牛蒡子苷对ARDS细胞模型的抗炎作用及PI3K-AKT-NF-кB信号通路的影响

The Anti-inflammatory Effect of Arctiin on Acute Respiratory Distress Syndrome(ARDS)vitro Model and the Effect of PI3KAKT-NF-кB Signaling Pathway

[目的]研究牛蒡子苷(Arctiin,Arc)对急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)细胞模型的抗炎作用及其相关机制。[方法]将RAW 264.7细胞分为4组:对照组,模型组和Arc高、低剂量组。Arc高、低剂量组在脂多糖(lipopolysaccharide,LPS)刺激24 h前用Arc预处理24 h。采用噻唑蓝法(methy thiazolyl tetrazolium,MTT)测定Arc对RAW 264.7细胞的毒性,采用共聚焦显微镜观察细胞形态变化,以酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测细胞上清液中白介素-6(interleukin-6,IL-6)、白介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis-α,TNF-α)水平,免疫印迹法检测细胞中磷酸酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)、蛋白激酶B(protein kinase B,AKT)、核因子-κB(nuclear factor-κB,NF-κB)、核因子-κB抑制蛋白(inhibitor of NF-κB,IкBα)磷酸化水平。[结果]Arc对RAW 264.7细胞增殖活性无明显抑制作用(P>0.05),Arc高、低剂量组细胞形态与对照组相似。与对照组比较,模型组IL-6、IL-1β、TNF-α水平显著升高(P<0.001);与模型组比较,Arc高、低剂量组IL-6、IL-1β、TNF-α水平降低(P<0.05)。与对照组比较,模型组PI3K、AKT、NF-κB、IкBα磷酸化水平显著升高(P<0.001);与模型组比较,Arc高、低剂量组PI3K、AKT、NF-κB、IкBα磷酸化水平显著降低(P<0.05)。[结论]Arc对LPS诱导的ARDS细胞模型具有抗炎作用,其机制可能与抑制PI3K-AKT-NF-кB信号通路的活化有关。

[Objective]To study the effect of Arctiin(Arc)on lipopolysaccharide(LPS)-triggered acute respiratory distress syndrome(ARDS)in RAW 264.7 cells and its related mechanism.[Methods]The RAW 264.7 cell fell to 4 groups:control group,model group,Arc high-dose group and Arc low-dose group.Arc high-dose group and Arc low-dose group were pre-treated with Arc for 24 h to LPS 24 h stimulation.At the end of the experiment,the relative cell viability of RAW 264.7 cells were determined by methy thiazolyl tetrazolium(MTT)assay and the morphological changes were observed by confocal microscope.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of interleukin-6(IL-6),interleukin-1β(IL-1β)and tumor necrosis-α(TNF-α)in the serum.Western blot was used to detect the phosphorylation levels of phosphoinositide 3-kinase(PI3K),protein kinase B(AKT),nuclear factor-κB(NF-κB)and inhibitor of NF-κB(IкBα)in cells.[Results]Arc did not significantly inhibit the proliferation of RAW 264.7 cells(P>0.05),and the cell morphology of Arc high-dose group and Arc low-dose group was similar to that of control group.Compared with control group,the level of IL-6,IL-1βand TNF-αin model group increased significantly(P<0.001);compared with model group,the level of IL-6,IL-1β,and TNF-αin Arc high-dose and low-dose groups decreased(P<0.05).Western blot showed that compared with control group,the level of phosphorylation PI3K,AKT,NF-κB and IкBαin model group increased significantly(P<0.001);compared with model group,the level of phosphorylation PI3K,AKT,NF-κB and IкBαin Arc high-dose and low-dose group decreased(P<0.05).[Conclusion]Arc has anti-inflammatory effect on LPS-triggered ARDS model,it may be related to the inhibition of the activation of PI3K-AKT-NF-кB signaling pathway.