Explore The Formation Of Osteoclasts And Mechanisms Of 1,25-dihydroxy Vitamin D₃ To Fibroblast-like Synoviocytes Of IL-22-mediated Rheumatoid Arthritis

Background Rheumatoid Arthritis（Rheumatoid Arthritis,RA）is a common chronic autoimmune disease characterized by synovial inflammation and irreversible bone destruction.Osteoclast play a key role in the joint destruction of RA patients,and increase the secretion of RANKL,which is the essential condition of osteoclast differentiation.1,25（OH）₂D₃can modulate immune function and bone metabolism.However,vitamin D deficiency is widespread phenomenon in RA patients.The IL-22 in RA synovial play a important role in proinflammatory and bone damage.IL-22 can promote the expression of RANKLthrough the “JAK-2 / STAT-3 and p38MAPK/nf-kappa B”lightning signaling pathways,thereby to facilitate the formation of osteoclasts.Our previous study found that 1,25（OH）₂D₃ can reduce the levels of IL-22,and inhibition the expression of RANKL in dose dependent.So we speculated that 1,25（OH）2 d3 may reduce the formation of osteoclast by blocking " JAK-2 / STAT-3 and p38MAPK/nf-kappa B" lightning signal pathways in RA patients.Objective:1.To explore the role of 1,25（OH）₂D₃ in rheumatoid arthritis.2.To explore whether 1,25（OH）₂D₃ reduce osteoclasts formation by blocking IL-22-mediated “JAK-2 / STAT-3 and p38MAPK/nf-kappa B”lightning signaling pathways in RA patients.Method:1.Retrospective analysis of 1,25（OH）₂D₃ role in RA.2.Isolate FLS from RA synovial tissue of patients and culture them to generation 4⁸,add IL-22 to stimulate the secretion of RANKL.Isolate PBMCs from the peripheral blood of healthy volunteers,add the M-CSF pretre atment after 24 h when the cells adherent,coculture the two kind cells.At the same time,add RA NKL（30ng/ml）,M-CSF（25ng/ml）,IL-22（10ng/ml）,1,25（OH）₂D₃（1nM）and different signal transduction protein inhibitors according to the experimental groups.We div ided all the sample into the following five groups: group a: MN+M-CSF+RA-FLS+1,25（OH）₂D₃+IL-22;group b: MN+M-CSF+RA-FLS+1,25（OH）₂D₃+IL-22+PD9805;group c: MN+M-CSF+RA-FLS+1,25（OH）₂D₃+IL-22+parthenolid;group d: MN+MCSF+RA-FLS;group e: MN+M-CSF+RA-FLS+RANKL.Terminate the culture on t he 14 th day.Fix the cells and stain them with TRAP,then find if there’s any effe ct of chemical intervention and time on differentiation of the osteoclasts;detected the level of the osteoclasts’ marker TRAP,cathepsin K,matrix metall oproteinase（MMP）-9 mRNA by RT-PCR.Results:1.（1）The serum 25-（OH）D levels were lower than that of the healthy control group（t=-3.87,P<0.05）.（2）Serum 25-（OH）D levels were negatively correlated with BMI index,DAS28 score,ESR,CRP（r=-0.735,-0.539,-0.343,-0.539,P<0.01）,and positively correlated with Ts（r=0.636,P<0.01）.（3）The disease activity was classified into three groups: highly active disease,moderately active disease,slightly active disease,and significant differences of serum25-（OH）D levels were found by comparasion with each droup（F=132.839,P<0.01）.The serum 25-（OH）D levels of highly active disease were negatively correlated with ESR and DAS28 score（r=-0.381,-0.567,P<0.01）.（4）BMI was classified into four groups: low BMI,normal BMI,overweight,obese,and significant differences of serum 25-（OH）D levels were found by comparasion with each droup（F=31.41,P<0.01）.The serum 25-（OH）D levels of overweight and obese were negatively correlated with ESR（r=-0.395,-0.385,P<0.05）.（5）The 25-（OH）D levels of anti-CCP pisitive group were lower than negativegroup（t=-3.08,P<0.01）,and anti-CCP positive group’s serum 25-（OH）D levels were negatively correlated with DAS28 score（r=-0.464,P<0.01）.2.Real-time PCR results（1）In the experimental groups,the expression of the target genes in the groups b,c are higher than the group a,a statistically significant difference.（P<0.05）.（2）In the control groups,the expression of the target genes in the groups d are higher than the group e,a statistically significant difference.（P<0.05）.（3）The expression of the target genes in the groups b and c have a statistically significant difference.（P<0.05）.TRAP staining results（4）In the experimental groups,the number of group b are higher than group a,and have a statistically significant difference.（P<0.05）.（5）In the experimental groups,the number of group c are higher than group a,and have a statistically significant difference.（P<0.05）.（6）In the control groups,the number of group d are higher than group e,and have a statistically significant difference.（P<0.05）.Conclusion1.1,25（OH）₂D₃ is closely associated with RA,which paticipate in the occurrence and development of RA.2.1,25（OH）₂D₃ can inhibit the formation of osteoclasts by blocking the IL-22-mediated JAK-2/ STAT-3 signaling pathway.