Akt信号通路在二氧化硅诱导RAW264.7细胞磷酸化的作用

Effect of Akt signaling pathway on phosphorylation of RAW264.7 cells induced by SiO_2

目的观察蛋白激酶B(Akt)信号通路在游离二氧化硅(SiO_2)诱导小鼠单核巨噬细胞株RAW264.7细胞发生Akt磷酸化的情况,探讨Akt信号通路在矽肺早期炎症反应中的作用。方法①常规培养RAW264.7细胞,分为5个不同时间点的SiO_2刺激组,采用终质量浓度为100 mg/L的SiO_2混悬液刺激15、30、60、120和240 min,另设不予SiO_2处理的对照组。染尘结束后,收集细胞,采用蛋白免疫印迹法检测磷酸化(丝氨酸/苏氨酸位点)Akt(p-Akt)表达水平,以选择最佳染尘时间。②将RAW264.7细胞分为对照组(不予任何处理)、SiO_2组(予终质量浓度为100 mg/L SiO_2混悬液处理)和干预组(予Akt活化抑制剂Deguelin预处理1 h后,予终质量浓度为100 mg/L SiO_2混悬液处理)。孵育60 min后,收集细胞,以细胞免疫荧光法观察细胞内p-Akt表达及定位情况,以蛋白免疫印迹法检测细胞中p-Akt相对表达水平,以酶联免疫吸附实验检测细胞上清液中肿瘤坏死因子-α(TNF-α)和转化生长因子-β1(TGF-β1)水平。结果①离体RAW264.7细胞染尘模型最佳处理时间为60 min。②细胞免疫荧光结果显示,RAW264.7细胞经SiO_2刺激后,Akt磷酸化作用被激活;SiO_2组细胞p-Akt荧光表达量较对照组增强,干预组细胞p-Akt荧光表达量弱于SiO_2组。SiO_2组、干预组细胞内p-Akt蛋白相对表达水平和细胞上清液中TNF-α、TGF-β的水平均高于对照组(P<0.05),干预组细胞上述3个指标均低于SiO_2组(P<0.05)。结论 Akt信号通路可能通过参与SiO_2诱导巨噬细胞磷酸化而参与矽肺早期炎症反应的过程。

Objective To observe the status of protein kinase B(Akt) signaling pathway in Akt phosphorylation induced by free silica(SiO2) in mouse monocyte macrophage cell RAW264.7, and the role of Akt signaling pathway in early inflammatory response of silicosis. Methods i) RAW264.7 cells were routinely cultured and divided into SiO2 stimulation groups at 5 different time points, and were stimulated for 15, 30, 60, 120 and 240 minutes with SiO2 suspension with a final concentration of 100 mg/L, and a control group without SiO2 treatment. At the end of treatment, the cells were collected and the expression of phospho-(Ser/Thr) Akt(p-Akt) was detected by Western blotting to select the optimal time of treatment. ii) RAW264.7 cells were divided into control group(no treatment), SiO2 exposure group(previous concentration of 100 mg/L SiO2 suspension) and intervention group(pre-treated with Akt activation inhibitor deguelin for one hour and then treated with 100 mg/L SiO2 suspension), samples were collected after incubation for 60 minutes. The p-Akt expression and distribution in cells were detected by cellular immunofluorescence assay, the relative expression of p-Akt in cells was detected by Western blotting, and the levels of tumor necrosis factor-α(TNF-α) and transforming growth factor-β1(TGF-β1) in the supernatant of cells were detected by enzyme-linked immunosorbent assay. Results i) The optimal treatment time of RAW264.7 cells for SiO2 exposure model was 60 minutes in vitro. ii) The results of cellular immunofluorescence assay showed that Akt phosphorylation was activated in RAW264.7 cells after stimulant with SiO2, and the fluorescence of p-Akt was enhanced in the SiO2 exposure group than the control group, and in the intervention group it was relatively weaker than the SiO2 exposure group. The relative expression of p-Akt as well as the levels of TNF-α and TGF-β1 in the SiO2 exposure group and the intervention group were higher than that in the control group(P<0.05), and the above three idexes in the intervention group were lower than the SiO2 exposure group(P<0.05). Conclusion Akt signaling pathway is involved in the process of SiO2-induced macrophages phosphorylation, and participates in the early inflammatory response of silicosis.