The Research Of FuZheng JieDu Decoction Interventing And Regulating The Tumor Associated Macrophages Activated In Vitro

Relapse and metastasis of malignant tumor have always been the key problem that restricts the clinical efficacy in cancer treatment. In cancer patients after resection of primary tumor, some immune regulating cells and cytokines have close relation with tumor relapse and metastasis, in which the important one is the tumor-associated macrophages(TAMs). In the past, researchers thought that macrophages played an important role in anti-cancer immune regulation in that macrophages could directly kill cancer cell, or eliminate cancer cells by presenting certain tumor antigen to activate body immune response. However, as more researches are devoted to tumor stroma and cancer inflammation, it comes to realized that tumor-associated macrophages (TAMs) have the property of M2-polarized macrophages and play a role in promoting cancer development, invasion, metastasis, suppressing immune response and promoting tumor neovascularization and microlymphatic formation rather than anti-cancer effect. At present, macrophages are supposed to be at least two different activated phenotype and functional characteristics:(1)classically activated macrophages, caMphi, M1type;(2) alternatively activated macrophages, aaMphi, M2type.Type of M1macrophages can directly or indirectly secrete a variety of proinflammatory cytokines to kill pathogens and tumor cells; and type of M2macrophages characterized by low antigen presenting ability, and can produce inhibitory cytokines such as IL-10, TGFβto down-regulate the immune response, promote the growth of tumor cells and distant metastasis. M1and M2type are the two extremes state of macrophages activation in a row. According to the research results of thyroid papillary carcinoma, ovarian cancer and breast cancer, researchers have pointed out that the TAMs may occur alternative activation and TAMs prefer to M2polarization.TCM has a significant effect in the prevention and treatment of tumor relapse and metastasis. Fu Zheng Jie Du (FZJD) method is the major principle to prevent cancer metastasis after surgery in TCM cancer treatment. Our previous study has already confirm that:Fu Zheng Jie Du decoction had a better effect in controlling tumor size,reducing lung metastasis of tumor-bearing mice, and improving the prognosis of tumor-bearing mice.The mechanism was to reduce CD68+macrophage infiltration in tumor tissue, reduce M2macrophages induced cytokine IL-4, IL-10, IL-13, TGF-(3,and protect the immune function by reducing immunosuppressive cells (CD11b+F4/80+macrophages, MDSC) in spleen. Based on the previous research, we conducted experiments in vitro, using the in vitro activated tumor associated macrophage as the target, with co-cultureing with MFC cells,built the co-culture model in normoxic and hypoxic conditions.We observed the influence of FuZheng JieDu decoction on TAM’s cell surface markers,on the regulatory effect of immunosuppressive factors such as IL-10,and the intervention in TAM’s related chemotactic genes mRNA in the co-culture system, to further elucidate the molecular mechanisms of FZJD in regulating tumor microenvironment mediated by tumor associated macrophages.Objectives:1.To explore the role of macrophages in tumor microenvironment.2.To elucidate the molecular mechanisms of FZJD in regulating tumor microenvironment mediated by tumor associated macrophages.Methods:1. Preparation of rat blank serum and serum containing FZJD decoction, as the experimental medication of this research.2. Using LPS and IL-4activating RAW264.7cells in vitro, respectively inducing to M1and M2-type macrophages and identified the activated phenotypes.3. Build the non-contact co-culture system of M2macrophages with MFC cells in vitro.4.Using flow cytometry to observe the effect of FZJD on TAM’phenotypes in12h、24h、36h、48h both in normoxic and hypoxic conditions. 5. Using ELISA to observe the effect of FZJD on the expression of IL-10、IL-13and TGFfβ in co-culture system both in normoxic and hypoxic conditions in12h、24h、36h、48h.6. Using PCR to observe the effect of FZJD on TAM mRNA gene expression of CCL22、 CCL3、 Arg I、iNOS and HIF1-α both in normoxic and hypoxic conditions in36h.Result:1.RAW264.7cells induced by IL-4after24h, the expression of M2macrophages marker CD206increased significantly compared with no tempted group (p<0.05);while induced by LPS after24h, the expression of Ml macrophages marker CD16/32increased significantly compared with IL-4induced group (p<0.05).After induction,the expression of M2type: aaMphi (IL-4induced)> caMphi (LPS induced)>RAW264.7(p<0.05), the expression of Ml type:caMphi (LPS induced)>aaMphi (IL-4induced)>RAW264.7(p<0.05)2.Successfully build the co-culture system of M2macrophages with MFC cells both in normoxic and hypoxic conditions.3.Both in normoxic and hypoxic conditions, FZJD can significantly increase the proportion of Ml-type TAM (P<0.05),and it has the trend of reducing the proportion of M2-type TAM (P>0.05).4. In normoxic conditions at each time point, FZJD can reduce the expression of IL-10,especially at24h,36h and48h (p<0.05), and can reduce the expression of IL-13,especially at12h and24h (p<0.05). Both in normoxic and hypoxic conditions, FZJD can reduce the expression of TGF-(3at each time point (p<0.05)5.In normoxic conditions,FZJD can reduce the expression of CCL22,which is the chemokine gene of M2-type TAM. In hypoxic conditions, FZJD can reduce the expression of Arg I, which is the specific expression gene of M2-type TAM. Both in normoxic and hypoxic conditions, FZJD can regulate the expression of CCL3and iNOS,which are associated with the Ml-type TAM (p<0.05) 6. In normoxic conditions,the mRNA expression of HIFl-α in FZJD and control group was not statistically significant (p>0.05). In hypoxic conditions, the mRNA expression of HIFl-α in FZJD is lower than the control group,but the difference is not statistically significant (p>0.05).Conclusion:1.Activating in vitro can successfully establish the M2and Ml macrophages model, and then build the co-culture system of M2macrophages with MFC cells both in normoxic and hypoxic conditions.2.FZJD can reduce the ratio of M2macrophages and increase the proportion of M1macrophages in co-culture system both in normoxic and hypoxic conditions, promote the transformation of TAM from M2to M1polarization.3.FZJD in vitro co-culture system can reduce the expression of negative regulatory cell factors IL-10、IL-13and TGF β both in normoxic and hypoxic conditions, regulate the tumor microenvironment.4.FZJD in vitro co-culture system can reduce the mRNA expression of M2macrophages specific gene Arg I and the chemotaxis gene CCL22, improve the mRNA expression of M1macrophages specific gene iNOS and the M1chemotaxis gene CCL3both in normoxic and hypoxic conditions.5. FZJD in vitro co-culture system can reduce the mRNA expression of HIFl-α in hypoxic conditions, improve the state of hypoxia of local tumor microenvironment to a certain extent.