基于NF-κB通路探讨六神胶囊对脂多糖诱导巨噬细胞RAW264.7炎症反应的影响

Effect of Liushen Capsules (六神胶囊) on Lipopolysaccharide-induced Inflammatory Response in RAW264.7 Macrophages: based on NF-κB Pathway

目的探讨六神胶囊的抗炎作用及机制。方法RAW264.7细胞分为六神胶囊组和地塞米松组,六神胶囊按9.76、4.88、2.44、1.22、0.61、0.31、0.15μg/ml梯度稀释;地塞米松按250、125、62.5、31.25、15.63、7.81、3.91μg/ml梯度稀释,选择细胞存活率大于90%的最大3个六神胶囊浓度和最大无毒剂量的地塞米松进行后续实验。RAW264.7细胞分为空白组,地塞米松组,模型组及六神胶囊高、中、低剂量组。除空白组外,其余各组采用脂多糖诱导建立炎症模型。造模后六神胶囊高、中、低剂量组分别加入筛选浓度的高、中、低浓度六神胶囊溶液各1 ml;地塞米松组加入筛选浓度的地塞米松溶液1 ml;模型组和空白组加入1 ml培养液。24 h后收集细胞上清液检测一氧化氮(NO)的浓度,ELISA法检测细胞中白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)的含量,实时荧光定量PCR法检测细胞中IL-1β、TNF-α、IL-6和诱导型一氧化氮合酶(iNOS)mRNA表达,蛋白印迹实验检测核因子κB p65(NF-κB p65)、磷酸化核因子κB p65(p-NF-κB p65)、核因子抑制蛋白κB(IκBα)和磷酸化核因子抑制蛋白κB(p-IκBα)表达。结果选择六神胶囊1.22、0.61、0.31μg/ml浓度,地塞米松31.25μg/ml浓度进行后续实验。与空白组比较,模型组细胞中TNF-α、IL-1β、IL-6及NO水平,TNF-α、IL-1β、IL-6和iNOS mRNA表达,p-IκBα、p-NF-κB p65蛋白表达显著升高,IκBα蛋白表达显著降低(P<0.01);与模型组比较,地塞米松组及六神胶囊中、高剂量组细胞TNF-α、IL-1β、IL-6及NO水平,TNF-α、IL-1β、IL-6和iNOS mRNA表达,p-IκBα和p-NF-κB p65蛋白表达均显著降低,IκBα蛋白表达升高(P<0.05或P<0.01);与地塞米松组比较,六神胶囊高剂量组IL-1β水平降低、IL-6水平升高,TNF-αmRNA及p-NF-κB p65蛋白表达显著降低(P<0.01);六神胶囊高剂量组TNF-α、IL-1β含量,IL-6、TNF-αmRNA表达,p-NF-κB p65蛋白表达低于六神胶囊中、低剂量组(P<0.01)。结论六神胶囊在体外具有良好的抗炎活性,可能通过抑制NF-κB信号通路的激活,降低iNOS的表达,进一步抑制NO的合成和炎性细胞因子的产生,从而改善炎症反应,以高剂量效果较佳。

Objective To explore the anti-inflammatory effect of Liushen Capsules(六神胶囊)and the underlying mechanism.Methods The RAW264.7 cells were divided into Liushen Capsules group and dexamethasone group.Liushen Capsules solution at the concentration of 9.76,4.88,2.44,1.22,0.61,0.31,and 0.15μg/ml were used for gradient dilution,and dexamethasone solution at the concentration of 250,125,62.5,31.25,15.63,7.81,and 3.91μg/ml were administered for gradient dilution.The top three maximum concentrations of Liushen Capsules solution with cell viability over 90%,as well as the maximum non-toxic concentration of dexamethasone solution were selected for subsequent experiments.RAW264.7 cells were divided into blank group,dexamethasone group,model group and high-,medium-and low dose Liushen Capsules groups.Except for the blank group,the rats were induced by lipopolysaccharide to establish the inflammation model.After modeling,1 ml of the selected concentrations of Liushen Capsules solutions were added into the corresponding high-,medium-and low-dose Liushen Capsules groups,while 1 ml of the dexamethasone solution with selected concentration was added in the dexamethasone group;and 1 ml of culture medium was given in the model group and the blank group.After 24 hours,the cell supernatant was collected to detect the concentration of nitric oxide(NO),and the ELISA method was used to detect the levels of interleukin-1β(IL-1β),tumor necrosis factorα(TNF-α),and interleukin-6(IL-6)in the cells.The mRNA expressions of IL-1β,TNF-α,IL-6 and inducible nitric oxide synthase(iNOS)in the cells were detected by real-time fluorescence quantitative PCR,while the expressions of nuclear factorκB p65(NF-κB p65),phosphorylated NF-κB p65(p-NF-κB p65),inhibitor of nuclear factorκBα(IκBα)and phosphorylated IκBα(p-IκBα)were detected through western blotting.Results The selected concentrations of Liushen Capsules solution were 1.22,0.61,and 0.31μg/ml for subsequent experiments,and the selected concentration of dexamethasone solution was31.25μg/ml.Compared to those in the blank group,the levels of TNF-α,IL-1(3,IL-6 and NO in the cells,the mRNA expressions of TNF-α,IL-1β,IL-6 and iNOS,and the protein expressions of p-IκBαand p-NF-κB p65 significantly increased in the model group,and the protein expression of IκBαwas significantly reduced(P<0.01).Compared to those in the model group,the levels of TNF-α,IL-1β,IL-6 and NO,the mRNA expressions of TNF-α,IL-1β,IL-6 and iNOS,the protein expre-ssions of IκBαand p-NF-κB p65 were significantly reduced,while the protein expression of IκBαwas increased(P<0.05 or P<0.01).Compared to the dexamethasone group,the high-dose Liushen Capsules group had lower level of IL-1β,higher level of IL-6,and decreased expressions of TNF-αmRNA and p-NF-κB p65 protein(P<0.01).The levels of TNF-αand IL-1β,the mRNA expressions of IL-6 and TNF-α,and the protein expression of p-NF-κB p65 in the high-dose Liushen Capsules group were significantly lower than those in the medium-and low-dose groups(P<0.01).Conclusion Liushen Capsules has good anti-inflammatory activity in vitro,which may improve the inflammatory responses by inhibiting the activation of NF-κB signaling pathway,reducing the expression of iNOS,further inhibiting NO synthesis and inflammatory cytokines productions,and higher dose shows better effect.