血红素氧合酶-1/一氧化碳系统对胰岛素样生长因子-Ⅰ诱导的兔血管平滑肌细胞增殖的抑制作用

Heme oxygenase-1 and carbon monoxide are key mediators for vascular smooth muscle cells proliferation induced by insulin-like growth factor-Ⅰ

目的研究血红素氧合酶1/一氧化碳(HO1/CO)系统对胰岛素样生长因子Ⅰ(IGFⅠ)诱导的血管平滑肌细胞(VSMCS)增殖的影响及分子机制。方法体外培养兔主动脉VSMCS,用IGFⅠ诱导其增殖,用氯化血红素和锌原卟啉9,分别诱导和阻断HO1的表达,从而促进和抑制CO生成,通过RTPCR及WESTERNBLOT检测HO1MRNA和蛋白的表达,采用酶联免疫法测定培养上清液中碳氧血红蛋白含量,应用同位素3HTDR掺入试验检测VSMCS增殖,采用流式细胞技术检测细胞增殖周期。结果氯化血红素显著诱导VSMCSHO1MRNA及蛋白的表达,显著增加VSMCS培养上清液中CO的生成量,呈剂量依赖趋势;梯度浓度的氯化血红素对IGFⅠ增高的VSMCS3HTDR掺入量抑制率分别为29.6%、45.0%和54.1%,显著抑制细胞周期进程,导致G0/G1细胞显著增多,S期和G2/M期细胞显著减少(P均<0.01),与加入的氯化血红素及上清液中碳氧血红蛋白含量呈剂量依赖趋势;锌原卟啉9则导致相反的结果。结论内源性CO通过抑制VSMCS的DNA合成及细胞周期进程直接参与VSMCS的增殖调节;HO1表达上调是细胞对抗氧化应激和损伤的保护性反应;HO1MRNA及蛋白表达增加是内源性CO抑制VSMCS增殖的基础。

Objective To determine the role and related mechanisms of heme oxygenase-1/carbon monoxide （HO-1/CO） on VSMCs proliferation induced by insulin-like growth factor- Ⅰ （IGF- Ⅰ ）. Methods VSMCs isolated from rabbit aorta were cultured in vitro and proliferation was induced by IGF- Ⅰ. Hemin （a substrate and inducer of HO-1 ） or zinc protoporphyrin-Ⅸ （Znpp-Ⅸ, an inhibitor of HO-1 ） was added to stimulate or inhibit the expression of HO-1. The mRNA and protein expressions of HO-1 were detected by RT-PCR and Western blot analysis. CO released into the culture media was quantitated by measuring carbon monoxide hemoglobin （COHb）, VSMCs proliferation and cell cycle were determined by ^3H-TdR incorporation assay and flow cytometry, respectively. Results The HO-1 mRNA and protein expressions in VSMCs and the amount of COHb in the culture media were significantly increased and the IGF- I -induced ^3 H-TdR incorporations of VSMCs significantly reduced by hemin in a dose-dependent manner （P 〈 0. 01 ）. Furthermore, VSMCs in the G0/Gt phase were increased and in the S and G2/M phase decreased by hemin （P 〈 0. 01 ）. Opposite results were observed in VSMCs treated with Znpp-Ⅸ. Condusions Endogenous HO-1 and CO are important mediators for inhibiting IGF-Ⅰ induced VSMCs proliferation by reducing VSMCs DNA synthesis and decelerating cell cycle progression.