Effects Of Human Umbilical Cord Mesenchymal Stem Cell-derived Exosomes On The Phenotype,Function And Mechanisms Of B Cells In Collagen-induced Arthritis Mice

Background:Rheumatoid Arthritis(RA)is a common,long-term,systemic autoimmune disease characterized by synovial tissue hyperplasia,pannus formation and cartilage destruction as the main clinical features.Immune imbalance is an important pathway for the pathogenesis of RA.Immune cells such as B cells accumulate and infiltrate in the synovium of the joint,and then secrete inflammatory factors and autoantibodies,causing chronic inflammation of the synovium,cartilage tissue,and bone destruction.At present,the treatment of RA is mainly based on anti-rheumatic drugs such as non-steroidal anti-inflammatory drugs,hormones,and biological agents.However,long-term use of these drugs has many side effects.Therefore,it is very necessary to find safe and effective alternative drugs.Human umbilical cord mesenchymal stem cell-derived exosomes(h UCMSC-Exos)are extracellular vesicles secreted by h UCMSCs,with a diameter of about 30-150 nm.Compared with h UCMSCs,it is characterized by stable physical and chemical properties,no heterologous risk,easy storage and transportation,and can be used for the treatment of RA.In vitro studies have shown that h UCMSCs can inhibit B cell proliferation,differentiation,secretion of immunoglobulins,and induce B cells to differentiate into regulatory B cells(Bregs),thereby promoting the production of the anti-inflammatory factor IL-10.This study intends to further explore the effect and possible mechanism of h UCMSCs and h UCMSC-Exos on B cells in CIA mice,and provide theoretical and experimental basis for the clinical treatment of RA with exosomes.Objective:(1)To observe the therapeutic effects of h UCMSC-Exos on CIA mice.(2)To explore the effects of h UCMSC-Exos on B cells in CIA mice.(3)To study the mechanism of h UCMSC-Exos regulating B cell activation and differentiation.Methods:(1)DBA/1J male mice were selected to establish the CIA mouse model,and they were randomly divided into 5 groups:normal group,CIA group,MTX group,h UCMSCs group and h UCMSC-Exos group.(2)After 7 weeks of intervention,the body weight,the arthritis index,the number of swollen joints of the mice,and the average thickness of the hind paw were recorded.(3)HE staining was performed on mouse ankle joints to observe the histopathological changes of mouse ankle joints.(4)The levels of BAFF and RF in mouse serum were detected by enzyme-linked immunosorbent assay.(5)The ratios of total B cells,activated B cells,plasma cells,BAFFR~+and TACI~+B cells in the peripheral blood of mice were measured by flow cytometry.(6)The expressions of TRAF2,IκBαand p-P65 in mouse spleen B cells were detected by Western blotting.Results:(1)Compared with normal mice,the body weight of CIA mice was significantly decreased,and the arthritis index,paw swelling number and hind paw thickness were significantly increased(P<0.01).Intervention of CIA mice with MTX,h UCMSCs and h UCMSC-Exos can significantly increase their body weight and reduce their arthritis index,paw swelling number and hind paw thickness(P<0.05).(2)Compared with normal mice,the bone volume/total volume,the number of trabecular bone and the thickness of trabecular bone in the ankle joint of CIA mice were significantly decreased(P<0.05),and the bone surface area/total volume and the degree of trabecular bone separation were significantly increased(P<0.01);Intervention of CIA mice with h UCMSC-Exos increased their bone volume/total volume and the number of trabecular bone in the ankle joint(P<0.05),and decreased the degree of trabecular bone separation(P<0.01).(3)Intervention of CIA mice with MTX,h UCMSCs and h UCMSC-Exos can reduce their degree of inflammatory cell infiltration at the ankle joint.(4)Compared with normal mice,the serum levels of BAFF and RF in CIA mice were significantly increased(P<0.05).After the intervention of CIA mice with h UCMSC-Exos,the levels of serum BAFF and RF could be reduced(P<0.05).The comparison between groups showed that there was no significant difference in serum BAFF and RF levels between MTX group,h UCMSC group and h UCMSC-Exos group.(5)Compared with normal mice,the ratios of total B cells,activated B cells,plasma cells,BAFFR~+and TACI~+B cells in peripheral blood leukocytes of CIA mice were significantly increased(P<0.05).After the intervention of CIA mice with h UCMSC-Exos,the ratios of total B cells,activated B cells,plasma cells,and BAFFR~+B cells in peripheral blood leukocytes were significantly reduced(P<0.05).The comparison between groups showed that there was no significant difference in the ratios of total B cells,activated B cells,plasma cells,BAFFR~+and TACI~+B cells in the peripheral blood leukocytes of the MTX group,h UCMSC group,and h UCMSC-Exos group.(6)Compared with normal mice,the expression levels of TRAF2 and p-NF-κB65 in spleen B cells of CIA mice were significantly increased,and the expression levels of IκBαwere significantly decreased(P<0.01).After the intervention of CIA mice with hUCMSC-Exos,the expressions of TRAF2 and p-NF-κB65 signaling protein molecules in B cells in spleen of CIA mice were significantly decreased(P<0.05),and the expression of IκBαwas increased(P>0.05).The comparison between groups showed that there was no significant difference in the expression levels of TRA F2,IκBα,p-NF-κB65 in B cells in spleen of MTX group,h UCMSC group and h UCMSC-Exos group.Conclusion:(1)hUCMSC-Exos can exert anti-inflammatory and tissue repair effects by increasing the body weight of CIA mice,reducing their arthritis index,paw swelling number and hind paw thickness,and improving the degree of ankle bone destruction and joint histopathological changes.(2)hUCMSC-Exos can inhibit the differentiation of B cells and reduce the ratio of BAFFR~+and the secretion of RF in CIA mice(3)hUCMSC-Exos can inhibit B cell activation,differentiation and antibody secretion in mice by regulating BAFF/TRAF2/NF-κB signaling pathway.