冬凌草甲素通过NF-kapppaB信号通路对LPS刺激树突状细胞的影响

Effect of Oridonin on LPS-Stimulated Dendritic Cells via NF-κB Pathway

目的探讨冬凌草甲素(ORI)通过核因子NF-kappaB(NF-κB)炎症信号通路对脂多糖(LPS)刺激树突状细胞(DC)的影响。方法CCK8法检测ORI对DC活性的影响,并根据CCK8检测法结果,筛选出12.50、25.00μM ORI为干预浓度,实验分为空白组、LPS组、12.50μM ORI+LPS组、25.00μM ORI+LPS组。采用ELISA法检测通路相关炎症因子肿瘤坏死因子-α(TNF-α)及白介素-12(IL-12)浓度,RT-PCR法检测细胞因子白介素-6(IL-6)mRNA的表达,免疫荧光法检测磷酸化(p)-NF-κB p65蛋白(p-NF-κB p65)的表达水平。结果40.00、50.00μM ORI干预DC 24 h后,DC活性降低(P<0.05或P<0.01),50.00μM ORI干预DC 48 h后,DC活性降低(P<0.01);ORI及LPS干预DC 24 h后,与LPS组对比,12.50μM ORI+LPS组、25.00μM ORI+LPS组的TNF-α质量浓度减少(均P<0.01),IL-12的质量浓度差异无统计学意义(P>0.05),IL-6 mRNA的表达下降(P<0.01),显微镜下观察发现12.50μM ORI+LPS组、25.00μM ORI+LPS组细胞内荧光标记p-NF-κB p65的表达减少。结论ORI可能通过抑制NF-κB p65炎症通路的活化从而对LPS刺激DC炎症反应起到抑制作用。

Objective To explore the effect of oridonin(ORI)on lipopolysaccharide(LPS)-stimulated dendritic cells(DC)through NF-κB inflammatory pathway.Methods The viability of DC was detected by CCK8 assay.According to the results of CCK8 assay,12.50 and 25.00μM ORI were selected as the intervention concentrations.Cells were divided into four groups:blank group,LPS group,12.50μM ORI+LPS group,and 25.00μM ORI+LPS group.The concentrations of tumor necrosis factor-α(TNF-α)and interleukin-12(IL-12)were measured by ELISA.The expression of interleukin-6(IL-6)mRNA was detected by RT-PCR.The expression of phosphorylated NF-κB p65(p-NF-κB p65)was determined by immunofluorescence assay.Results The viability of DC was significantly decreased by treatment with 40.00 and 50.00μM ORI for 24 h,as well as by 50.00μM ORI intervention for 48 h(P<0.05 or P<0.01).Compared with LPS group,TNF-αconcentration and IL-6 expression de creased in both 12.50μM ORI+LPS group and 25.00μM ORI+LPS group(P<0.01).No obvious change was observed in the level of IL-12(P>0.05).Microscopy showed that the expression of fluorescently labeled p-NF-κB p65 decreased in both 12.50μM ORI+LPS group and 25.00μM ORI+LPS group.Conclusion ORI may suppress the activation of NF-κB p65 pathway,thereby inhibiting the inflammatory response in LPS-stimulated DC.