The Effect Of Low-dose Glucocorticoid On Bone Metabolism In Rheumatoid Arthritis

Background:Rheumatoid arthritis(RA)is a chronic, progressive, systemic, autoimmune disease. Thepathogenesis is synovitis, cellular infiltration and pannus in early RA, involvement ofcartilage and bone,destruction of joint leading to deformity and functional impairment andosteoporosis(OP) with increasing disorder in mid-term and late. Bone metabolism disordersinvolve in many kinds of inflammatory cytokines leading to persistence synovitis, activitedosteoblast and increased resorption of bone in RA, which play a key role of more rapid boneloss and joint destruction at the onset of3months of disease. Within the past few years,studies have shown that periarticular bone loss as well as irreversible damage is an earlyfeature of the disease with an increased risk of generalized OP complication of60%-80%resulting in decreased quality of life and huge economic burden. Over the past decades,glucocorticoids (GC) are the most widely used anti-inflammatory and immunomodulatoryagents in RA owing to their strong anti-inflammatory and immunosuppressor effect.However, there have been controversial for inlovement of osteoporosis and increased risk offractures.The effect of low-dose glucocorticoid (LGC) in RA includes inhibiting inflammatoryresponse, decreasing disease activity and reducing the joint damage, which brings newexpect about efficacy and prognosis of RA. The benefits anti-inflammatory effect of LGCmay partially courteract their negative effect on bone.GC exert their effects mainly through the glucocorticoid receptor (GR). Recent studiesdemonstrate that GR is involved in pathogenesis of RA. MicroRNAs (miRNAs) are a kind ofendogenous, small, noncoding single-stranded RNA molecules that negatively regulate geneexpression at the post-transcriptional level. Recent studies have uncovered dysregulatedmiRNA expression in RA in the afflicted synovium,synovial fluid and the peripheralcirculation, suggesting that abnormalities in miRNA expression may contribute to themolecular mechanisms of the inflammation,bone loss and cartilage damage.Therefore, the aim of the present study is to evaluate the bone metabolism, GR mRNAexpression, miRNA expression and the activity of NK cell in early activity RA patientsreceiving LGC short-term therapy, which will explore the mechanisms of LGC and provide data for clinic.Objective:1. To investigate the effects of short-term LGC on bone metabolism in RA;2. To investigate the effects of short-term LGC on expression levels of hGRα and hGRβmRNA in RA;3. To investigate the effects of short-term LGC on expression levels of miRNA andtarget gene in RA;4. To investigate the influence of RA disease on bone metabolism;5. To investigate the mechanisms of LGC in RA and provide data for clinic.Methods:Postmenopausal female patients of early activity RA and healthy postmenopausalfemale controls sampled at medical examination center at the same time period were studied.Patients were randomly assigned to low-dose glucocorticoid therapy group (LGC group) ornon-glucocorticoid therapy group (NGC group). Biomedical markers including inflammationmarkers (IL-6, IL-1α, TNF-α, CRP and ESR), bone metabolism markers (bone formationmarkers: BGP, BAP and PICP; bone resorption markers: uDPD and CTX) and bone massdensity (BMD) were measured for both RA groups at baseline and3-month trail. Expressionlevels of hGRα and hGRβ mRNA in peripheral blood mononuclear cells was determinedwith real time fluorescent quantitative PCR for both RA groups at baseline and3-month trail.All the detections were compared with healthy controls at baseline. microRNA expressionlevels were detected with gene microarrays for LGC group at baseline and3-month trail.Cluster analysis using microRNA expression data was also performed. And, target genes ofdifferentially expressed microRNAs were predicted and analyzed