TNF-α增强主动脉平滑肌细胞内1型1,4,5-三磷酸肌醇受体表达参与感染性休克的发生机制

Expression of Inositol 1,4,5-trisphophate Receptor type 1 in the Vascular Smooth Muscle Cells Induced by Tumor Necrosis Factor Alpha

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 investigate the effect of TNF - ct on the expression of inositol 1,4,5 - trisphophate receptor type 1 （IP3RI） in vascular smooth muscle cells （ VSMC）, in order to delineate the mechanism of. TNF - a causing the change of VSMC contraction function in septic shock. Methods We primarily cuhured VSMC, divided them into TNF - α - treated Oh, 4h, 8h, 24h groups. We i- dentified the effect of TNF - ct on the expression of IP3R ｜ mRNA, protein and the activity of IP3R I promoter by immunofluorescence staining, Western blot, RT- PCR and dual luciferase reporter gene assay. Results TNF- eL could increase the IP3RI protein level （WB） （4h：1.059±0.005 vs 1.000±0.002, P =001 ;8h：2.416±0.042 vs 1.000±0.002, P 〈0.01 ;24h：2. 138±0.010 vs 1.000 ± O. 002, P 〈 0. O1, n = 9）, but no expression on the distribution of IP3RI in VSMC （IF） was seen, and increase the IP3RI mRNA lev-el （RT-PCR） （4h：2.260±0.889 vs 1.00±0.02, P=0.193;8h：5.449±2.279 vs 1.00±O.02,P=0.000;24h：3.049 ± l.684 vs 1. 000 ± 0. 002, P = 0. 042, n = 9）. After PGL3 - IP3 R I promoter plasmid was transient transfect into VSMC, the activity of IP3 R I promoter could be increased by TNF - α （3.56 ± 0. 65 vs 1.00 ± 0. 05, P = 0.02, n = 9）. Conclusion TNF - α can increase the activ- ity of IP3R I promoter, and increase the release of Ca2＋ in VSMC mediate by IP3Rs. This function of TNF - α may affect the contraction function of VSMC and participate in vascular control in septic shock.