Withaferin A Inhibits M2 Polarization Of Macrophage By STAT6 Signaling Pathway

IntroductionMacrophages are one of the important components of innate immunity.According to the functional and morphological differences,macrophages can be divided into two types,including classically activated M1 macrophages(M1 macrophages)and alternatively activated M2 macrophages(M2 macrophages).M2 macrophages are stimulated and activated by Th2 cytokines IL-4,IL-13 and other cytokines IL-10,IL-33.M2 macrophages can express Arg-1,MR,Fizz1,YM1 and other cytokines.M2 macrophages also have the functions of anti-inflammatory,promoting tumor growth,metastasis,invasion,tissue repair,angiogenesis and so on.Macrophages in tumor microenvironment were called tumor associated macrophages(TAMs),most of which are M2 macrophages.TAMs as a target for cancer treatment is expected to become a new therapeutic strategy.Withaferin A(WA)is a steroid lactone compound extracted from medicinal plant withania somnifera.It has been proved to have anti-inflammatory,anti-tumor,immune regulation,anti-angiogenesis and other functions.Recent studies have shown that WA may become a potential therapy for non-small cell lung cancer,liver cancer,breast cancer and other cancers.Previous results of our group have showed that LPS-stimulated macrophage products could enhance WA-induced apoptosis of human breast cancer cells by inhibiting NF-?B pathway.In view of the anti-inflammatory and immunomodulatory effects of WA which were reported in the literature,whether WA has effect on the polarization of TAMs in tumor microenvironment has not been reported yet.Signal transducer and activator of transcription 6(STAT6)is one of the most important signaling pathways in the M2 polarization of macrophages.Therefore,we intend to investigate the effect of WA on the M2 polarization of macrophages in tumor microenvironment.We also investigate the role of STAT6 signaling pathway in the influence of WA on polarization.In this study,mouse peritoneal macrophages and mouse monocyte macrophage strain Raw264.7 were selected as research objects.Firstly,IL-4 was used to stimulate polarized two kinds of cells to observe the effect of WA on polarization.Then the conditioned medium(CM)of human breast cancer cells line MDA-MB-231 and MCF-7 was selected to simulate the tumor microenvironment.To observe the morphological changes of macrophages exposed to tumor conditioned medium;to observe the effect of WA on macrophages in tumor microenvironment.At the same time,the changes of STAT6 signaling pathway under the above-mentioned influencing factors were detected.The mechanism of WA's influence on the polarization of macrophages was preliminarily discussed.Methods(1)Firstly,the effect of WA on the proliferation and survival rate of mouse peritoneal macrophages and Raw264.7 cells was observed.The cells were treated with the different concentration of WA(1,2.5,5,10 ?M)for 12 hours.The absorbance was measured to observe the effect of WA on the proliferation and survival rate of these two macrophages.(2)To observe the effect of WA on polarization of IL-4 treated mouse peritoneal macrophages,DMSO control group(Control group),IL-4 group(20ng/ml),WA group(1?mol/ml)and WA+IL-4 group were established.Western blotting and qRT-PCR were used to detect the effect of WA on M2 macrophage-related cytokines Arg-1 and MR.(3)To observe the effect of WA on polarization of MDA-MB-231 CM treated mouse peritoneal macrophages,Control group,MDA-MB-231 CM group(100%),WA group and WA+MDA-MB-231 CM group were established.To observe the effect of WA on polarization of MCF-7CM treated mouse peritoneal macrophages,Control group,MCF-7CM group(100%),WA group and WA+MCF-7CM group were established.Western blotting and qRT-PCR were used to detect the effect of WA on M2 macrophage-related cytokines Arg-1 and MR.(4)To observe the effect of WA on polarization of IL-4 treated mouse monocyte macrophages Raw264.7,Control group,IL-4 group,WA group and WA+IL-4 group were established.To observe the effect of WA on polarization of Raw264.7 cells treated with two kinds of CM,Control group,MDA-MB-231 CM group,MCF-7CM group,WA group,WA+MDA-MB-231 CM group and WA+MCF-7CM group were established.The effects of WA on M2 macrophage marker Arg-1 and MR/CD206 were detected by Western blotting,qRT-PCR and flow cytometry.(5)To observe WA's effects on STAT6 signaling pathway of IL-4 treated two macrophages,Control group,IL-4 group,WA group and WA+IL-4 group were established.Western blotting was used to detect the effect of WA on STAT6 and p-STAT6 proteins in STAT6 signaling pathway.(6)To observe WA's effects on STAT6 signaling pathway of CM treated two macrophages,Control group,MDA-MB-231 CM group,MCF-7CM group,WA group,WA+MDA-MB-231 CM group and WA+MCF-7CM group were established.Western blotting was used to detect the effect of WA on STAT6 and p-STAT6 proteins in STAT6 signaling pathway.Results(1)When the WA concentration were above 1?mol/ml,WA have no obvious effect on the survival rate of two macrophages.When the WA concentration were above 2.5?mol/ml,the survival rate of two macrophages were obviously decreased.(2)Compared with the Control group,the expression of M2 macrophage-related cytokines Arg-1 and MR in IL-4 group of mouse peritoneal macrophages increased significantly;compared with the IL-4 group,the expression of Arg-1 and MR in WA+IL-4 group decreased significantly.(3)Compared with the Control group,the expressions of Arg-1 and MR in MDA-MB-231 CM group and MCF-7CM group of mouse peritoneal macrophages were increased;compared with the MDA-MB-231 CM group and MCF-7CM group,the expressions of Arg-1 and MR in WA+MDA-MB-231 CM group and WA+ MCF-7CM group were decreased significantly.(4)Compared with the Control group,the expression of Arg-1 and MR/CD206 in IL-4 group,MDA-MB-231 CM group and MCF-7CM group of Raw264.7 cells increased;compared with the IL-4 group,MDA-MB-231 CM group and MCF-7CM group,the expression of Arg-1 and MR/CD206 in WA+IL-4 group,WA+MDA-MB-231 CM group and WA+ MCF-7CM group decreased significantly.(5)STAT6 protein in each group did not change significantly.Compared with the Control group,the expression of p-STAT6 protein in IL-4 group of two macrophages increased obviously;compared with the IL-4 group,the expression of p-STAT6 protein in WA+IL-4 group decreased obviously.(6)STAT6 protein in each group did not change significantly.Compared with the Control group,the expression of p-STAT6 proteins in MDA-MB-231 CM group and MCF-7CM group of the two macrophages increased significantly;compared with the MDA-MB-231 CM group and MCF-7CM group,the expression of p-STAT6 proteins in WA+MDAMB-231 CM group and WA+MCF-7CM group decreased significantly.Conclusions(1)MDA-MB-231 CM and MCF-7CM can induce the M2 polarization of mouse peritoneal macrophages and mouse monocyte macrophages Raw264.7 cells;WA inhibits IL-4,MDA-MB-231 CM and MCF-7CM mediated the M2 polarization of mouse peritoneal macrophages and mouse monocyte macrophages Raw264.7 cells.(2)WA inhibits IL-4,MDA-MB-231 CM and MCF-7CM mediated the M2 polarization of mouse peritoneal macrophages and mouse monocyte macrophages Raw264.7 cells by STAT6 signaling pathway.