| Objective Rheumatoid arthritis(RA) is a synovial tissue inflammation characterized by systemic autoimmune response, which can lead to bone and cartilage destruction and degeneration, joint deformities and stiffness. RA pathogenesis is complex and remains unclear. The condition can often cause protraction, serious influence to lif e and health of the patients. So, discussion of RA risk factors and pathogenesis for the treatment of RA has important research value. This study focused on expression relationship of Cadherin-11(Cad-11) and miR-144 in RA by fibroblast-like synovial cells in order to investigate the role of mi R-144 overexpression lentivirus in mouse RA synovial tissue pathology process.Methods 24 BALB / c 6-8 week mice were(male and female), body weight(20 ± 2) g were selected. 12 randomly selected mice were randomly divided into three groups, the normal group, the adjuvant group and the model group, the type Ⅱ collagen emulsion was injected into BALB / c mice tail base subcutaneously to construct type Ⅱ collagen-induced arthritis(CIA) mouse model. In vitro groups were divided into control mice(CT) FLSs, rheumatoid arthritis model group(RA) FLSs, RA + miR-144 and RA + Anti-miR-144, q PCR technique were adopted to test mi R-144, Cadherin-11, TNF-α, IL-1, IL-2, IL-6 levels. Further ELISA measurements of cell culture supernatant content of inflammatory cytokines, including IL-1, IL-2, IL-6 and other express TNF-α, Western blot was used to detect the expression of Cadherin-11. ELISA was adopted for further measurements of inflammatory cytokines in the culture supernatants including TNF-α, IL-1, IL-2, IL-6 and so on. For in vivo experimental section, the use of 12 CIA model mice were randomly divided into three groups, namely saline group, control plasmid group, and mi R-144 group, which involves mi R-144 overexpression lentivirus tail vein injection process. Synovial tissue of mice with HRP-labeled antibody method and immunohistochemistry to detect the expression and distribution of Cadherin-11 and TNF-α, and the pathological changes were observed by HE staining of synovial tissue.Results(1) The three groups of mice were divided based on NES treatment, emulsion adjuvant treatment and type Ⅱ collagen treatment process, the knee joint synovial tissue biopsy was observed by the light microscope showing: normal group and adjuvant the different treatment of mice, whose knee synovial tissue cells distributed uniformly and regularly arranged without local inflammatory cell infiltration, and pannus formation, cartilage cells arranged regularly knee, cartilage surface smooth. Model mice knee joint showed local inflammatory reaction, mononuclear cells proliferation, neutrophil and lymphocyte infiltration, edema and synovial layer cell hyperplasia and pannus formation, synovial tissue fibrosis occurs and fibrous exudation and deposition of collagen fibers, articular cartilage was severely damaged. Real-time PCR was used to detect mi R-144, Cadherin-11 and inflammatory cytokine levels of the CT group and RA group, the results showed in the RA group mi R-144 was significantly lower than CT group, Cad-11, TNF-α, IL-1, IL-6 levels were significantly higher than the CT group(p <0.05), IL-2 expression showed no significant difference between the two groups(p> 0.05).(2) Cytology in vitro analysis showed that in the RA group and RA + mi R-144 group, Cad-11 level showed no statistically significant difference(p> 0.05), but the value was significantly higher than the CT group and RA + Anti-miR-144 group(p < 0.05). RA + miR-144 group showed that TNF-α, IL-1, IL-6 levels were significantly lower than RA group(p <0.05). No significant difference(p> 0.05) was detected for IL-2 in the four groups. Western-Blot assay results suggested that CT group and RA non-transfected miR-144 group observed a significant blot, transfected miR-144 in RA mouse fibroblast-like synovial cells with no obvious Western blotting, and RA + Anti-mi R-144 group can also see a significant blot. PCR results showed that in the RA group Cadherin-11 levels were significantly higher than CT group(p <0.05), while RA mice by transfection of miR-144 FLSs of Cadherin-11 levels were significantly lower than RA group and RA + Anti-mi R-144 group(p <0.05). ELISA results suggested that in the RA + miR-144 group, TNF-α, IL-1, IL-6 levels were significantly lower than RA group and RA + Anti-miR-144 group(p <0.05), while RA group and RA + Anti- mi R-144 group showed TNF-α, IL-1, IL-6 was no significant difference(p> 0.05). IL-2 was no significant difference(p> 0.05) in the four groups.(3) In vivo studies of pathological synovial tissue of each group in the light microscope showed: saline group and the no-load group of mice treated knee inflammation with obvious inflammatory infiltration and synovial cell proliferation, pannus formation and cartilage damage. Synovial cells in the miR-144 mice knee were evenly located with no typical inflammation, cartilage damage. Immunohistochemical staining showed: TNF-α scattered in knee joint synovial layer. Image-Pro Plus 6.0 software analysis suggested that in the saline group, no-load group, mi RNA group, TNF-α expression percentage were 0.79 ± 0.05,0.73 ± 0.07,0.28 ± 0.04; In the saline group and the control plasmid group mouse, expression level of TNF-α is close(P> 0.05). RA model mice treated with miR-144 overexpression lentivirus, synovial tissue expression of TNF-α levels were significantly decreased, the difference was statistically significant(P <0.05).mi RNA overexpression lentivirus treated mice with RA synovial tissue under a fluorescence microscope showed: consistent Cadherin-11 protein is evenly distributed on the knee joint synovial layer of cells, cell morphology was consistent with previously description. Image-Pro Plus 6.0 software analysis suggested that the saline group, no-load group, miRNA group Cadherin-11 expression percentage were 0.45 ± 0.07,0.36 ± 0.06,0.12 ± 0.03. Saline group and the control plasmid group were similar on Cadherin-11 protein expression(P > 0.05); but after injection of the experimental mice mi-RNA overexpression lentivirus, Cadherin-11 protein expression in synovial layer of cells decreased significantly compared with the other groups, miR-144 overexpression lentivirus mouse and other Cadherin-11 protein expression differences synovial layer of cells was statistically significant(P <0.05) between the two groups of mice.Conclusions mi RNA-144 overexpression may weaken or suppress arthritis in a mouse model of synovial tissue inflammation, and decrease the expression of TNF-α. The effect was based on the inhibition of Cadherin-11. mi R-144 overexpression make mice with RA synovial inflammation reaction tends to ease and improve the pathological process, and reduce the local synovial tissue TNF-α, IL-1, IL-6 expression level. Therefore, based on the "mi RNA-144 expression lentivirus " strategy, it is expected to become an important means of treatment of RA, but its clinical application may still need to be more in-depth study of the detailed mechanism. |
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