Investigating The Effect Of KGM On RAW264.7 Polarized M1/M2 Subtype Of Macrophages Based On NF-κB And JAK-STAT6 Pathways

Objective:She Liugu is a tuber of the perennial herb of the genus Amorphaceae,which is an antitumor Chinese medicine commonly used in traditional Chinese medicine.Its main effective ingredient,glucomannan(KGM),accounts for about 50 % to 60 % of She Liugu.the early stage of the research study had found that KGM can enhance the phagocytosis ability of macrophages,promoted the release of cytokines,improved the body’s immune ability,and had a clear immune and antitumor effect.The polarization of macrophages is the main link in the development of immune function against tumor.Targeting measures such as inhibiting the differentiation of M2 macrophages and eliminating M2 macrophages has become one of the important areas of tumor treatment research.It has not been reported whether KGM immune antitumor effect can exert immunomodulatory effects by affecting macrophage M1/M2 subtypes.This study intends to take the macrophage cell line RAW264.7 as the research object,establish a M1/M2 subtype macrophage polarization model,and investigate the regulatory effect of KGM on macrophage polarization and related signaling pathways,so as to provide a scientific basis for the research of anti-tumor drugs related to KGM immunity.Method:(1)Macrophage cell line RAW264.7 was cultured in vitro,and RAW264.7 was polarized into M1 subtype macrophages by LPS(1 μg/m L)+ IFN-γ(20 ng/m L).Flow cytometry was used to detect the positive expression rates of membrane surface proteins CD16/32 and CD206,and real-time fluorescence quantitative PCR was used to detect the relative expression levels of TNF-α,i NOS,IL-1β,IL-6,IL-12,Fizz1,Arg1 and TGF-βm RNA,so as to verify the success of the M1 polarization model.(2)KGM interferes with successfully polarized M1 subtype macrophages.Flow cytometry was used to detect the effect of KGM on the positive expression rates of macrophage membrane surface proteins CD86 and CD206.ELISA was used to detect the secretion of inflammatory factors TNF-αand IL-1β.Griess method was used to detect nitric oxide release,q RT-PCR method was used to investigate the effect of KGM on M1 subtype macrophage-related cytokine m RNA,and Western blotting was used to was used to detect the effect of KGM on the expression of NF-κB pathway TLR4,Myd88,IKKα,P-IκBα,NF-κB P65 and P-NF-κB P65 in M1 subtype macrophages.(3)IL-4(20 ng/m L)was used to induce RAW264.7 polarization into M2 subtype macrophages,the positive expression rates of CD206 and CD16/32 were detected by flow cytometry,and the relative expression levels of TNF-α,i NOS,IL-1β,Fizz1,Arg1 and TGF-β m RNA were detected by real-time quantitative PCR,so as to verify the success of the M2 subtype macrophage model.(4)KGM interfered with successfully polarized M2 subtype macrophages,the effect of KGM on the surface molecules of macrophage membrane CD86 and CD206 was detected by flow cytometry.ELISA was used to detect the secretion of IL-10 and TGF-β inflammatory factors,q RT-PCR was used to detect the effect of KGM on M2 subtype macrophage-related cytokine m RNA expression.Western blotting was used to detect the effect of KGM on the expression of IL-4R α,STAT6,P-STAT6,SOCS1,KLF4,PPAR γ and PPAR δ proteins in the JAK-STAT6 pathway of M2 subtype macrophages.Results:(1)LPS(1 μg/m L)+ IFN-γ(20 ng/m L)can effectively induce the macrophage cell line RAW264.7 to polarize into M1 subtype macrophages.After detection,the positive expression rate of M1 subtype specific membrane surface molecule CD16/32 increased from 46.58 % to 71.89 %,and the positive expression rate of M2 subtype specific membrane surface molecule CD206 decreased from 10.65 % to 8.59 %,all of which have significant differences(P<0.01);Compared with the control group,M1 subtype macrophage-specific markers IL-6,IL-12,IL-1β,i NOS,TNF-α m RNA expression increased(P<0.05),and the expression of Fizz1 and Arg1 m RNA specific markers of M2 subtype macrophages decreased(P<0.01),which verified the successful polarization of M1 subtype macrophages.