芒柄花素调控核因子-κB信号通路缓解软骨细胞炎症损伤作用

Effect of Formononetin on Alleviating Inflammatory Injury of Chondrocytes via NF-κB Signaling Pathway

目的观察芒柄花素对C28/I2软骨细胞炎症损伤的影响,研究芒柄花素干预核因子κB(NF-κB)信号通路缓解C28/I2软骨细胞炎症损伤的机制。方法筛选芒柄花素给药浓度,建立脂多糖(LPS)诱导C28/I2细胞炎症损伤模型。实验分组:正常对照组、LPS组、芒柄花素小剂量组(12.5μmol·L^(-1))、芒柄花素中剂量组(25μmol·L^(-1))、芒柄花素大剂量组(50μmol·L^(-1))。采用细胞计数试剂盒-8(CCK-8)检测芒柄花素对软骨细胞活力的影响。采用酶联免疫吸附测定(ELISA)检测C28/I2细胞培养上清液白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平;蛋白质印迹法检测C28/I2细胞内核因子κB抑制蛋白α(IκBα)、磷酸化核因子κB抑制蛋白α(p-IκBα)、核因子κB p65(NF-κB p65)、磷酸化核因子κB p65(p-NF-κB p65)蛋白表达。结果CCK-8实验结果显示,与正常对照组比较,10μg·mL^(-1) LPS组软骨细胞活力显著下降,给予芒柄花素后软骨细胞活力明显提高(P<0.01)。ELISA实验结果显示,与LPS组比较,给予芒柄花素可降低C28/I2细胞上清液IL-6和TNF-α水平(P<0.01或P<0.05)。蛋白印迹实验结果显示,与LPS组比较,芒柄花素可以剂量依赖性地下调NF-κB通路的磷酸化水平(P<0.01或P<0.05)。结论芒柄花素可缓解软骨细胞炎症损伤,其机制与调控NF-κB信号通路磷酸化活化有关。

Objective To explore the effect of formononetin on alleviating inflammatory injury of C28/I2 cell,and to explain its mechanism of alleviating inflammatory injury of chondrocytes via NF-κB signaling pathway.Methods The treating concentrations of formononetin were screened,and the model of LPS-induced inflammatory injury in C28/I2 cells was established.Experimental group:normal control group,LPS group,low-dose group(12.5μmol·L^(-1)),medium-dose group(25μmol·L^(-1)),and high-dose group(50μmol·L^(-1)).The cell counting kit 8(CCK-8)was used to detect the effect of formononetin on chondrocyte viability.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory cytokine interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in the supernatant of C28/I2 cell culture.Western blotting assay was used to measure the protein expressions of nuclear factor kappa B inhibitory proteinα(IκBα),phospho-nuclear factor kappa B inhibitory proteinα(p-IκBα),nuclear factor kappa B p65(NF-κB p65)and phospho-nuclear factor kappa B p65(p-NF-κB p65)in C28/I2 cells.Results The results of CCK-8 assay showed the chondrocyte viability decreased in the 10μg·mL^(-1) LPS group and increased in formononetin group(P<0.01).The results of ELISA experiments showed that the administration of formononetin could reduce the levels of IL-6 and TNF-αin the supernatant of C28/I2 cells(P<0.01 or 0.05)when compared with the LPS group.The results of Western blotting showed that compared with the LPS group,the phosphorylation level of the NFκB pathway could be down-regulated in a dose-dependent way(P<0.01 or 0.05).Conclusion The formononetin can alleviate the inflammatory injury of C28/I2 cell,which is related to the modulation of the phosphorylation activation of NF-κB signal pathway.