| Objective:In this study,we propose to establish a collagen-induced arthritis(CIA)mouse model to investigate the mechanism of mesenchymal stem cell(MSC)to improve T-lymphocyte differentiation through regulation of glucose metabolism in the treatment of CIA joint inflammation,and to provide a theoretical basis for clinical of rheumatoid arthritis(RA).Methods:1.Male 7-8 weeks DBA/1 mice were injected intradermally with type II collagen and adjuvants to establish a CIA model.CIA mice were randomly divided into CIA group(n=10)and MSC group(n=10),and unimmunized DBA/1 mice were set as the control group(n=6).Each mouse in MSC group was injected with 1×10~6 MSCs through tail vein on D28 after initial immunization,while mice in the CIA group were given the same volume of PBS,then the general condition,feet thickness and arthritis index(AI)of mice were observed and detected at different time points.2.Mice were sacrificed on D60 and spleens were collected and spleen index was measured in each group.The morphology of synovial membrane and spleen was observed by HE staining.The expression of serum tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-17(IL-17)and transforming growth factorβ(TGFβ)were detected by ELISA assay.Immunohistochemical staining was performed to detect the changes of Ki67expression in spleen;FOXP3,ROR-γt,PU.1,glucose transporter 1(GLUT1),fructose-2,6-biphosphatase 3(PFKFB3)and glucose-6-phosphate dehydrogenase(G6PD)in spleen were measured by q RT-PCR assay and correlation analyses were performed as well.Results:1.Compared with the control group,the mice in the CIA group were in poor mental condition with rough hair and lower body weight,while the above conditions improved in MSC group.The joint inflammation in CIA group reached peak on D38,and gradually regressed.AI scores in MSC group were significantly lower than those in CIA group on D45(P<0.05).Compared with the control group,the foot paw thickness of CIA mice began to increase on D27 and then slowly decreased on D38,and the foot claw thickness in MSC group showed a gradually decreased on D49(P<0.05).2.HE staining showed that the knee joint surface of the control group was smooth and flat,with no synovial hyperplasia,inflammatory cell infiltration and bone erosion;In the CIA group,there was a demonstrable synovial hyperplasia,increased pannus and severe cartilage destruction;while the above in the knee joint of mice was reduced in MSC treatment group.Compared with the control group,serum TNFα,IL-6 and IL-17 levels in mice of the CIA group were significantly increased(P<0.05),in addition TGFβlevels were significantly reduced(P<0.05).In contrast,the serum inflammatory cytokines levels(TNF-α,IL-6 and IL-17)in the MSC group were significantly lower than those in the CIA group(P<0.05),while the TGFβlevels was significantly higher than those in the CIA group(P<0.05).3.The spleen weight,spleen index and immunohistochemical staining of Ki67 expression were entirly higher in the CIA group compared with the control group(P<0.05).The spleen weight and Ki67 expression in the MSC treated group were significantly lower than those in the CIA group(P<0.05),and the spleen index tended to decrease,however,there was no statistical difference(P>0.05).Compared with the control group,the HE staining results showed increased spleen germinal centers,massive red marrow and hyperplasia of white marrow and lymphoid follicles in the mice of CIA group,while in the MSC group,there were improved changes of the above.4.Compared with the control group,the results of different T cell transcription factors by q RT-PCR showed that the expression levels of ROR-γt m RNA and PU.1 m RNA were significantly higher(P<0.05)and FOXP3m RNA expression level was lower(P<0.05)in the mice of CIA group.In the MSC group,the expression level of ROR-γt m RNA was decreased(P<0.05),FOXP3 m RNA was significantly increased(P<0.05),and the level of PU.1showed decreased trend,but there was no statistical difference(P>0.05)compared with the CIA group.The correlation analysis suggested that AI was significantly positively correlated with ROR-γt m RNA and PU.1 m RNA(P<0.05)and negatively correlated with FOXP3 m RNA(P<0.05).5.The results of splenic glycometabolism related protein suggested that GLUT1 m RNA and G6PD m RNA expression were elevated in the CIA group compared with the control group(P<0.05);GLUT1 m RNA expression was reduced in the MSC group compared with the CIA group(P<0.05),and G6PD m RNA levels tended to be decreased but with no statistical difference(P>0.05).And PFKFB3 m RNA was unchanged.The analysis of the correlation between splenic T cell transcription factors and protein in glucose metabolism showed that splenic GLUT1 m RNA was significantly positively correlated with ROR-γt m RNA and PU.1 m RNA in mice(P<0.05)and negatively correlated with FOXP3 m RNA(P<0.05);G6PD m RNA was significantly positively correlated with ROR-γt m RNA and negatively correlated with FOXP3 m RNA(P<0.05),but had no correlation with PU.1m RNA(P>0.05);PFKFB3 m RNA was positively correlated with FOXP3m RNA(P<0.05),and had no correlation with ROR-γt m RNA and PU.1m RNA(P>0.05).Conclusions:1.MSC improved general condition of CIA mice,reduced the AI scores,decreased joint inflammation and levels of pro-inflammatory cytokines,and increased anti-inflammatory cytokine levels as well.2.MSC inhibited the proliferation of splenic lymphocytes,decreased the expression of Th17 transcription factor(Th9 transcription factor showed decreased trend,but with no significant difference),and increased the levels of Treg transcription factor of CIA mice.Of note,all the above transcription factors correlated with AI,suggesting that MSC improve CIA via regulating the proliferation and differentiation of T lymphocytes.3.MSC reduced GLUT1 m RNA expression and cause a downward trend in G6PD m RNA expression in splenocytes of CIA mice.In addition,splenic glucose metabolism related proteins were correlated with T lymphocyte transcription factors,revealing that MSC may ameliorate joint inflammation by reducing glucose uptake of T lymphocytes,affecting their glucose metabolism levels,and participating in regulating T cell proliferation and differentiation. |
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