Experimental Study Of Subacromial Bursitis In Rotator Cuff Disease Treated With Inhibition Of Il-1β And Tnf-α By SiRNA

Rotator cuff disease (RCD) is the most common cause of shoulder pain anddysfunction. Pathophysiology and biochemical mechanism of RCD have been identified.With the development of cell biology and molecular biology,we know Subacromial bursitiswas regarded as the major souce of pain and dysfunction in rotator cuff disease. By thenon-specific stimuli such as mechanical stretch, hypoxia, inflammatory cytokines, or otherinflammatory cells nearby, bursal cells can be activated to secre IL-1、TNF-α，MMP-1、MMP-9、COX-1、COX-2、SDF-1、substance P and VEGF. These inflammatory cytokinesform a long-term chronic inflammatory network of subacromial bursitis. Unfortunately,despite multiple investigations have supported the role of these cytokines in subacromialbursitis, these cytokines received relatively little emphasis in the treatment of subacromialbursitis.RNA interference (RNAi) exerts its silencing effect by mediating sequence-specificmRNA degradation. In this process, double-stranded RNA (dsRNA) molecules are cleavedby a ribonuclease (Dicer) into21–23bp fragments called “short interfering RNA”(siRNA).The siRNA in turn binds to a protein complex called “RNA-induced silencing complex(RISC),” and targets it to mRNAs that have complementary sequence with the siRNA.Then, the targeted mRNAs are destroyed through cleavage by RISC. siRNA has recentlybecome an important tool for suppressing the expression of specific genes. To date, therehas been no report of applying siRNA to reduce inflammatory cytokines in the subacromialbursa in patients with rotator cuff disease. In the present study, siRNAs targeting IL-1β andTNF-α were delivered into the bursal cells dually to assess the effects of siRNA therapy ofsubacromial bursitis.ObjectiveIn this study,we expect to clear phenotype and pathological feature of subacromialbursa cell, explore the feasibility of silence IL-1β and TNF-α of subacromial bursa cell byusing RNAi, and value the effects of knock down of IL-1β,TNF-α and other inflammatoryfactors in the inflammatory network.. further more, we whish to confirm the pathogenesisof subacromial bursitis, and role of IL-1β and TNF-α in the inflammatory network. At last,we can provides an important theoretical basis for the clinical treatment, gene therapy anddrug screening of rotator cuff disease. Methods1. Subacromial bursa specimens were derived from patients with rotator cuff disease.Modified double enzyme digestion method was used to isolate cells from synovial tissuesof Subacromial bursa specimens. Cells were cultured and amplifed in vitro in flat plates.HE staining, immunofluorescence of Vimentin and CD68to identify cell phenotype.2. IL-1β specific siRNA(1-3) and TNF-α specific siRNA(a-b) were designed andsynthesized. The subacromial bursa synoviocytes were transfected by using cationic lipidvetors. Determined transfection efficiency by the FAM fluorescent labeling. Cellmorphology after transfection was observed by invert microscope. Screened effectivesiRNA sequences by Western Blot.The level of genes expression were valued usingRealtime-PCR.3. Subacromial bursa synoviocytes were treated with two siRNAs dually and theexpression levels of related genes were examined by Realtime-PCR.Results1. Prime subacromial bursa synoviocytes were adherent well in culture,and themorphology of cells were spindle-shaped after3passages. With H-E staining, cells werespindle, nuclei were round and large, nucleoli were prominent. Immunofluorescencerevealed cells cultured after3passages surface antigens Vimentin positive expression, butCD68negative. So it is indicated that subacromial bursa synoviocytes cultured after3passages are pure fibroblast-like synoviocytes.2. The experiment showed that the transfection efficiency was more than80%. Itwas observe morphology cells and growth after transfection had no significant effect. Theresults of Western Blot showed IL-1β protein level was inhibited most significantly bysiRNA-2and TNF-α protein level was inhibited by siRNA-c most significantly.siRNA-2and siRNA-3inhibit the expression of IL-1β protein effectly Compared with the controlgroup. The inhibition of the IL-1β and TNF-α had similar dose-dependent andtime-dependent manner. The effects of inhibition increased with the increase of siRNAfinal concentration. A significant decrease in mRNA levels of gene were observed after24hours, which rebounded after48hours.3. In current study, siRNA-2and siRNA-c were finally chosen to transfectfibroblast-like synoviocytes at the concentration of200nM dually.24hours Aftertransfection, The results of realtime PCR confirmed that the expressions of the IL-1β and TNF-α genes were inhibited significantly compared with controls. Additionally, themRNA levels of MMP-1, MMP-9, COX-1and COX-2decreased significantly after thedown-regulation of IL-1β and TNF-α. We also found the expression of SDF-1genedecreased after transfection.Conclusion1. Subacromial bursa synoviocytes were abundant after modified double enzymedigested cultivation.2. It was identified that subacromial bursa synoviocytes cultured after3passages arepure fibroblast-like synoviocytes.3. Specific siRNA can effectively inhibit the the expression of the IL-1β and TNF-αgenes of fibroblast-like synoviocytes transfected by using cationic lipid vetors.4. The inhibition of both IL-1β and TNF-α by siRNA had dose-and time-dependentmanners.5. The levels of MMP-1,MMP-9,COX-1,COX-2and SDF-1denes down-regulatedafter the dual knockdown of IL-1β and TNF-α genes of subacromial bursa fibroblast-likesynoviocytes.It is clear that RNAi has an interrupting effect to inflammatory network insubacromial bursitis.6. It proved that there is a positive feedback relationship between IL-1β, TNF-α andother factors in inflammatory network of subacromial bursitis.7. It could be a new promising therapeutic approach to heal subacromial bursitis andeven rotator cuff disease in the future by knockdown of target genes in inflammatorynetwork by siRNA.