紫草素调节结核分枝杆菌诱导THP-1细胞炎症反应的机制研究

A Mechanism Study on Shikonin Regulating Inflammatory Reaction of THP-1 Cells Induced by Mycobacterium Tuberculosis

目的观察紫草素对结核分枝杆菌诱导THP-1细胞炎性因子分泌的影响,探究其作用机制。方法检测不同浓度紫草素(0.50、2.50、12.50、62.50μg/ml)处理THP-1细胞的抑制率和肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、巨噬细胞炎性蛋白-3α(MIP-3α),筛选紫草素最适浓度为2.50μg/ml。用脂质体法将si-NC、si-ROCKⅠ、pcDNA、pcDNA-ROCKⅠ分别转染结核分枝杆菌诱导的THP-1细胞,将pcDNA、pcDNA-ROCKⅠ转染2.50μg/ml紫草素处理的THP-1细胞。检测不同处理方式的THP-1细胞中TNF-α、IL-1β、MIP-3α、磷酸化细胞核转录因子(P65)、NF-κB抑制蛋白α(IκBα)mRNA表达水平,同时检测细胞中ROCKⅠ蛋白表达。结果成功构建结核分枝杆菌诱导THP-1细胞炎症损伤模型。紫草素(0.50、2.50、12.50、62.50μg/ml)呈浓度依赖性降低THP-1炎性因子的分泌,并选用2.50μg/ml的紫草素用于后续实验。与si-NC组比较,si-ROCKⅠ组THP-1细胞中ROCKⅠ、TNF-α、IL-1β、MIP-3αmRNA表达水平均显著降低(P<0.05)。与实验+pcDNA组比较,实验+pcDNA-ROCKⅠ组THP-1细胞中ROCKⅠ、TNF-α、IL-1β、MIP-3αmRNA表达水平均明显升高(P<0.01)。与对照组和si-NC组比较,实验组和si-ROCKⅠ组THP-1细胞中P65、IκBα、ROCKⅠmRNA和ROCKⅠ蛋白表达均显著降低(P<0.01)。结论紫草素通过ROCKⅠ/NF-κB信号通路调节结核分枝杆菌诱导THP-1细胞的炎性因子分泌。

Objective To observe the effect of Shikonin on secretion of inflammatory factors in THP-1 cells induced by mycobacterium tuberculosis and to explore its mechanism.Methods Inhibitory rates of THP-1 cells treated with different concentrations of Shikonin(0.50,2.50,12.50,62.50μg/mL),levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and macrophage inflammatory protein 3α(MIP-3α)were detected,and the optimal concentration was found to be 2.50μg/mL.The si-NC,si-ROCKⅠ,pcDNA and pcDNA-Rho kinaseⅠ(ROCKⅠ)were respectively transfected into THP-1 cells induced by mycobacterium tuberculosis,and pcDNA and pcDNA-ROCKⅠwere transfected into THP-1 cells treated with 2.50μg/mL Shikonin.The mRNA expressions of TNF-α,IL-1β,MIP-3α,phosphorylated nuclear transcription factor(P65)and NF-κB inhibitory proteinα(IκBα)in THP-1 cells treated with different methods were detected,and ROCKⅠprotein expression in the cells was detected synchronously.Results The inflammatory injury models of THP-1 cells induced by mycobacterium tuberculosis were successfully established.Shionin(0.50,2.50,12.50,62.50μg/mL)reduced the secretion of inflammatory factor in mycobacterium tuberculosis-induced THP-1 cells in a concentration-dependent manner,and 2.50μg/mL Shionin was selected for subsequent experiments.The mRNA expressions of ROCKⅠ,TNF-α,IL-1βand MIP-3αin THP-1 cells were significantly decreased in si-ROCKⅠgroup compared with those in si-NC group(P<0.05).The mRNA expressions of ROCKⅠ,TNF-α,IL-1βand MIP-3αin mycobacterium tuberculosis-induced THP-1 cells in experimental+pcDNA-ROCKⅠgroup were significantly increased compared with those in experimental+pcDNA group(P<0.01).The mRNA and protein expressions of P65,IκBαand ROCKⅠin THP-1 cells in experimental and si-ROCKⅠgroups were significantly decreased compared with those in control and si-NC groups(P<0.01).Conclusion Shikonin may regulate the secretion of inflammatory factors in THP-1 cells induced by mycobacterium tuberculosis through the ROCKⅠ/NFκB signaling pathway.