| Rheumatoid arthritis(RA)is a refractory autoimmune disease characterized by immune dysfunction,autoantibody production,synovial inflammatory hyperplasia,and joint bone erosion.Bone erosion caused by abnormal activation of osteoclasts(OC)is a key pathological change in being teratogenic and disabling of RA.Regulatory T cells(Tregs)can slow down the bone erosion process by mediating bone immunity via interacting with OC.The intestinal mucosal immune system and gut microbiota constitute the intestinal microenvironment.And by influencing each other,they maintain the balance of the intestinal microenvironment jointly.Numerous studies have confirmed the disorders of intestinal microenvironment in patients with RA.And the previous experiments of our research team have also showed that unbalance of intestinal microenvironment exists in RA animal model,named Collagen-induced arthritis(CIA).Furthermore,studies have found that the aryl hydrocarbon receptor(AhR)signaling pathway plays an important role in bone immunity and intestinal microenvironment,and it is a key node between connecting immunity and bone balance,intestinal mucosal immunity and gut microbiota.Human umbilical mesenchymal stem cells(HUMSCs)have the potential for the treatment of RA,but the underlying mechanism is still unclear.Based on the CIA model and the Tregs-OC co-cultured system to explore,this study is aimed at exploring the effect of HUMSCs on bone immunomodulatory and intestinal microenvironment,and the possible mechanism of the AhR signaling pathway involved.1.Effect of human umbilical mesenchymal stem cells on collagen-induced arthritis rats by regulating bone immunity and intestinal microenvironmentObjective:With the in-depth study in relationship between gut microbiota and diseases,numerous studies have gradually found that disorder of intestinal microenvironment can affect RA.And previous studies have also verified that bone immune mediated by Tregs affectes process of RA via regulating bone destruction of OC.Furthemore,AhR is not only a key factor in regulating intestinal microenvironment,but also a impotant role in regulating differentiation and function of Tregs and OC.HUMSCs are accompanied by extremely low immunogenicity and show the therapeutic potential against RA,but the underlying mechanisms have not been fully elucidated.The purpose of this study was to explore the effects and mechanisms of HUMSCs in rats with CIA based on bone immunity and intestinal microenvironment.Methods:HUMSCs were isolated and cultured from neonatal umbilical cord and identified by flow cytometry.Third generation of HUMSCs(1×106)were transplanted into rat with CIA via intravenous injection through the tail.Methotrexate(MTX)was used as a positive drug for 28 days and arthritis score was evaluated by five-level grading system every 3 days during the period.At the end of the experiment,HUMSCs homing tissues were identified by immunofluorescence with NuMA protein as the marker.H&E staining and Micro-CT were used to assess pathology and bone destruction in ankle joint.The number of OC in ankle joint was detected by tartrate-resistant acid phosphatase(TRAP)staining.The percentage of Tregs and helper T cell 17(Th17)and the gene expressions of related cytokines were detected by flow cytometry and RT-PCR in popliteal lymph nodes,respectively.The percentages of Tregs,Th17 and B cells in the mesenteric lymph nodes,small intestine Peyer's patch(PP)and lamina propria lymphocyte(LPL)were detected by flow cytometry.The gene expressions of related cytokines in mesenteric lymph nodes and ileum were detected by RT-PCR and the expression of related proteins in ileum was detected by immunohistochemistry.Furthermore,the levels of immune-related factors in serum and the changes in gut microbiota in the ileum were detected by Luminex multi-cytokine analysis technique and 16s sequencing technology.The levels of indole and its derivatives in plasma were detected by liquid chromatography tandem-mass spectrometry(LC-MS/MS).At last,the gene and protein levels of AhR in ileum were evaluated via RT-PCR and immunohistochemistry,respectively.Results:HUMSCs were successfully isolated and cultured.HUMSCs homed to the spleen,inguinal lymph nodes,popliteal lymph nodes,mesenteric lymph nodes,ankle cartilage,and ileum mucosa in rats with CIA.The MTX and HUMSCs treatment reduced the arthritis score,pathology score,bone volume/bone surface area ratio and OC number in the ankles(P<0.05).The ratio