Immunomodulatory Activity Of Periplaneta Americana Extract CⅡ-3 And Its Chemical Components Based On M2 Polarization Of Macrophages

Objective:The M2 polarization model of macrophages in vitro was established to effectively inhibit the M2 polarization of macrophages as the activity index.To screen the best immunomodulatory active parts in Periplaneta americana and further explore the active components.To study whether the immune regulation function of the body has a certain impact on its tumor metastasis,and to explore the new anti-tumor mechanism of Periplaneta americana extract from the perspective of immunology.Methods:1.LPS（1 μg·mL-1）combined with IFN-γ（20 ng·mL-1）induced macrophages cell line RAW264.7 to polarize into M1 phenotype for 24 hours,and IL-4（20 ng·mL1）induced macrophages cell line RAW264.7 and Ana-1 to polarize into M2 phenotype for 48 hours.2.MTT assay was used to test the cytotoxic activity of the extracts of Periplaneta americana and its twelve compounds on the experimental cell line,and the cytotoxic activity of the extracts of the effect of conditioned medium on CT26.WT cell.3.RT-PCR was used to detect the effect of Periplaneta americana extract on the mRNA expression of M1 marker iNOS and M2 marker ARG-1 and CD206 in M1/M2 polarized macrophages.4.ELISA was used to detect the effect of the CII-3 and the compound pericanaside on the expression of M2 macrophage cytokine markers TGF-β and IL-10.5.The effects of the CII-3 and pericanaside on the expression of M2 macrophage marker CD206 by flow cytometry.6.The effect of the CII-3 and the pericanaside on the fluorescence intensity of M2 macrophage marker CD206 by cellular immunofluorescence.7.The macrophages were incubated with IL-4,the CII-3 and pericanaside alone or together,the conditioned medium（CM）was collected,and the CT26.WT cells were treated with different CM culture groups,and the migration ability of tumor cells was detected by cell scratch and transwell migration assay.Result:1.The CII-3 of Periplaneta americana was the most effective extract for inhibiting the mRNA expression of ARG-1 and CD206 markers of RAW264.7 polarized M2 macrophages induced by IL-4（P<0.05）.2.The pericanaside could down-regulate the mRNA expression of ARG-1 and CD206 of RAW264.7 polarized M2 macrophages induced by IL-4（P<0.05）.3.RT-PCR,ELISA,flow cytometry,and immunofluorescence results showed that CII-3 and the pericanaside inhibited the expressions of IL-4-induced M2 macrophage marker genes ARG-1 and CD206 and related cytokines TGF-βand IL-10（P<0.05）.4.The cell scratch and transwell assays showed that the CM of CII-3 and pericanaside could inhibit the migration of tumor cells CT26.WT（P<0.05）.Conclusion:1.The pericanaside had the best inhibitory effect on M2 macrophages,but had no significant effect on M1 macrophages.2.CII-3 and pericanaside play an anti-metastatic effect by interfering with the M2-type polarization of macrophages.