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

目的研究血红素氧合酶-1／一氧化碳（HO-1／CO）系统对胰岛素样生长因子-I（IGF-I）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IGF-I诱导其增殖,用氯化血红素和锌原卟啉-9,分别诱导和阻断HO-1的表达,从而促进和抑制CO生成,通过RT．PCR及Western blot检测HO-1mRNA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^3H-TdR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VSMCs HO-1 mRNA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IGF-I增高的VSMCs ^3H-TdR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;HO-1表达上调是细胞对抗氧化应激和损伤的保护性反应;HO-1 mRNA及蛋白表达增加是内源性CO抑制VSMCs增殖的基础。

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

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 (3)H-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 G(0)/G(1) phase were increased and in the S and G(2)/M phase decreased by hemin (P < 0.01). Opposite results were observed in VSMCs treated with Znpp-IX. CONCLUSIONS: 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.