Effect Of Yupingfeng Powder On The Function Of Skin T Lymphocyte And Macrophage Mediated By Thymic Epithelial Cells In Mice

Objective Yupingfeng Powder can regulate the immune function of the body,and it has definite therapeutic on the skin immune system dysfunction which is the key cells of skin resident T lymphocytes and macrophages.Thymic epithelial cells(TECs)provide the necessary microenvironment for the development,differentiation and maturation of T lymphocytes.Previous researches in our group have found that it would cause serious subcutaneous tumor metastasis because of the loss of thymus function in breast cancer mice.Therefor,we researched the effect of Yupingfeng Powder on the function and differentiation of T lymphocyte and macrophage in BALB/c mice skin mediated by TECs,and discussed the possible mechanism of TECs.Methods Section One:The skin tissue of BALB/c mice was isolated,and we cultured skin tissue which added Yupingfeng Powder 25,50,100 μg/mL(YPF-L,M,II)and thymic stromal lymphopoietin 2.5,5,10 ng/mL(TSLP-L,M,H)for 72 h;TECs were isolated and co cultured with skin tissue,the co cultured system was added same concentrations Yupingfeng Powder and TSLP as alone cultured skin tissue for 72 h.The number of CD3,CD4,CD8 positive cells and the expression of TSLP in the skin were detected by immunohistochemistry.Section Two:Yupingfeng Powder(25,50,100 μg/mL)directly intervened TECs for 48 h,Thiazolyl blue tetrazolium bromide(MTT)was used to detecte the effects of Yupingfeng Powder on the proliferation of TECs.TSLP,STAT3,IL-6,IL-7,IL-10,IL-12,IL-17 and TNF-α mRNA in TECs were detected by qPCR,Enzyme linked immunosorbent assay(ELISA)was used to detect IL-7 levels secreted by TECs,the expression of TSLP and STAT3 in TECs was detected by Western blot.Section Three:Mice spleen lymphocytes and thymocytes were isolated,the effects of Yupingfeng Powder(6.25-800 μg/mL)on the proliferation of spleen lymphocytes and thymocytes were detected by MTT;TECs co cultured with spleen lymphocytes and thymocytes respectively,Yupingfeng Powder(25,50,100 μg/mL)intervened the co cultured system for 48 h,the changes of T lymphocyte subsets in spleen lymphocytes and thymocytes were detected by flow cytometry,and ELISA was used to detect the levels of interferon(IFN)-γ,Interleukin(IL)-2 and IL-4 secreted by the spleen lymphocytes and thymocytes.Section Four:The effects of Yupingfeng Powder on the proliferation of RAW264.7 were detected by MTT;TECs co cultured with RAW264.7 and 25,50,100 μg/mL Yupingfeng Powder was treated with the co culture system for 48 h,fluorescence micro sphere phagocytosis assay combined with flow cytometry was used to detecte the phagocytosis rate and phagocytosis index of RAW264.7,the expression levels of TNF-α,IL-6,IL-1β mRNA of RAW264.7 were detected by qPCR.Section Five:The effects of Yupingfeng Powder on the proliferation of THP-1 was detected by MTT;TECs co cultured with THP-1 and 25,50,100μg/mL Yupingfeng Powder was treated with the co cultured system for 48 h,the mRNA levels of TNF-α,IL-6,IL-1 β of THP-1 were detected by qPCR.Results Section One:Compared with the control group,the number of CD3,CD4,CD8 positive cells in skin tissue increased after TECs co cultured with skin tissue after 72 h,it was more obvious after giving drug,and the number of CD4 positive cells increased significantly after giving drug.Section Two:After Yupingfeng Powder directly intervened,MTT results shown that Yupingfeng Powder had no significant effect on the proliferation of TECs;compared with the control group,the levels of TSLP,STAT3,IL-6,IL-10,IL-12,IL-17 and TNF-α in Yupingfeng Powder each dose group were significantly decreased,while the levels of IL-7 were significantly increased.The results of ELISA shown that IL-7 levels in Yupingfeng Powder each dose group increased(P<0.05,P<0.01),Western blot assay shown that the expression of TSLP and STAT3 decreased in Yupingfeng Powder low and middle dose group.Section Three:12.5-200 μg/mL Yupingfeng Powder could promote the proliferation of spleen lymphocytes and thymocytes after 48 h(P<0.01);Compared with the control group,the percentage of CD3+,CD4+,CD3+CD4+,CD8+and CD3+CD8+T cells of the co culture each dose groups in spleen lymphocytes and thymocyte increased significantly,and CD4/CD8 ratio of the co culture high does groups in spleen lymphocytes and the co culture each does groups in thymocytes increased significantly,the results of ELISA shown that IFN-y levels of the co culture each does groups and IL-2 levels of the co culture middle and high dose groups in spleen lymphocytes and thymocyte incrcascd(P<0.05,P<0.01),IL-4 levels were no change.Section Four:12.5-200 μg/mL Yupingfeng Powder could promote the proliferation of RAW264.7 cells after 48 or 72 h(P<0.01).Compared with the control group,the phagocytosis rate(29.27±0.76)%,(36.76±2.08)%,(37.04±0.36)%and the phagocytosis index 0.49±0.00,0.61 ±0.01 and 0.59±0.03 of RAW264.7 in co cultured each dose groups were significantly increased(P<0.05,P<0.01),the TNF-α levels of the co cultured groups and co cultured each dose groups in RAW264.7 cells were significantly increased,the levels of IL-6 and IL-1β of the co cultured each dose groups were significantly increased(P<0.05,P<0.01).Section Five:12.5-200 μg/mL Yupingfeng Powder could promote the proliferation of THP-1 cells after 48 h(P<0.01).Compared with the control group,the TNF-α levels of the co cultured groups and co culture each dose groups in THP-1 cells were significantly increased,the levels of IL-6 and IL-1β of the co culture each dose groups were significantly increased(P<0.05,P<0.01).Conclusion The presence of TECs could increase the number of T lymphocytes in BALB/c mice skin,while Yupingfeng Powder can promote the proliferation and differentiation of T lymphocytes in mice skin mediated by TECs,which is related to Yupingfeng Powder promoting the proliferation and differentiation of spleen lymphocytes and thymocytes in vitro,promoting the differentiation of T cells into Th1 cells,and enhancing the level of IL-7 secreted by TECs.In addition,Yupingfeng Powder can promote the proliferation and function of mice macrophages mediated by TECs.It is suggested that Yupingfeng powder can enhance the function of skin immune cells mediated by TECs in many ways.