of Tregs and the gene expression levels of IL-10 and TGF-?1 were increased and the ratio of Th17 and the gene expression level of IL-17A were decreased in the popliteal lymph nodes and mesenteric lymph nodes(P<0.05;P<0.01),which were the lymph tissue closest to the nidus and one of the gut-associated lymphoid tissues,respectively.The proportion of Tregs and B cells were increased and the proportion of Th17 were decreased in other gut-associated lymphoid tissues,namely,PP and the LPL after interventing of MTX and HUMSCs(P<0.05;P<0.01).In ileum,gene expression of TGF-?1 and protein expressions of IL-10,TGF-?1,IL-22 and IgA were increased(P<0.05;P<0.01)and expressions of IL-17A gene and protein were decreased after MTX and HUMSCs intervention(P<0.05;P<0.01).MTX and HUMSCs intervention were able to respond to abnormal serum levels of IL-10,TGF-?1,IL-17A,IL-1?,TNF-?in CIA rats(P<0.05).The OUT and Chao1 diversity of gut microbiota in ileum was increased in CIA group(P<0.05),while this anormal Alpha diversity was reversed after MTX and HUMSCs intervention(P<0.01).The partial least square discriminant analysis(PLS-DA)showed that the gut microbiota colony structure was changed after CIA model,MTX and HUMSCs intervention caused counterclockwise changes based on CIA colony structure distribution.Both CIA and treatments altered the relative abundance of the dominant bacteria in ileum compared with Control rats.The relative abundances of the genera Bacteroides and Bacillus were increased in the HUMSCs-treated rat with CIA.Besides,the level of indole,indoleacetic acid and indole-3-lactic acid were consistently upregulated compared with CIA group(P<0.05).Finally,compared with the Control group,the expression of the ileum AhR gene and protein was decreased in the CIA group;compared with the CIA group,the levels of ileum AhR gene and protein were significantly increased in HUMSCs group(P<0.05;P<0.01).Summary:HUMSCs had therapeutic effect on CIA rats.Mechanisms were as follows:on the one hand,HUMSCs targeted to popliteal lymph nodes and joints.By regulating the immune status of Tregs and Th17 in popliteal lymph nodes,HUMSCs showed the bone immunity effect and reduced the bone erosion in ankle joints.On the other hand,HUMSCs targeted to mesenteric lymph nodes and ileum.By regulating the immune status of Tregs,Th17 and B cells in PP and LPL,HUMSCs improved the intestinal mucosal immunity and influenced the intestinal microenvironment homeostasis,and arthritis was indirectly improved.The mechanism was related to the upregulation of AhR agonistic ligand indole and its derivatives followed by AhR activation.2.Effect and related mechanism of AhR agonist Tapinaraf on Tregs-OC co-cultured systemObjective:RA is characterized by the progressive damage of articular cartilage and secondary bone erosion.OC plays a key role in development of bone erosion because of function in bone resorption.And Tregs participate in bone immunity to inhibit bone destruction mediated by OC.Based on Tregs-OC co-cultured system established in the early stage,this study aimed to investigate the possible role and mechanism of AhR signaling pathway in RA bone immune by Tapinaraf as the tool medicine.Methods:Mouse bone marrow from the tibia,femur and spleen cells were isolated and induced into OC and Tregs respectively.Then,the co-culture system of Tregs-OC was established.The AhR agonist Tapinaraf with a concentration gradient was used to intervene OC,Tregs and Tregs-OC co-culture system.Cytotoxicity in Tregs and OC was evaluated by CCK-8 and number of OC differentiation under single culture and co-culture was detected by TRAP.Gene expressions were detected by RT-PCR of OC differentiation pathway under single culture and co-culture condition and Tregs cytokines under single culture condtion.Finally,Genc levels of AhR pathway in both cells were detected by RT-PCR.Results:In this study,OC and high-purity Tregs were successfully induced and differentiated.Tapinaraf at 10-3-103nM had no effect on the survival rate of OC and Tregs,while the survival rate was reduced at 104nM(P<0.05)in both OC and Tregs,indicating cytotoxicity of Tapinaraf at this concentration.Then,TRAP staining showed that Tapinaraf decreased the number of OC differentiation via concentration-dependent manner.Compared with the Control group,the minimum effective concentrations of Tapinaraf were 100nM and 10-1nM in OC group and Tregs-OC group,respectively(P<0.05).Furthermore,at the same Tapinaraf