Key Words Rheumatoid arthritis • 1,25-dihydroxyvitamin D 3 • MicroRNA-22 • Fibroblast-like synoviocytes • Pro-inflammatory cytokine • Collagen induced arthritis Abstract Objective: This study aims to investigate the regulatory mechanism of 1,25-(OH) 2 D 3 on the proliferation of fibroblast-like synoviocytes (FLS) and expressions of pro-inflammatory cytokines in rheumatoid arthritis (RA) rats via . Methods: A rat model of RA was established with a subcutaneous injection of type II collagen.

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... treated with different concentrations of 1,25-(OH) 2 D 3 the proliferation of FLS was estimated by the MTT method, and the optimal concentration of 1,25-(OH) 2 D 3 was selected for further experiments. Cell proliferation was detected by MTT. Cell cycle and apoptosis wereanalyzed by FCM.The IL-1ß, IL-6, IL-8, and PGE2 protein expressions were determined by ELISA, and MMP-3, INOS, and Cox-2 mRNA expressions were measured by qRT-PCR. Results: The rat model of RA was successfully established. Compared with the blank group, the 1,25-(OH) 2 D 3 and miR-22 inhibitors groups exhibited higher proliferation inhibition and apoptosis rates, lower levels of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, and PGE2), and decreased mRNAexpressions of MMP-3, INOS, and Cox-2. The miR-22 mimics group had lower proliferation inhibition and apoptosis rates, elevated expressions of pro-inflammatory cytokines and MMP-3, INOS, and Cox-2 than the blank group. In contrast to the 1,25-(OH) 2 D 3 group, the proliferation inhibition and apoptosis rates were down-regulated, and the expressions of pro-inflammatory cytokines and MMP-3, INOS, and Cox-2 were up-regulated in the 1,25-(OH) 2 D 3 + miR-22 mimics group. Conclusion: Our study demonstrated that 1,25-(OH) 2 D 3 inhibits the proliferation of FLS and alleviates inflammatory response in RA rats by down-regulating miR-22. Comparison of cell cycle and apoptosis among the seven groups The results of cell cycle and apoptosis revealed that there was no notable difference among the blank, miR-22 mimics NC, miR-22 inhibitors NC and 1,25-(OH) 2 D 3 + miR-22 mimics groups (all P > 0.05). Compared to the blank group, the 1,25-(OH) 2 D 3 and miR-22 inhibitors groups had more cells at G0/G1 phase anda higher apoptosis rate, andthe miR-22 mimics group exhibited less cells at G0/G1 phase and a lower apoptosis rate (P<0.05). Compared to the 1,25-(OH) 2 D 3 group, less cells at G0 / G1 phase and decreased apoptosis rate were observed in the 1,25-(OH) 2 D 3 + miR-22 mimics group (P< 0.05) (Fig. 5) . Comparisons of the protein expressions of IL-1ß, IL-6, IL-8 and PGE2 among the seven groups Based on the results of ELISA, no significant difference was observed in the protein expressions of pro-inflammatory cytokines (IL-I ß, IL-6, IL-8, and PGE2) among the blank, miR-22 mimics NC, miR-22 inhibitors NC, and 1,25-(OH) 2 D 3 + miR-22 mimics groups (all P > 0.05). Compared with the blank group, the 1,25-(OH) 2 D 3 and miR-22 inhibitors groups had down-regulated protein expressions of L-1ß, IL-6, IL-8 and PGE2, andthe miR-22 mimics group had higherprotein expressions of these cytokines (all P< 0.05). Compared with the 1,25-(OH) 2 D 3 group, protein expressions of L-1ß, IL-6, IL-8, and PGE2 were up-regulated in the 1,25-(OH) 2 D 3 + miR-22 mimics group (P < 0.05). It can be concluded that 1,25-(OH) 2 D 3 could down-regulate the expressions of pro-inflammatory cytokines in FLS of rats with RA, which can be reversed by miR-22 mimics (Fig. 6) . Comparisons of mRNA expressions of miR-22, MMP-3, INOS and cox-2 among the seven groups According to the qRT-PCR, there was no significant difference in the mRNA expressions of miR-22, MMP-3, INOS, and Cox-2 among the blank, miR-22 mimics NC, miR-22 inhibitors NC, and 1,25-(OH) 2 D 3 + miR-22 mimics groups (all P > 0.05). Compared with the blank group, the 1,25-(OH) 2 D 3 and miR-22 inhibitors groups had decreased mRNAexpressions of miR-22, MMP-3, INOS, and Cox-2, while mRNA expressions of miR-22, MMP-3, INOS, and Cox-2 were Fig. 6. Comparisons of pro-inflammatory cytokines protein expressions in fibroblast-like synoviocytes among the seven groups: *, P< 0.05 in comparison tothe blank group, #, P< 0.05 in comparison tothe 1,25-(OH) 2 D 3 group.