The Role Of Human Umbilical Cord Mesenchymal Stem Cells On Rats With Adjuvant Arthritis And The Effects On Macrophage Function

Rheumatoid arthritis(RA)is a systemic inflammatory autoimmune disease involving the small joints.The clinical symptoms are generally chronic,symmetrical,progressive polyarthritis.The basic pathological features are a large number of abnormal hyperplasia of synovial cells and abnormal stenosis of the joint cavity,which ultimately leads to limited joint activity and destruction of joint function.Its etiology and pathogenesis are still not fully understood.Early diagnosis and early effective treatment can prevent joint damage and achieve better long-term results.In the absence of effective therapeutic drugs in the clinic,in recent years,with the rise of cellular immunotherapy,a large number of researchers have made useful explorations for the use of mesenchymal stem cells for the treatment of rheumatoid arthritis.Macrophages are an important part of the body's innate immune response,expressing adhesion molecules,chemokine receptors and other surface antigens,as well as secreting chemokines,cytokines,growth factors,proteases and other mediators by recruiting other immune cells.,presenting antigens,promoting autoimmune responses,etc.,play an important role in the pathogenesis of RA.Macrophages are generally classified into classically activated macrophages(M1 macrophages)and selectively activated macrophages(M2 macrophages)according to their activation pathways.M1macrophages are characterized by increased microbicidal activity and production.Proinflammatory mediators promote the Th1 response of CD4+lymphocytes,but M2macrophages secrete anti-inflammatory cytokine effects,and M2 macrophages play an important role in inhibiting Th1 immune responses and enhancing tissue remodeling.Mesenchymal stem cells(MSCs)are non-hematopoietic progenitor cells derived from bone marrow.They are abundant in source,easy to culture in vitro,self-differentiated and low in immunogenicity,and do not involve ethical and related conditions.advantage.MSCs have been studied and reported in a variety of inflammation-related diseases by direct contact with macrophages or secretion of soluble factors to regulate macrophage function,and have a certain therapeutic effect.Whether human umbilical cord mesenchymal stem cells(hUC-MSCs)have therapeutic effects on adjuvant arthritis rats and their effects on macrophage-associated immune function in rats is not clear.Objective:To investigate the therapeutic effect of intravenous administration of human umbilical cord mesenchymal stem cells(hUC-MSCs)on adjuvant arthritis rats and the effect of hUC-MSCs on the function of rat macrophages mechanism.Methods:Adjuvant induced arthritis(AA)was established by complete Freund's adjuvant method.Model animals were randomly divided into model group and hUC-MSCs(2×10~6,5×10~6 cells/ml,tail vein injection).The group,Etancercept group(2.8 mg/kg,subcutaneous injection),another normal rats as control group.After modeling,the body weight and paw volume were recorded weekly,the whole body score and arthritis index score were calculated,and the joint swelling number was calculated.The administration of d21 was started.After 2 weeks of administration,the animals were sacrificed,and the weight of thymus and spleen were weighed.The pathological changes of the ankle joint and spleen were observed by HE staining.The levels of TNF-?,IL-1?,IL-6,IL-10 and IL-17 in serum of AA rats were detected by ELISA.CCK-8 The detection of T and B lymphocyte proliferation function,neutral red detection of rat phagocytosis of macrophages;flow cytometry to detect the percentage of T cell and macrophage related subpopulation.Peritoneal macrophages at the peak of AA rat inflammation were isolated and co-cultured with different numbers of hUC-MSCs for 48 hours to detect macrophage polarization,macrophage phagocytosis and levels of TNF-?,IL-1?,IL-6,IL-10 in co-culture supernatant.Results:1.Effects of hUC-MSCs on body weight changes and arthritis indexes in AA ratshUC-MSCs(2×10~6,5×10~6 cells/ml)and Etancercept(2.8 mg/kg)were treated for 2weeks after the d21 after CFA injection in AA rats,hUC-MSCs and Etancercept treatment group,the paw swelling degree,arthritis index,systemic system score and joint swelling number of AA rats decreased significantly,and the weight of rats in the treatment group increased significantly compare to model group AA rats.2.Effect of hUC-MSCs on the pathology of ankle joint in AA ratsThe pathological examination of the ankle joint showed that the joint structure of the AA model was significantly damaged,the synovial cells proliferated significantly,the inflammatory cells infiltrated,and the formation of neovascular and vasospasm;hUC-MSCs and the Etancercept treatment group had significant improvement in the ankle joint pathology.3.Effects of hUC-MSCs on the pathology of spleen in AA ratsHistopathological examination showed that the number of spleen germinal centers increased,lymphocytes were severely infiltrated,and the boundary between white pulp and red pulp was blurred in the model group AA rats.Compared with the model group,the number of germinal centers in the spleen tissues of the hUC-MSCs and the Etancercept treatment group decreased,the lymphocyte infiltration was reduced,and the score was significantly reduced.4.Effects of hUC-MSCs on thymus index and spleen index in AA ratsThe volume of spleen and thymus organs in the model group was significantly larger than that in the normal group,and the indices of the two groups were correspondingly increased.The spleen and thymus index of AA rats were significantly decreased after