紫杉醇水蛭素支架涂层复合物对LPS诱导的HCASMC炎性活化过程中核转录因子NF-κ B p65及其下游炎症因子的调控作用

Regulating effect of paclitaxel-hirudin compound on NF-κ B p65 and its downstream inflammatory factors during HCASMC inflammatory activated process induced by lipopolysaccharide

目的探讨支架涂层复合物紫杉醇水蛭素对脂多糖(LPS)诱导人冠状动脉平滑肌细胞(HCASMC)炎性活化过程中核转录因子NF-κ B p65及其下游炎症因子表达的影响。方法选用4~6代的HCASMC,将细胞分为空白对照组(正常HCASMC,未做任何处理)、LPS模型组(LPS干预)、紫杉醇水蛭素高浓度组(1μmol/L紫杉醇+0.2 mg/ml水蛭素+LPS干预)、紫杉醇水蛭素低浓度组(1μmol/L紫杉醇+0.0125 mg/ml水蛭素+LPS干预)。每组设置3个复孔。ELISA法检测细胞上清肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)和白介素-1β(IL-1β)的表达水平,用Q-PCR法对各组细胞NF-κ B p65、TNF-α、IL-6和IL-1βm RNA进行检测,用Western Blotting检测NF-κ B p65蛋白表达水平。结果与空白对照组比较,LPS模型组NF-κ B p65、TNF-α、IL-6和IL-1β的m RNA表达水平明显升高,差异有统计学意义(P均<0.05);HCASMC经高、低浓度紫杉醇水蛭素复合物预处理,LPS刺激后,上述指标低于LPS模型组,差异有统计学意义(P均<0.05)。LPS模型组NF-κ B p65蛋白表达水平明显高于空白对照组,而紫杉醇水蛭素预处理组NF-κ B p65蛋白表达水平较LPS模型组明显降低,其中高浓度处理组降低更为显著。与空白对照组比较,LPS模型组TNF-α、IL-1β和IL-6表达水平升高,差异有统计学意义(P均<0.05)。与LPS模型组比较,紫杉醇水蛭素低浓度与高浓度组TNF-α、IL-1β和IL-6水平明显降低,差异有统计学意义(P均<0.05)。其中高浓度紫杉醇水蛭素干预后IL-1β表达下降较低浓度更为显著,差异有统计学意义(P<0.05)。结论紫杉醇水蛭素支架涂层复合物对LPS诱导的HCASMC炎性活化过程中NF-κ B p65的激活具有明显的抑制作用,有效下调核转录因子NF-κ B p65的转录活性,显著抑制下游炎症因子TNF-α、IL-6和IL-1β的表达。

Objective To investigate the influence of stent coating drug, paclitaxel-hirudin compound, on expressions of NF-κB p65, a nuclear transcription factor, and its downstream inflammatory factors during inflammatory activated process of human coronary artery smooth muscle cells （HCASMC） induced by lipopolysaccharide （LPS）. Methods HCASMC were divided into blank control group （normal HCASMC）, LPS model group （intervened by LPS）, high-dose paclitaxel-hirudin compound group （intervened by 1 μmol/L paclitaxel＋0.2 mg/mL hirudin＋LPS） and low-dose high-dose paclitaxel-hirudin compound group （intervened by 1 μmol/L paclitaxel＋0.0125 mg/mL hirudin＋LPS）. The expressions of supernatant tumor necrosis factor-α （TNF-α）, interleukin-6 （IL-6） and interleukin-1β （IL-1β） were detected by using ELISA. The mRNA expressions of NF-κB p65, TNF-α, IL-6 and IL-1β were detected by using Q-PCR, and protein expression of NF-κB p65 was detected by using Western blotting assay. Results The mRNA expressions of NF-κB p65, TNF-α, IL-6 and IL-1β increased in LPS model group compared with blank control group （all P〈0.05）, and all of them were lower in high-dose and low-dose paclitaxel-hirudin compound groups than those in LPS model group （all P〈0.05）. The protein expression of NF-κB p65 was significantly higher in LPS model group than that in blank control group, and was lower in high-dose and low-dose paclitaxel-hirudin compound groups than that in LPS model group, and the decrease was more significant in high-dose paclitaxel-hirudin compound group. Compared with blank control group, levels of TNF-α, IL-1β and IL-6 increased in LPS model group （all P〈0.05）. Compared with LPS model group, levels of TNF-α, IL-1β and IL-6 decreased significantly in high-dose and low-dose paclitaxel-hirudin compound groups （all P〈0.05）. The decrease of IL-1β was more significant in high-dose paclitaxel-hirudin compound group compared with low-dose paclitaxel-hirudin compound group （P〈0.05）. Conclusion Paclitaxel-hirudin compound has significant inhibitory effect on NF-κB p65 activation, down-regulates effectively transcriptional activity of NF-κB p65, and inhibits significantly expressions of downstream inflammatory factors-TNF-α, IL-6 and IL-1β during HCASMC inflammatory activated process induced by LPS.