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目的研究TNF-α对主动脉平滑肌细胞(VSMC)内1型1,4,5-三磷酸肌醇受体(IP3RⅠ)表达的影响,揭示TNF-α影响VSMC收缩功能参与感染性休克的发生机制。方法原代分离培养大鼠VSMC。按TNF-α处理的不同时间点(0、4、8、24h)分4组。分别应用Western blot、免疫荧光、RT-PCR、双荧光素酶检测方法,观察TNF-α对IP3RⅠmRNA、蛋白表达及其启动子活性的影响。结果 TNF-α处理组细胞内IP3RⅠ分布未见变化,8、24h组荧光强度增强提示IP3RⅠ蛋白含量增加,IP3RⅠ蛋白表达升高(4h:1.059±0.005 vs 1.000±0.002,P=0.010;8h:2.416±0.042 vs 1.000±0.002,P<0.01;24h:2.138±0.010vs 1.000±0.002,P<0.01,n=9),IP3RⅠmRNA表达明显增加(4h:2.260±0.889 vs 1.00±0.02,P=0.193;8h:5.449±2.279 vs1.00±0.02,P=0.000;24h:3.049±1.684 vs 1.00±0.02,P=0.042,n=9)。转染PGL3-IP3RⅠpromoter质粒后TNF-α组IP3RⅠ启动子活性明显增强(3.56±0.65 vs 1.00±0.05,P=0.020,n=9)。结论 TNF-α可上调IP3RⅠ基因启动子活性,从而引起IP3RⅠ蛋白表达升高,增强VSMC内IP3Rs系统介导的Ca2+释放作用,这可能是TNF-α影响VSMC收缩功能参与感染性休克血管调控的机制之一。
Objective To study the effect of TNF-α on the expression of inositol 1,4,5-triphosphate receptor (IP3RⅠ) 1 in aortic smooth muscle cells (VSMCs) and to elucidate the mechanism by which TNF-α affects VSMC contractile function in septic shock . Methods Primary cultured rat VSMCs were isolated and cultured. At different time points (0, 4, 8, 24 h), TNF-α was divided into 4 groups. Western blot, immunofluorescence, RT-PCR and dual-luciferase assay were used to observe the effect of TNF-α on IP3RⅠmRNA, protein expression and promoter activity. Results There was no change in the distribution of IP3RⅠ in TNF-α-treated group. The enhanced fluorescence intensity in 8 and 24h group indicated that the content of IP3RⅠ protein increased and the expression of IP3RⅠ protein increased (4h: 1.059 ± 0.005 vs 1.000 ± 0.002, P = 0.010; 8h: 2.416 (P <0.01), the expression of IP3RⅠmRNA increased significantly (4h: 2.260 ± 0.889 vs 1.00 ± 0.02, P = 0.193; 8h : 5.449 ± 2.279 vs 1.00 ± 0.02, P = 0.000; 24h: 3.049 ± 1.684 vs 1.00 ± 0.02, P = 0.042, n = 9). The IP3RⅠ promoter activity of TNF-α group was significantly enhanced after transfection with PGL3-IP3RⅠpromoter plasmid (3.56 ± 0.65 vs 1.00 ± 0.05, P = 0.020, n = 9). Conclusion TNF-α may up-regulate the promoter activity of IP3RⅠ gene, resulting in the increase of IP3RⅠ protein expression and the enhancement of IP3Rs system-mediated Ca 2+ release in VSMCs. This may be the mechanism of TNF-α affecting VSMC contractile function in the regulation of septic shock one.