concentrationl the number of OC differentiation in Tregs-OC group was significantly reduced compared with that in OC group(P<0.05;P<0.01).Compared with the Control group,Tapinaraf had no effect on the gene expression of RANK in both OC group and Tregs-OC group.But Tapinaraf inhibited c-fos gene expression from concentrations of 100nM and 10-1nM respectively(P<0.05),and Tapinaraf inhibited NFATc1 gene expression at concentrations of 10-3nM,101nM.103nM and from 10-1nM respectively(P<0.05).Furthermore,at the same Tapinaraf concentration,the gene expressions of RANK,NFATc1 and c-fos in Tregs-OC group were significantly lower than that in OC group(P<0.05;P<0.01).In addition,the gene expressions of Tregs related gene such as IL-10 and TGF-?1 were upregulated by Tapinaraf in concentration-dependent manner at the minimum effective concentrations of 100nM and 101nM respectively(P<0.05).Finally,the gene expressions of AhR and CYP1A1 were enhanced by Tapinaraf in concentration dependence among Tregs group,OC group and Tregs-OC group and there were statistical differences at the concentration of 10-1nM(P<0.05).Besides,at the same Tapinaraf concentration,the gene expressions of AhR and CYP1A1 in Tregs-OC group were significantly higher than that in OC group(P<0.05;P<0.01).Summary:Tapinaraf activated AhR and downstream CYP1A1 in Tregs,OC and Tregs-OC co-culture system.On the one hand,the expressions of c-fos and NFATc1 in OC were reduced after intervened by Tapinaraf,which suppressed the differentiation of OC.On the other hand,Tapinaraf indirectly exerted the inhibitory effect to the differentiation of OC by increasing the expression of IL-10 and TGF-?1 in Tregs.3.Study on the regulation of Tregs-OC co-culture system by human umbilical mesenchymal stem cells through aryl hydrocarbon receptorObjective The effects of mesenchymal stem cells on Tregs and OC in vitro have been reported,but the effects and related mechanisms of mesenchymal stem cells on Tregs-OC co-cultured system have not been elucidated.The purpose of this study was to investigate the possible role and mechanisms of HUMSCs on Tregs-OC co-cultured system.Methods:Mouse bone marrow and spleen cells were isolated and induced into OC and Tregs,respectively.And the Tregs-OC co-culture system was established.The HUMSCs culture supernatant was used to intervene the cells.Cell proliferation rates of both Tregs and OC were detected by CCK-8 assay.Then,number of OC differentiation was evaluated by TRAP staining.Besides,gene expression and protein level of cytokines in Tregs were detected by RT-PCR and Luminex multi-cytokine analysis technique respectively.Finally,AhR gene expression of Tregs and OC were detected via RT-PCR.Results:The HUMSCs culture supernatant increased the cell proliferation rate of Tregs and OC(P<0.05).Compared with Control group,HUMSCs culture supernatant decreased the number of OC differentiation(P<0.01).The number of OC differentiation in Tregs-OC group was less than that in OC group,but it was no statistical difference observed.Furthermore,HUMSCs culture supernatant increased the gene expressions of IL-10,TGF-?1 and protein level of TGF-?1 in Tregs(P<0.05;P<0.01).Finally,the gene expressions of AhR and CYP1Alwere enhanced in OC and Tregs after HUMSCs culture supernatant intervention(P<0.05).Summary:HUMSCs inhibited the differentiation of OC through AhR.HUMSCs increased the expression of IL-10 and TGF-?1 in Tregs through CYP1A1 to exert an inhibitory effect on OC differentiation.Conclusion:HUMSCs had therapeutic effect on CIA rats.HUMSCs exerted joint bone immunity by regulating the immune state of popliteal lymph nodes to reduce bone erosion of ankle joint in CIA rats.By regulating the intestinal mucosal immune and changing the ileal gut microbiota,HUMSCs exerted its effect on regulating the intestinal microenvironment and improve arthritis indirectly.The mechanism might be related to AhR activation caused by the up-regulation of the AhR agonistic ligand indole and its derivatives.In the Tregs-OC co-culture system,Tapinarof-mediated AhR and downstream signaling pathway activation could inhibit the differentiation of OC,which also improved the bone immune function of Tregs,and further exerting the inhibitory effect on OC.The inhibitory effect of HUMSCs to OC differentiation was achieved by activating AhR in OC and by activating downstream gene CYP1A1 of AhR in Tregs to increase bone immunity of Tregs. |
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