| Objective:Rheumatoid arthritis(RA)is a chronic autoimmune disease characterised by aggressive arthritis and bone destruction,with osteoclast-mediated bone erosion leading to irreversible joint destruction.Currently,non-steroidal anti-inflammatory drugs,glucocorticoids and disease-modifying anti-rheumatic drugs including biologics(DMARDs)etc.can help relieve inflammation in RA patients in the early and active stages of the disease,but cannot repair the cartilage and bone destruction that develops in patients with advanced disease.Therefore,targeting osteoclasts to protect bone holds promise as a viable option for the treatment of RA.Mesenchymal stem cells(MSCs)produce a heterogeneous membrane secretion system,Mesenchymal stem cell exosomes(MSC-Exos),at rest or under stress,which has been found to be a major source of immunomodulatory and tissue repair capacity in MSCs.It has been found to be a major source of immunomodulatory and tissue repair capacity for MSCs.MSC-Exos has been used to treat various disease models.Our group has demonstrated that human umbilical cord-derived mesenchymal stem cell exosomes(HUCMSC-Exos)transplantation can inhibit collagen induced arthritis(CIA),an animal model of RA.arthritis(CIA),an animal model of RA,and improved joint imaging and pathological changes.In this study,we established an in vivo CIA mouse model and an in vitro culture system of Receptor Activator of Nuclear Factor κβLigand(RANKL)to induce osteoclast formation,and investigated whether HUCMSC-Exos could affect osteoclast differentiation to alleviate bone destruction in RA.Methods:Part I Acquisition and identification of HUCMSC-Exos.Healthy neonatal umbilical cord tissues from the Department of Obstetrics and Gynecology,Baiqiu’en Hospital,Shanxi,China,which underwent cesarean delivery,were selected for primary culture,and when the cell apposition was greater than 90%,the cells were passaged,and P3-P6 generation cells were selected for three-way differentiation induction assay,and the surface markers of HUCMSCs were identified by flow cytometry.When the growth density of HUCMSCs was greater than 70%,the cells were washed three times with PBS to remove the residual serum,and then the cells were cultured for48 hours with the addition of serum-free medium.The cell supernatant was then collected and HUCMSC-Exos was obtained by ultra-filtration tube concentration and differential centrifugation,and the extracted HUCMSC-Exos was identified in terms of morphology and protein concentration.Part II In vivo experimental study of the effect of HUCMSC-Exos on RA osteoclasts.In vivo,a CIA mouse model was established,and methotrexate(MTX)intervention was used as a positive control group.The effects of HUCMSC-Exos on the gross and joint swelling of mice were examined by arthritis index and body weight changes;The effects of HUCMSC-Exos on joint structure of mice were examined by Micro-CT and HE histopathological staining The effect of HUCMSC-Exos on the destruction of joint structures in mice was investigated by Micro-CT and HE histopathological staining;The effect of HUCMSC-Exos on articular cartilage and bone in mice was investigated by staining with red solid green.The effect of HUCMSC-Exos on the expression of the cytokines Interleukin6(IL-6),Interleukin10(IL-10),Tumor necrotic factorα(TNF-α)and Interleukin1β(IL-1β)in mouse serum was measured enzyme linked immunosorbent assay(ELISA);The effects of HUCMSC-Exos on osteocast differentiation and function-related genes(Mmp-9,Trap,Nfatc1)and proteins(Mmp-9,Trap,Nfatc1)were investigated by Tartrate-resistant acid phosphatase(TRAP)staining,Quantitative Real-time PCR(RT-q PCR)and immunohistochemical experiments.Part III Experimental in vitro study of the effect of HUCMSC-Exos on RA osteoclasts.In vitro,a model of RANKL-induced differentiation of RAW264.7 cells to form osteoclasts was established.The effect of HUCMSC-Exos on the proliferation ability of RAW264.7 cells was detected by CCK8 assay;MTX intervention was used as a positive control,HUCMSCs and HUCMSC-Exos were used as experimental groups and co-cultured for 72 h.The effect of HUCMSC-Exos on osteoclast differentiation was detected by TRAP staining assay;the effect of HUCMSC-Exos on osteoclast differentiation was detected by RT-q PCR and The effect of HUCMSC-Exos on RANKL-induced osteoclast differentiation formation and function-related gene(TRAP,MMP-9,NFATc1)and protein(TRAP,MMP-9,NFATc1)expression was examined by RT-q PCR and Western blot assay.Results:1.HUCMSCs and HUCMSC-Exos appraisal results:HUCMSCs were long shuttle-shaped under the microscope,and the results of flow cytometry showed that HUCMSCs highly expressed CD105,CD73,CD90,but hardly expressed CD34,CD14,HLA-DR,which met the identification criteria of HUCMSCs;HUCMSC-Exos had a "teato-like" structure with a particle size of 110.5 nm,which met the identification criteria of HUCMSC-Exos.The HUCMSC-Exos was in the shape of a "teatro-like" structure,with a particle size of 110.5 nm,and met the criteria for the identification of HUCMSC-Exos at a concentration of 0.8μg/μL.2.Results of in vivo animal experiments:In vivo,HUCMSC-Exos intervention was found to significantly reduce foot swelling in arthritic mice by arthritis index and number of swollen joints;HUCMSC-Exos intervention significantly reduced the level of joint bone erosion in mice by Micro-CT;HUCMSC-Exos intervention was found to inhibit inflammatory cell infiltration and bone and cartilage destruction in joints by HE and red solid green staining;HUCMSC-Exos intervention was found to HUCMSC-Exos intervention inhibited inflammatory cell infiltration and destruction of bone and cartilage in the joints.HUCMSC-Exos intervention was found to down-regulate the expression of genes related to osteoclast differentiation and secretion(Trap,Mmp9,Nfatc1);HUCMSC-Exos intervention was found to down-regulate the expression of proteins related to osteoclast differentiation and secretion(Trap,Mmp9,Nfatc1)by immunohistochemistry assay.3.Results of in vitro cellular assay:In vitro,HUCMSC-Exos was found to be non-proliferative and toxic to osteoclast precursors(RAW264.7 cells)at a range of concentrations by CCK8 assay;osteoclast formation could be successfully induced using RAW264.7 cells;HUCMSC-Exos intervention could be observed to significantly inhibit osteoclastogenesis by TRAP staining,and by RT-q PCR assay revealed that HUCMSC-Exos intervention could down-regulate the expression of genes related to osteoclast differentiation and secretion(TRAP,MMP-9,NFATc1);Western blot assay revealed that HUCMSC-Exos intervention could down-regulate the expression of proteins related to osteoclast differentiation and secretion(TRAP,MMP-9,NFATc1).Conclusions:HUCMSC-Exos ameliorates RA bone destruction by inhibiting osteoclast differentiation and secretory function. |
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