The Research Of The Regulation Mechanism Of Fuzheng Jiedu Decoction On The Expression Of TEMs Cells In Tumor-burdened Mice Model Stimulated By M-CSF

Background:Known tumor immune microenvironment is composed of tumor cells and immune inflammatory cells in interstitial cells,which is the special environment for the survival of gastric cancer cells,and is the basis for the recurrence and metastasis of gastric cancer.Traditional treatment is aimed at "killing the last one tumor cell."Recent studies have found that the tumor immune microenvironment has gradually become an important target for the prevention and treatment of recurrence and metastasis of gastric cancer.Tumor stroma,the main components of inflammatory cells,most of these cells are not have antitumor activity,but and endothelial cells,fibroblasts and extracellular matrix interaction,secrete a variety of immune suppressor IL-10,inducing angiogenesis factor,more show the immunosuppression,promote the tumor cell immune escape,thus accelerating the transformation of tumor recurrence.Tumor associated macrophage(Tumor associated macrophage,TAM)is the immune microenvironment of Tumor is one of the most important immune inflammatory cells,and it's an important subgroup of expressing the Tie2 mononuclear macrophage(Tie2 expressing monocyte/macrophages,TEMs),than TAM polarization tend to M2 type,have a stronger role in vascular remodeling and promote Tumor tissue viability,thus became a focus in the treatment of gastric carcinoma in recent years.TEMs cells were discovered and defined by De Palma et al.in 2005 through studies of breast cancer mice and are widely present in solid tumor tumor stroma and peripheral blood.TEMs cells are derived from peripheral blood mononuclear cells and recruited to tumor tissues under the chemotaxis of angiopoietin-2(Ang2).TEMs can up-regulate immunosuppressive factors such as IL-10,Mrcl,Stabilin-1Msr2,Argl,and down-regulate the expression of anti-angiogenic molecules and Nos2,such as tnf-alpha,CXCL11,PTGS2/COX-2,TNF-a,IL-12,IL-1?,CCL5,CXCL10,etc.In the development of gastric cancer,this cell plays a role in promoting the growth,recurrence and metastasis of tumor tissue by inhibiting the proliferation of T cells,promoting the infiltration of regulatory T cells,suppressing the anti-tumor immune response and promoting angiogenesis,and regulating microvascular remodeling and other mechanisms.The principle of uprighting and detoxification is a basic rule for gastric cancer based on the basic pathogenesis of stomach cancer,which is the "virtual cancer" and the "virtual standard".It is in line with the complex and changeable clinical features of gastric cancer..Under the guidance of this principle,combined with long-term experience,our department has put forward the principle prescription of Fuzheng Jiedu Fang for treating gastric cancer,which has been widely used in clinical treatment and has achieved a good therapeutic effect.In order to further improve the curative effect and analyze the molecular mechanism of Fuzheng Jiedu Decoction in treating gastric cancer,we conducted a series of experimental studies.Completed project prior to confirm the centralizer detoxification medicine can regulate the transformation and activation of TAM phenotypes,inhibit TAM mediated the mTOR signaling pathway,the expression of angiogenesis factors,related regulation of tumor microvascular remodeling,thus effectively inhibiting gastric cancer before a tumor-burdened model of transplanted tumor growth in mice.This experiment based on this,further study on TAM cells subgroup TEMs the expression and function of cells in gastric cancer,and explore the centralizer detoxification party regulation M-CSF spurred a tumor-burdened TEMs cells expressing mechanism in mice,and study out the centralizer detoxification molecular mechanism of the inhibition of gastric cancer cell proliferation.Methods:Model:a mouse model of pre-transplant gastric cancer was established by M-CSF,40 of the SPF male 615 mice were taken,and the weight was between 18 and 22g,with 0.2mlmfc cell suspension per inoculation(the concentration was 1×106/ml).When the tumor was reached in 7 days,each mouse was injected into the transplanted tumor with 50?l M-CSF(concentration of 100ng/ml).With the experimental animals were randomly divided into four groups:model control group(c),Fuzheng Jiedu group(FZJD),5-Fu group(5-Fu),5-Fu+FZJD group(5-Fu+FZJD).the model control group given normal saline lavage,three groups were given corresponding drugs,after the intervention treatment time for two weeks.Effect observation:to observe the growth and inhibition of the transplanted tumor of the mouse model of the pretransplant gastric cancer in the patients with the stimulation of M-CSF.Mechanism to explore:both FACS flow cytometry to detect the M-CSF portability tumor-burdened mice gastric cancer before each group before and after the stimulus TEMs(F4/80+CD202+)accounted for the