(2)Different doses of KGM interfere with the successfully polarized M1 subtype macrophages.Compared with the model group,100 μg/m L KGM can further increase the expression of the molecular marker CD86 on the surface of M1 subtype membrane,and the positive expression rate of CD86 increased from 59.87 % to 72.95 %(P<0.01),reduced the expression of the molecular marker CD206 on the surface of M2 subtype membrane,and the positive expression rate of CD206 decreased from 10.40 % to 3.53 %(P<0.01);Griess results showed that KGM can significantly increase the expression of NO,compared with the model group,there is a statistical difference(P<0.01);ELISA results showed that compared with the model group,KGM can induce the expression and release of TNF-α and IL-1β(P<0.01);The results of q RT-PCR showed that 100 μg/m L KGM could effectivelyincrease the expression of cytokine TNF-α,i NOS 、 IL-6 、 IL-1β,and IL-12 m RNA of M1-type macrophages(P<0.01)and decrease the expression of M2-type macrophages’ marker cytokine Fizz1 and Arg1 m RNA(P<0.01).It was preliminarily shown that KGM could enhance the activity of M1 subtype macrophages and induce the M2 subtype macrophages to change to M1 type.Western blotting results showed that LPS and IFN-γcan activate the TLR4/Myd88 signal transduction-mediated NF-κB pathway.After KGM intervention,100 μg/m L KGM can promote the NF-κB pathway-related proteins IKKα,and P-IκBα protein expression(P<0.01)promotes the release of NF-κB from the cytoplasm into the nucleus,and can effectively activate the NF-κB pathway mediated by TLR4/Myd88 signal transduction.(3)IL-4(20 ng/m L)can effectively induce RAW264.7 to be polarized into M2 subtype macrophages.The positive expression rate of M2-subtype specific membrane surface molecule CD206 increased from 7.19 % to 48.62 %(P<0.01),and the positive expression rate of M1-subtype specific membrane surface molecule CD16/32 decreased from 40.87 % to 28.31 %(P<0.01).The expressions of Arg1、TGF-β、Fizz1 m RNA of M2 subtype macrophages were increased(P<0.05),the expressions of TNF-α,i NOS,and IL-12 m RNA of M1 subtype macrophages were decreased(P<0.05),and M2 subtype macrophages were successfully polarized.(4)Different doses of KGM interfered with the successfully polarized M2 subtype macrophages.Compared with the model group,100 μg/m L KGM reduced the expression of the molecular marker CD206 on the surface of the M2 subtype membrane.The positive expression rate of CD206 decreased from 33.04 % to 14.97 %(P<0.01),increased the expression of the molecular marker CD86 on the surface of M1 subtype membrane,and the positive expression rate of CD86 increased from 39.63 % to 55.46 %(P<0.01);ELISA results showed that KGM reduced the expression and release of cytokines IL-10 and TGF-β,which were significantly different from the model group(P<0.01).q RT-PCR results showed that compared with the model group,the KGM group can effectively increase M1 macrophages significant cytokine TNF-α,i NOS and IL-6 m RNA expression(P<0.01),reduced M2 macrophages iconic cytokine Fizz1 m RNA expression(P<0.01),further proof that KGM can enhance the activity of M1 subtype macrophages and induced the transformation of M2 to M1 type;Western blotting results showed that IL-4 induced the activation of M2-type macrophages through the JAK-STAT6 pathway.KGM inhibited this process and reduced the expression of IL-4Rα,SOCS1,P-STAT6,KLF4,PPAR γ and PPAR δ of proteins in the JAK-STST6 signaling pathway,all of which were statistically significant(P<0.01),JAK-STAT6 may be a potential mechanism for KGM to inhibit the activation of M2 subtype macrophages.Conclusion:KGM has an immunomodulation effect on M1 and M2 subtypes of activated mouse macrophage RAW264.7,which can further promote the activation of M1 subtype macrophages,inhibit the activation of M2 subtype macrophages,enhance the immunomodulation function of M1 subtype macrophages,and promote the transformation of M2 subtype macrophages into M1 phenotype.The potential mechanism of KGM regulating M1 subtype macrophages may be the TLR/Myd88/NF-κB pathway,and the JAK-STAT6 pathway may be involved in the immunosuppressive effect of KGM regulating M2 subtype macrophages.