treatment with hUC-MSCs and Etancerceptgroup.5.The proliferation of thymus T lymphocytes and spleen B lymphocytes in AA rats by hUC-MSCsCompared with the normal group,the proliferation of T lymphocytes and B lymphocytes in the model group AA rats was strong.The proliferation of T and B lymphocytes in the AA rats treatment with hUC-MSCs and Etancercept were inhibited and decreased.6.Changes of the ratio of hUC-MSCs to spleen CD4+T cell subsets in AA ratsAfter spleen lymphocyte separation,flow cytometry showed that the percentage of CD3~+T cell,helper T cell(CD3~+CD4~+),cytotoxic T cell(CD3~+CD8~+),??T cells(CD3~+TCR~+)and unsensitized T cells(CD4~+CD62L~+)increased in the spleen of the inflammatory model group compared with the normal group.The percentage of double negative T cells(CD3~+CD4~-CD8~-)and memory T cells(CD4~+CD44~+)was reduced.Compared with the inflammatory model group,the treatment group with hUC-MSCs and Etancercept the proportion of double negative T cells(CD3~+CD4~-CD8~-)and unsensitized T cells(CD4~+CD62L~+)cells was significantly increased;however,the percentage of CD3~+T cell,helper T cell(CD3~+CD4~+),cytotoxic T cell(CD3~+CD8~+),??T cells(CD3~+TCR~+)and memory T cell(CD4~+CD44~+)subsets decreased significantly.7.Effect of hUC-MSCs on the percentage of peritoneal macrophages in AA ratsFlow cytometry analysis of peritoneal macrophages showed significant differences in M2 macrophages(CD11b~+CD163~+)and M1 macrophages(CD11b~+CD86~+)in model rats;hUC compared with model group-The percentage of M2 macrophages(CD11b~+CD163~+)was significantly increased in the hUC-MSCs group and the Etancercept group;however,the percentage of M1 macrophages(CD11b~+CD86~+)is significantly decreased.8.Effect of hUC-MSCs on phagocytosis of peritoneal macrophages in AA ratsThe detection of macrophage phagocytosis by neutral red phagocytosis showed that macrophage phagocytosis was enhanced in model group AA rat compared with normal group rats;compared with model group AA rats,hUC-MSCs and Etancercept treatment group AA rat macrophages phagocytosis function was inhibited to varying degrees.9.Effects of hUC-MSCs on the serum levels of TNF-?,IL-1?,IL-6,IL-10 and IL-17 in AA ratsThe results of ELISA showed that the serum levels of TNF-?,IL-1?,IL-6 and IL-17 in the model group AA were significantly increased,but the serum levels of IL-10 levels were significantly decreased,however the cytokines of TNF-?,IL-1?,IL-6 and IL-17 levels were decreased but the level serum of IL-10 is increased in serum of AA rats treated with hUC-MSCs and Etancercept group.10.Morphology and identification of hUC-MSCsThe cell morphology is long fusiform,and the cells are arranged in a spiral distribution.The identification of shunt cell cytometry showed that the positive expression levels of CD73,CD90 and CD105 on the surface of hUC-MSCs were99.54%,99.78%and 97.21%,respectively,while the positive markers on the cell surface were CD14,CD34,CD45 and HLA-DR which positive expression rates were0.52%,0.07%,0.01%,and 0.02%,respectively.11.Morphology and polarization of rat macrophages after 48 hours of co-culture of hUC-MSCsUnder the microscope,the macrophage morphology of the model group was different,and the cell morphology showed pseudopods and was dendritic and rod-like.The model group macrophages and hUC-MSCs were co-cultured for 48 hours,and the rods,stars and dendrites were observed.The number of macrophage morphology is significantly reduced,with most cells appearing round.Flow cytometry showed that the percentage of M2 macrophages(CD11b~+CD163~+)decreased and the percentage of M1 macrophages(CD11b~+CD86~+)increased in the model group compared with the normal group macrophages.And compared with the inflammatory model group the percentage of M2 macrophages increased after macrophages co-culture with hUC-MSCs in proportion(1:1,1:5,1:10,1:50),but the percentage of M1macrophages decreased.The percentage of macrophages varied with the proportion of hUC-MSCs,and the ratio of M2/M1 increased with the number of hUC-MSCs.12.Effect of hUC-MSCs on phagocytic function of rat macrophages after 48hours of co-cultureThe results showed that macrophage phagocytosis detected by neutral red,the macrophage phagocytosis was enhanced in the model group compared with the normal group;hUC-MSCs were proportional to the model group macrophages(1:1,1:5,1:10,1:50)which phagocytic function of co-cultured macrophages was inhibited,and the effect of inhibiting macrophage phagocytosis increased with the proportion of hUC-MSCs.13.Effects of hUC-MSCs on cytokine levels in rat macrophage co-culture supernatant after 48 hours of co-cultureLevels of TNF-?,IL-1?,IL-6 and IL-10 in the co-culture supernatant were determined by ELISA.Compared with the normal group,the levels of TNF-?,IL-1?and IL-6 in the supernatant of model rats were significantly increased,level of IL-10were significantly decreased.The ratio of hUC-MSCs to macrophage co-culture with(1:1.1,5,1:10,1:50),levels of TNF-?,IL-1?and IL-6 in the co-culture supernatant were significantly decreased,while level of IL-10 was significantly increased.Conclusion:1.HUC-MSCs can improve the joint damage of AA rats and reduce the pathological scores of joints and spleen by injection of by tail vein.At the same time,it also can inhibit the proliferation of T and B lymphocytes and regulate the proportion of T lymphocyte subsets in AA rats.Therefore,it have effect on rats with AA by tail vein injection.2.HUC-MSCs can inhibit macrophage polarization from M1 to M2 in vitro and in vivo.macrophage phagocytosis be inhibited by hUC-MSCs.The levels of TNF-?,IL-1?and IL-6 be down-regulate and level of IL-10 be increased by hUC-MSCs.