with KEGG database.Results:1. The rate of low bone mass and osteoporosis were higher in postmenopausal femalepatients of early activity RA than in healthy controls (P＜0.05).2. In both RA groups, inflammation markers (IL-6, IL-1α and TNF-α) weresignificantly higher(P＜0.01), bone formation markers (BGP, BAP and PICP) weresignificantly lower (P＜0.05), and bone resorption markers (uDPD and CTX) weresignificantly higher (P＜0.05) compared with healthy controls at baseline.3. There was no significant difference observed for all measured indexes (P＞0.05)between LGC and NGC groups at baseline. At3-month trail, inflammation markers (IL-6, IL-1α, CRP and ESR) decreased dramatically in both NGC group (P＜0.05) and LGC group(P＜0.01); while IL-1α level was even lower in LGC group than in NGC group (P＜0.05).The levels of bone formation markers were higher, while the levels of bone resorptionmarkers were lower in LGC group than in NGC group, but no significant difference werefound (P＞0.05). At3-month trail, BMD increased slightly in both group, but no significantdifference were found (P＞0.05), either.4. At baseline, both levers of hGRα and hGRβ mRNA in two RA groups weresignificantly higher than that in healthy controls (P＜0.01).5. At3-month trail, the levers of hGRα mRNA in LGC group were significantlydecreased (P＜0.01), however, it was still higher than that in healthy controls (P＜0.05).While the levers of hGRα mRNA in NGC group were not obviously decreased comparedwith baseline (P＞0.05), and there was significant difference between LGC and NGC groups(P＜0.05).6. At3-month trail, there was no significant difference was found about the levers ofhGRβ mRNA in both LGC and NGC groups compared with baseline (P＞0.05).7. Thirteen differentially expressed microRNAs were identified according to foldchange and diffscore. They are hsa-miR-34a, hsa-miR-134, hsa-miR-200b, hsa-miR-106b*,hsa-miR-1281, hsa-miR-188-5p, hsa-miR-202*, hsa-miR-219-1-3p, hsa-miR-455-5p, hsa-miR-513b, hsa-miR-521, hsa-miR-548f and hsa-miR-645. After LGC treatment, all theabove listed microRNAs were up-regulated.8. Verification experiments replicated the result that hsa-miR-34a expression level wasup-regulated after3-month LGC therapy(P＜0.05), and further proved that two target genesof hsa-miR-34a (ULBP2and NKG2D) were down-regulated(P＜0.05).9. KEGG pathway analysis of target genes of the thirteen differentially expressedmicroRNAs revealed4most relevant pathways (adj P＜0.00001), they are focal adhesionkinase signaling pathway, mTOR signaling pathway, chemokine signaling pathway andapoptosis signaling pathway.Conclusions:1. The rate of low bone mass and osteoporosis were higher in postmenopausal femalepatients of early activity RA than in healthy controls.2. LGC short-term therapy can increase bone formation, inhibit bone resorption and reduce bone loss through alleviating inflammation respones in early RA patients.3. There is no negative effect of LGC short-term therapy on bone metabolism and thereis no increase on the rate of osteoporosis in RA patients.4. Increased expression of hGRα and hGRβ mRNAs may involved in the onset of RA.LGC can decrease hGRα mRNA expression in peripheral blood mononuclear cells in RApatients.5. LGC can up-regulate the expression of thirteen microRNAs (hsa-miR-34a,hsa-miR-134, hsa-miR-200b, hsa-miR-106b*, hsa-miR-1281, hsa-miR-188-5p,hsa-miR-202*, hsa-miR-219-1-3p, hsa-miR-455-5p, hsa-miR-513b, hsa-miR-521,hsa-miR-548f,hsa-miR-645), and the expression of hsa-miR-34a in peripheral blood andknee synovial fluid were up-regulated most in the all.6. LGC may down-regulate ULBP2expression, change NKG2D activity and reduceNK cell activity through up-regulation of hsa-miR-34a.7. LGC can up-regulate certain microRNAs in peripheral blood and inhibit signalingpathways which their target genes involved.8. LGC can decrease NK cell activity, reduce bone loss and prevent erosion of joint inRA.