| Objective: Glioma is a primary malignant tumor derived from glial cells in the brain and spinal cord.It has low cure rate and high mortality rate.During the occurrence and progression of glioma,the surrounding environment centered on the tumor plays an important role,including a part of immune cells,which secrete a large number of cytokines and affect the biological activity of tumor and other components,which is called the glioma immune microenvironment.As part of the immune system,macrophages have two subtypes,the pro-inflammatory subtype M1 and the immunosu-ppressive subtype M2,the latter of which was found to be highly infiltrated in glioma tissues.Several studies have shown that glioma associated macrophages(GAMs)are closely related to the differentiation and progression of glioma,but the mechanism remains unclear.The IL-10 secreted by GAMs also was confirmed to promote the proliferation and invasion of tumor cells.In addition,as a steroid compound extracted from ginseng,ginsenoside Rh2 has been confirmed to have significant anti-glioma activity,however,its effect on the immune microenvironment of glioma is still unclear.This study aims to explore the effect of M2-type macrophages on glioma and its corresponding mechanism,and to study the intervention of ginsenoside Rh2 on macrophages in the microenvironment of glioma,which provides certain reference significance for searching for immunotherapeutic drugs related to glioma and revealing the role played by immune cells in tumor progression.Methods: 1.Our study used mRNA sequencing data as analysis foundation,which were selected from 542 glioblastoma(GBM)tissues and 180 glioma tissues with Ⅰ-Ⅳ grades of Oncomine database.M2 macrophages were marked by CD206 and CD163.M1 macrophages were marked by HLA-DPB1/MHC Ⅱ(HLA-DPB1 was the beta chain of human MHC Ⅱ molecule,and it can directly reflect the expression level of the latter).We assume the mRNA expression values of these molecules can reflect their overall expression level in glioma tissues.Then the expression differences of macrophages of two subtypes in different grade glioma were compared.Kaplan-Meier survival curve was used to analyze the effect of two kinds of macrophages with different expression levels on the overall survival rate of GBM patients.In mechanism research,the mRNA sequencing data of 542 GBM tissues of TCGA-GBM dataset was used to as analysis model,and we collected 427 genes co-expressed with CD163 in GBM tissues to perform enrichment analysis.According to MCODE score and clinical significance of gene expression level,the hub genes group was screened out and then enriched into tumor pathogenic pathways of KEGG to establish a simple glioma promotion mechanism model with GAMs.2.In laboratory experiment,THP-1 cells were induced as M1 and M2 macrophages respectively.Then we used ginsenoside Rh2 with different concentrations to intervened the M2 macrophages cultured alone and macrophages co-cultured with U87 glioma cells respectively.Flow cytometry and cellular immunofluorescence staining were used to observe the subtypes of macrophage polarization state.Meanwhile,ELISA assay was used to test the protein content of IL-10 in glioma-macrophage microenvironment.CCK8 test was used to observe the effects of two subtypes of macrophages on the growth activity of U87 glioma cells,and then Rh2 was added to observe its intervention on the aforementioned effects.The effects of glioma and glial cells on the migration ability of M2 macrophages were observed by scratch test,and the intervention of Rh2 on the above-mentioned effect was observed.3.To investigate the expression of IL-10 in glioma tissues and the effect of ginsenoside Rh2 on it,we analyzed the mRNA expression of IL-10 in Ⅰ-Ⅳ grade of glioma,and the data was obtained from Oncomine platform.We also used the mRNA expression value,which was obtained from the GBM dataset of TCGA,to study the relationship relevance of IL-10 to the macrophage markers including CD163,CD206 and HLA-DPB1.Then we collected the photograph of immunohistochemical staining of IL-10,CD163 and HLA-DPB1 respectively,which was obtained from the database of The Human Protein Atlas,and we compared the difference of protein content of aforementioned molecule between normal cerebral cortex and high-grade glioma tissue.Next,we used ELISA to detect the changes in the protein content of IL-10 in the glioma-macrophage microenvironment after the intervention of ginsenoside Rh2,and analyzed the changes in the expression of IL-10 in the glioma tissues of relapsed patients by using the data from Oncomine,as well as the expression correlation between IL-10 and the genes coding glioma-promoting factors such as MMP-7,MMP-9 and VEGF-C.Results: 1.In comparison with normal brain tissues and low-grade glioma tissues,CD163 mRNA expression in Ⅳ-grade glioma tissues increased significantly,and the mRNA ratio of CD163/ MHC Ⅱ also raised obviously.GBM patients with higher CD163 expression level and mRNA ratio of CD163/ MHC Ⅱ had lower overall survival rate.Bioinformation analysis found that the family of chemokines and their receptors,which were co-expressed with CD163 in GBM tissues,participated in the progress of GBM with the closest interaction way.Four genes in this family including CCL20,CXCR4,C5AR1 and CXCL8 had significant influence on the overall survival rate of GBM patients,which might activate the calcium-MAPK and CXCR4-RHOA pathways to promote the biological activities of GBM.2.The experimental results showed that ginsenoside Rh2 can reduce the CD206 expression in M2 macrophages and increased the MHC Ⅱ expression meanwhile.After treating by Rh2,macrophages cultured in glioma microenvironment have shown the same as the aforementioned changes,and the protein content of IL-10 in this environment also decreased.Rh2 also inhibited the proliferation of U87 glioma cells induced by M2 macrophages and the migration ability of M2 macrophages mediated by glioma cells.3.Compared with non-tumor brain tissues,IL-10 mRNA and protein expression levels were higher in high-grade glioma tissues,and the mRNA expression values and protein content of macrophage related markers CD163 and HLA-DPB1 were also significantly increased.According to the analysis of mRNA expression,IL-10 had a significant positive correlation with CD163,CD206 and HLA-DPB1,respectively,and the correlation coefficient with CD206 was the highest.Compared with non-recurrent GBM patients,the expression of IL-10 mRNA was increased in the tumor tissues of recurrent GBM patients,and it was significantly positively correlated with the expression of glioma invasion and angiogenic factors such as MMP-7,MMP-9 and VEGF-C.Ginsenoside Rh2 can significantly reduce the protein content of IL-10 in glioma-macrophage microenvironment.Conclusions: 1.M2-type macrophage markers were highly expressed in GBM tissues.With the higher expression degree of M2 macrophages,the GBM patients had lower overall survival rate and poorer prognosis.By secreting a large number of chemokines and activating corresponding receptors,M2-type macrophages might cause cascading reactions in glioma cells or various cellular components of the whole tumor environment.They can promote the growth,invasion and escape of apoptosis of GBM through activation of calcium-MAPK,CXCR4-RHOA and c AMP pathways et al.2.Ginsenoside Rh2 can reduce the ratio of M2 macrophages in GAMs,and inhibit the migration of M2-type GAMs and the proliferation of glioma cells induced by M2 macrophages.3.The expression of IL-10 was significantly increased in GBM tissue,which may be related to the high infiltration of M2-type macrophages in GBM tissue,and the high expression of IL-10 may be associated with the recurrence of GBM and the increased secretion of tumor-promoting factors to a certain extent.Ginsenoside Rh2 can promote the M1 polarization of macrophage in glioma microenvironment and reduce the protein content of IL-10,which provides a new idea to reveal the mechanism of anti-glioma activity of Rh2. |
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