Wilforlide A Plays an Anti-lung Cancer Role by Inhibiting Macrophage M2 Polarization
Objective:To explore the effect of Wilforlide A(WA)on lung cancer cell proliferation and invasion and macrophage M2 polarization.Methods:(Experiment 1):LLC cells were divided into 0,0.01,0.1,1 and 10 μg/L groups according to WA concentra-tion.After incubation with WA for 48 h,the cell proliferation,apoptosis and invasion were detected by MTT method,AnnexinV-FITC/PI staining method and Transwell method,respectively,the protein expression levels of Bcl-2,Bax,MMP-2 and MMP-9 were detected by Western blot.(Experiment 2):RAW264.7 cells were divided into 0,0.01,0.1,1 and 10 μg/L groups according to WA concentration.The cell proliferation was detected by MTT assay.RAW264.7 cells were divided into Control group,IL-4 group,WA group and IL-4+WA group.The cells were cultured with 10 ng/mL of IL-4 and 1 μg/L of WA for 24 h.The levels of CD206,Arg-1 and IL-10 in cell culture supernatant were detected by ELISA.(Experiment 3):The cells were grouped as follows:Blank group,RAW264.7 group,IL-4RAW264.7 group,WA group and IL-4RAW264.7+WA group.LLC cells were co-cultured with RAW264.7 cells,IL-4-induced RAW264.7 cells and 10 μg/L WA for 24 h according to the grouping scheme.Cell proliferation,apoptosis,invasion,and M2 macrophage marker levels in cell culture supernatants were then detected by the above methods.The percentage of CD86 and CD206 positive were detected by flow cytometry.Results:(Experiment 1):Compared with the 0 µg/L group,the relative cell viability of the 0.1,1 and 10 μg/L groups de-creased(P＜0.05),the cell apoptosis rate and the relative protein expression level of Bax increased(P＜0.05),and the number of invasive cells and the relative protein expression levels of Bcl-2,MMP-2 and MMP-9 proteins decreased(P＜0.05).(Experiment 2):Compared with the 0 μg/L group,the relative cell viability of RAW264.7 cells in the 10 μg/L group decreased(P＜0.05).Compared with the Control group,the levels of CD206,Arg-1 and IL-10 in the IL-4 group increased(P＜0.05),while the levels of CD206,Arg-1 and IL-10 in the WA group decreased(P＜0.05).Compared with the IL-4 group,the levels of CD206,Arg-1 and IL-10 in the IL-4+WA group decreased(P＜0.05).(Experiment 3):Compared with the Blank group,the relative cell viability and number of invasive cells in the RAW264.7 group and the IL-4RAW264.7 group increased(P＜0.05),and the cell apoptosis rate decreased(P＜0.05).Compared with the RAW264.7 group,the rela-tive cell viability and number of invasive cells in the IL-4RAW264.7 group increased(P＜0.05),the cell apoptosis rate decreased(P＜0.05),and the levels of CD206,Arg-1 and IL-10 increased(P＜0.05),CD86 percentage decreased and CD206 percentage increased(P＜0.05).Compared with IL-4RAW264.7 group,the relative cell viability and number of invasive cells in the IL-4RAW264.7+WA group decreased(P＜0.05),the cell apoptosis rate increased(P＜0.05),and the levels of CD206,Arg-1 and IL-10 decreased(P＜0.05),CD86 per-centage increased and CD206 percentage decreased(P＜0.05).Conclusion:This study shows that WA has anti-NSCLC effects,and its mechanism is related to inhibiting the M2 polarization of macrophages.
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