Background/Purpose: In our previous study, several conclusions are reached like: (1) IL-22 can promote inflammation and osteoclast formation in the process of RA;(2) 1,25(OH)2D3 decreases serum IL-22 level in RA patients, and acts as a dose-dependent inhibition of expression of RANKL;(3) 1,25(OH)2D3 may inhibits the expression of RANKL by blocking IL-22-mediated JAK-2 /STAT-3and p38MAPK/ NF-ĸB transduction pathways.In this study, we will compare the formation of osteoclasts in different treatment groups to explore whether the 1,25(OH) 2D3 can inhibit the formation of osteoclasts by affecting the IL-22-mediated JAK-2 / STAT-3 and p38MAPK/ NF-ĸB signal transduction pathways.

Methods: 1. In this trial all the osteoclast precursors PBMCs came from the peripheral blood of healthy volunteers, the consentrations of RANKL, M-CSF, 1,25(OH)2D3 respectively: 30ng/mL、25ng/mL、1nM. All the samples were devided into the following groups: GroupA: PBMC+M-CSF；GroupB: PBMC+M-CSF+RANKL；GroupC: PBMC+M-CSF+RANKL+1,25(OH)2D3. Detected the levels of the osteoclasts’ marker TRAP, cathepsin K, matrix metalloproteinase (MMP)-9 mRNA by RT-PCR to assess the effect of the agents. 2.Isolate FLS from RA synovial tissue of patients and culture them to generation 4~8, add IL-22 to stimulate the secretion of RANKL. Isolate PBMC from the peripheral blood of healthy volunteers, add the M-CSF pretreatment after 24h when the cells adherent, co-culture the two kind cells. At the same time, add RANKL, M-CSF, IL-22, 1,25(OH)2D3 and different signal transduction protein inhibitors according to the experimental groups.The consent rations of RANKL and M-CSF are the same to part1, the consentration of IL-22 is 10ng/ml. We devided all the samples into the following 5 groups: Group a: PBMC+RA-FLS+M-CSF; Group b: PBMC+RA-FLS+M-CSF+RANKL; Group c: PBMC+RA-FLS+M-CSF+IL-22+1,25(OH)2D3; Group d: PBMC+RA-FLS+M-CSF+1,25(OH)2D3+IL-22+AG490; Group f: PBMC+RAFLS+M-CSF+1,25(OH)2D3+ IL-22+SB203580.

Results: 1.In the part1 the number of osteoclasts (multiple nuclear TRAP positive cells)in groupA, B, C respectively 7±>0.816, 30±2.944, 5±2.16. There was statistically significant difference between groups(P<0.05). 2.The mRNA of the three makers to osteoclast is higher in group B compared with the other two groups (P<0.05). But the difference between group A and C has no statistically significance(P>0.05). 3.In the part 2 the number of osteoclasts in the Groups a,b,c,d,f respectively 7.5±1.732, 38.5±3.416, 0.5±0.477, 21.75±3.775, 9±2.944. The number in the Group b is higher than in Group a, The difference was statistically significant(P<0.05); the difference between Groups c,d,f and Group b also has statistically significant(P<0.05); the difference between Groups d,f and Group c also has statistically significant (P<0.05). 4.The expression of the target genes in the Groups a,c,d and the Group f are lower than the Group b. There has no statistically significant difference between Group a and c, d and f (P>0.05). The differences between other groups are statistically significant(P<0.05).

Conclusion: 1,25(OH)2D3 can inhibit the formation of osteoclasts by blocking the IL-22-mediated JAK-2/STAT-3 signaling pathway.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/explore-the-possible-mechanisms-of-125oh2d3-on-the-formation-of-osteoclasts-in-rheumatoid-arthritis/