The Mechanism Of Yu Ping Feng San To Reshape The Immune Microenvironment Of Lung Cancer By Regulating Myeloid-derived Suppressor Cells

Objective:To observe the regulation of Yu Ping Feng San on tumor suppressor and myeloid-derived suppressor cells and T lymphocytes in Lewis lung cancer-bearing mice,and explain the possible mechanism of Yupingfengsan regulating immunity and inhibiting tumor from the whole,cell and molecular levels.Methods:In vivo experiments:1.Model preparation:6 weeks old,male C57BL/6 mice,subcutaneously injected1×10~6LLC cells into the right forelimb,and established a subcutaneous xenograft model of Lewis lung cancer-bearing mice.2.Group administration:The mice were divided into a model group(normal saline)and an experimental group(2340 mg/ml Yupingfeng San),200 ul was intragastrically administered once a day,and administration was started one week before the modeling,and 21 days were continuously administered.3.Observation of tumor volume and survival in mice.4.Flow cytometry to detect the proportion of mouse spleen and tumor immune cells.5.Quantitative real-time PCR was used to detect the expression of immunosuppressive genes in tumor tissues MDSCs.6.Western blotting was used to detect the expression of proliferation-related signaling pathway proteins in tumor tissues MDSCs.In vitro experiments:1.The mouse bone marrow cells of the model group were induced to culture in vitro for 48 hours,and were treated with 7mg/ml Yupingfengsan for 48h respectively,compared with the blank,The MDSCs and subpopulation ratio of bone marrow cells were detected by flow cytometry.2.The mouse bone marrow cells of the model group were induced to culture in vitro for 48 hours,and treated with 7mg/ml Yupingfeng Powder for 48h,compared with the blank,the CCK8 method was used to detect the proliferation of bone marrow cells,and the apoptosis of bone marrow cells was detected by flow cytometry.Quantitative real-time PCR The expression of immunosuppressive genes related to MDSCs in bone marrow cells was detected,and the expression of MDSCs proliferation-related signaling pathway proteins in bone marrow cells was detected by Western blotting.3.MDSCs were sorted from the spleens of model mice by magnetic mircobead.Lymphocytes were isolated from the spleens of blank mice with lymphocyte separation solution,and the purity of MDSCs was detected by flow cytometry.4.The establishment of MDSCs and lymphocyte co-culture system was divided into four groups:T,T+ypfs,MDSCs+T,MDSCs+T+ypfs with Yupingfengsan 7mg/ml intervention.The proportion of T cells subsets was detected by flow cytometry.Results:In vivo experiments:1.Compared with the model group,the tumor volume of the experimental group was significantly reduced(P<0.05),the spleen weight of the experimental group was significantly reduced(P<0.05),and the survival time of the experimental group was significantly prolonged(P<0.05).2.Compared with the model group,the proportion of spleen MDSCs in the experimental group decreased significantly(P<0.05).The proportion of spleen mononuclear MDSCs in the experimental group decreased significantly(P<0.05),and the proportion of spleen granulocyte MDSCs in the experimental group decreased significantly(P<0.05).The proportion of spleen CD3~+CD4~+T lymphocytes in the experimental group increased significantly(P<0.05).The proportion of spleen CD3~+CD8~+T lymphocytes in the experimental group increased significantly(P<0.05),and the proportion of spleen CD4~+CD25~+T lymphocytes in the experimental group decreased significantly(P<0.05).Compared with the model group,the proportion of tumor MDSCs in the experimental group was significantly decreased(P<0.05).The proportion of tumor CD3~+CD4~+T lymphocytes in the experimental group increased significantly(P<0.05).The proportion of tumor CD3~+CD8~+T lymphocytes in the experimental group increased significantly(P<0.05),and the proportion of tumor CD4~+CD25~+T lymphocytes in the experimental group decreased significantly(P<0.05).3.Compared with the model group,the expression levels of STAT3,ROS and Arg-1m RNA in the tumor tissues of the experimental group were significantly down-regulated(P<0.05).4.Compared with the model group,the expression of p-STAT3,p-AKT,p-MEK and p-ERK in the tumor tissues of the experimental group was down-regulated.In vitro experiments:1.Compared with the model group,the proportion of MDSCs in bone marrow cells of experimental groups decreased significantly(P<0.05),and the proportion of mononuclear MDSCs in the bone marrow cells of experimental groups decreased significantly.(P<0.05),the proportion of granulocyte MDSCs in the bone marrow of experimental groups decreased significantly(P<0.05).2.Compared with the model group,the number of bone marrow cells in the experimental group was significantly decreased(P<0.05),and the apoptosis rate of bone marrow cells in the experimental group was significantly increased(P<0.05).3.Yuping Fengsan intervenes in lymphocytes alone.There was no significant change in the proportion of CD3~+CD4~+,CD3~+CD8~+,CD4~+CD25~+T lymphocytes in the experimental group(P>0.05).Yupingfeng Powder intervened in the co-culture system of lymphocytes and MDSCs.Compared with the model group,the proportion of CD3~+CD4~+T lymphocytes in the experimental group increased significantly(P<0.05),and the proportion of CD3~+CD8~+T lymphocytes did not change significantly(P>0.05).),the proportion of CD4~+CD25~+T lymphocytes decreased significantly(P<0.05).4.Compared with the model group,the expression levels of STAT3,ROS and Arg-1m RNA in the bone marrow cells of the experimental group were significantly down-regulated(P<0.05).5.Compared with the model group,the expression of p-STAT3 was down-regulated in the bone marrow cells of the experimental group.Conclusions:1.Yupingfeng Powder can inhibit the growth of subcutaneous xenografts of Lewis lung cancer-bearing mice and prolong the survival of mice.2.Yupingfeng powder has the effect of inhibiting tumor and improving the immune function of Lewis lung cancer-bearing mice.It may be related to reducing the ratio of MDSCs and subpopulations,decreasing the proportion of CD4~+CD25~+T lymphocytes,and increasing the proportion of CD3~+CD4~+and CD3~+CD8~+T lymphocytes.3.The regulation of the number of T lymphocytes by Yupingfeng Powder may be related to inhibiting the proliferation of MDSCs and promoting the apoptosis of MDSCs.4.Yupingfeng Powder can down-regulate the expression of p-STAT3 and Arg-1m RNA.The targeted regulation mechanism of STAT3 and Arg-1 may be one of the ways of Yupingfeng Powder to inhibit the proliferation of MDSCs and regulate the number of T cells.5.The regulation effect of Yupingfengsan on MDSCs and T lymphocytes may be one of the ways of Yupingfengsan to inhibit tumor and immune regulation.