proportion of leucocyte CD45+ cells,immunohistochemical detection and Western-blot groups Tie2 expression.Immunohistochemistry and Western-blot were used to detect the expression of Tie2,Arg1,IL-10,TNF-a and COX-2 in each group.RT-PCR detected the gene levels of Arg1 IL-10,TNF-a and COX-2 in each group after drug intervention.Results:1.The effect of M-CSF on TEMs cell expression in the transplanted tumor of the former gastric cancer tumor.M-CSF group TEMs(F4/80+CD202+)cells accounted for the proportion of leucocyte CD45+ of 9.34±5.47%,control group TEMs(F4/80+CD202+)cells accounted for the proportion of leucocyte CD45+ of 1.53±0.28%.M-CSF group TEMs cells expressing quantity is higher than the control group,the difference was statistically significant(P<0.05).It is indicated that M-CSF can up-regulate the expression of TEMs cells in tumor tissues,thus affecting tumor recurrence and metastasis.2.M-CSF stimulated the tumor weight and the tumor rate in the mice with pretransplant gastric cancer.The tumor weight in the model control group was the highest,with 1.3467±0.3392.After the drug intervention,the tumor weight of each treatment group was reduced.and the tumor weight of the FZJD,5-Fu and 5-Fu+FZJD was 0.8935±0.2841,0.7021±0.3458,0.5748±0.3269.The tumor reinhibition rate was 33.65%,47.87%and 57.32%respectively in FZJD,5-Fu and 5-Fu+FZJD.The results showed that there were significant differences between the groups,and little difference in group.Transplantation tumor weight:control group>FZJD>5-Fu>5-Fu+FZJD.From the tumor weight and inhibitory rate data,by centering detoxification and 5-Fu combined with effective treatment group had better effect of inhibiting tumor cell growth,the difference has statistical significance(P<0.05).3.The effect of Fuzheng Jiedu Decoction on TEMs cell expression.Immunohistochemical results showed that the yellow brown area of the control group was less,and the color of Tie2 was darker after stimulation.The expression level of Tie2 protein was increased by M-CSF.Transplanted tumor control group Tie2 gene transcription level is highest,after treatment with drugs,each target gene transcription level have a lower trend,(P<0.05),the difference has statistical significance,combined with group of lower effect is most obvious.The expression of TEMs cells in tumor tissues can be down-regulated by Fuzheng Jiedu Decoction.4.The effect of Fuzheng Jiedu Decoction on the expression of immunosuppressive factors and anti-angiogenic factors.Immunohistochemical results show that the control yellow brown is rich,the FZJD,5-Fu and 5-Fu+FZJD after drug intervention yellow brown area is less,the color becomes shallow,display Tie2 expression level has obvious downtrend,confirmed the centralizer detoxification can cut TEMs cell content and distribution in the tumor stroma,and reduce the combination group was obviously,differences between groups was statistically significant(P<0.05).Furthermore,the correlation cytokines were further studied,and compared with the control group,Argl and IL-10 were down-regulated in each group,and TNF-? and COX-2 were up-regulated,and the 5-Fu+FZJD was the most obvious.The light density of Argl and I1-10 in the control group was higher.Compared with the control group,the expression level of Argl and IL-10 in the FZJD,5-Fu and 5-Fu+FZJD was decreased,and the combination group was the most obvious,and the difference was statistically significant(P<0.05).The light density of TNF-? and COX-2 protein in the control group was the lowest,and the expression level of TNF-?and COX-2 was increased after drug treatment,and the difference was statistically significant(P<0.05).Arg1 transplanted tumor in the control group,IL-10 gene transcription level is highest,after treatment with drugs,each target gene transcription level have a lower trend,(P<0.05),the difference is statistically significant,combined with group of lower effect is most obvious.Arg1 gene expression level was control group>FZJD>5-Fu>5-Fu+FZJD.The expression level of IL-10 was control group>FZJD>5-Fu>5-Fu+FZJD.Control the TNF-?,COX-2 gene transcription level minimum,after treatment with 5-Fu+FZJD,each target gene transcription level have a lower trend,(P<0.05),the difference is statistically significant,combining group reduced effect is most obvious.The level of TNF-? and COX-2 gene expression level was significantly higher than that in FZJD,5-Fu and 5-Fu+FZJD,and the difference was statistically significant(P<0.05).Conclusions:Fuzheng Jiedu Decoction can effectively inhibit the growth of transplanted tumor in the mouse model.Its mechanism may be that by reducing the TEMs cells in gastric cancer before a tumor-burdened amount of expression in the tumor tissue in mice,by immunosuppressive factor Arg1 and IL-10,increase anti-angiogenesis factor TNF-?,COX-2,inhibition of angiogenesis in the tumor immune microenvironment,thereby inhibiting the development of gastric cancer,reduce the relapse rate.