肺动脉平滑肌细胞氧化应激与增殖[J]. 生理学报 2020; 72 (5): 551-558.

HE Li-Nan, LAN Yu-Ru, HE Guang-Ming, GUO Shu-Jin, WEN Fu-Qiang, WANG Tao. Resveratrol inhibits hypoxia-induced oxidative stress and proliferation in pulmonary artery smooth muscle cells through the HIF-1α/NOX4/ROS signaling pathway. Acta Physiol Sin 2020; 72 (5): 551-558 (in Chinese with English abstract).