Effect of Akt signaling pathway on phosphorylation of RAW264.7 cells induced by SiO_2 / 中国职业医学

ABSTRACT

OBJECTIVE: To observe the status of protein kinase B(Akt) signaling pathway in Akt phosphorylation induced by free silica(SiO_2) 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 SiO_2 stimulation groups at 5 different time points, and were stimulated for 15, 30, 60, 120 and 240 minutes with SiO_2 suspension with a final concentration of 100 mg/L, and a control group without SiO_2 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), SiO_2 exposure group(previous concentration of 100 mg/L SiO_2 suspension) and intervention group(pre-treated with Akt activation inhibitor deguelin for one hour and then treated with 100 mg/L SiO_2 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 SiO_2 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 SiO_2, and the fluorescence of p-Akt was enhanced in the SiO_2 exposure group than the control group, and in the intervention group it was relatively weaker than the SiO_2 exposure group. The relative expression of p-Akt as well as the levels of TNF-α and TGF-β1 in the SiO_2 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 SiO_2 exposure group(P<0.05). CONCLUSION: Akt signaling pathway is involved in the process of SiO_2-induced macrophages phosphorylation, and participates in the early inflammatory response of silicosis.