活性氧不参与二氯化钴诱导的人肺动脉平滑肌细胞增殖

Reactive Oxygen Species May Not Be Involved in the Cellular Proliferation Induced by Cobalt Chloride in HPASMC

目的探讨活性氧是否参与二氯化钴诱导的人肺动脉平滑肌细胞(HPASMC)过度增殖。方法用化学性缺氧模拟剂二氯化钴处理HPASMC,建立缺氧性肺动脉高压血管重塑的细胞模型。用外源性活性氧供体过氧化氢处理HPASMC,观察活性氧对细胞增殖的影响;活性氧清除剂N-乙酰半胱氨酸(NAC)预处理HPASMC,观察其对二氯化钴或过氧化氢诱导的细胞增殖的改善作用;应用细胞计数试剂盒8检测细胞增殖,Western blot检测缺氧诱导因子1α(HIF-1α)蛋白的表达,双氯荧光素染色和荧光照相术检测细胞内活性氧的含量。结果在25～50μmol/L浓度范围内,二氯化钴处理24 h可诱导HPASMC增殖,50μmol/L二氯化钴处理24 h可使细胞内HIF-1α的水平明显增加;与二氯化钴的作用类似,过氧化氢在12～25μmol/L浓度范围内处理24 h也可引起细胞增殖,但不改变HIF-1α的水平;在二氯化钴或过氧化氢处理前,用不同浓度的NAC预处理,在1 500μmol/L浓度时,NAC预处理可明显抑制过氧化氢诱导的HPASMC过度增殖(P<0.05),而对二氯化钴诱导的细胞增殖则无明显影响(P>0.05);另外,50μmol/L二氯化钴处理6～24 h对细胞内活性氧的含量无明显影响(P>0.05)。结论化学性缺氧可诱导HPASMC过度增殖,其机制可能不依赖于活性氧。

Aim To investigate whether reactive oxygen species （ROS） were involved in cobalt chloride （CoCI2 ） induced cellular proliferation in human pulmonary artery smooth muscle cells （HPASMC）. Methods HPASMC were exposed to a chemical hypoxia agent COC12 to establish a cellular model of pulmonary arterial hypertension. An exogenous ROS donor, hydrogen peroxide （ H2O2 ）, was administered to examine the direct effect of ROS on HPASMC proliferation. Before the exposure of HPASMC to CoCI2 or H2O2, a ROS scavenger, N-acetylcysteine （NAC）, was used to assess the effect of inhibitory oxidative stress on the cellular proliferation induced by the two agents above. Cell prolif- eration was measured by cell counter kit 8, expression of hypoxia inducible factor-1 α （HIF-1 α） was tested by Western blot assay and intercellular ROS were observed by 2＇ ,7＇-dichlorfluorescein-diacetate staining followed by photofluorography. Results Treatment of HPASMC with COC12 for 24 h at concentrations ranging from 25 to 50 μmol/L induced significant cellular proliferation, and treatment with 50 txmol/L CoC12 for 24 h obviously increased intercellular HIF-1 ct level. Simi- larly, treatment with H202 for 24 h at concentrations ranging from 12 to 25 μmol/L triggered cellular proliferation, howev-er, treatment with H202 didn＇ t alter HIF-1 ct level in HPASMC. Pretreatment with NAC, prior to the treatment with CoCI2 or H202, statistically attenuated H2 02 -induced HPASMC proliferation （P 〈 0. 05 ）, but not CoC12-induced cellular prolifera- tion （P 〉 0. 05）. Exposure of HPASMC to 50 panol/L COC12 for 6-24 h didn＇ t alter intraceUular ROS content. Conclusion ROS may not be involved in CoC12-indueed cellular proliferation in HPASMC.