Regulation Of MascRNA On Osteoclast Generation And Its Therapeutic Effect On Animal Model Of Rheumatoid

Objective:Rheumatoid arthritis(RA)is a common autoimmune disease,characterized by synovial inflammation caused by the proliferation and infiltration of macrophages,lymphocytes and synovial cells and involving bone and cartilage,leading to joint dysfunction and even disability.There has been no specific treatment for RA.Since 1998,the birth of biological agents represented by tumor necrosis factor α(TNFα)blockers has made a breakthrough in the treatment of RA.However,such biological agents are only effective for some patients and may lead to infection and tumor due to immunosuppression,and cannot correct bone erosion,which is the most serious lesion of RA.Therefore,it is an important topic for drug research and development of RA to find drugs with high safety that can fully relieve or even reverse RA articular inflammation,especially bone erosion.mascRNA is a small molecular non-coding RNA with t RNA-like structure,which is the processing product of the 3 ’end of the primary transcript of long non-coding RNA MALAT1.Previous studies in our laboratory have shown that mascRNA can inhibit the two classical inflammatory pathways of NF-κB and MAPK.This project will further investigate whether mascRNA has a regulatory effect on osteoclast generation and differentiation,and evaluate the anti-inflammatory and anti-bone erosion efficacy of mascRNA in RA animal model.The results of this study are expected to provide new ideas for drug development and clinical treatment of rheumatoid arthritis.Methods:1.mascRNA-overexpressing plasmid was transfected into RAW264.7 cells by jet PEI-macrophage.RAW264.7 cell line with stable overexpression of mascRNA was obtained by G418 screening.In the presence of apoptosis inducers T5(TNFα+5Z-7-oxozeaenol),the important key proteins in the apoptosis pathway were detected by Western Blot.The effect of mascRNA on macrophage apoptosis was analyzed.2.Western blot analysis was conducted to determine whether the regulation of mascRNA on apoptosis depended on RIPK1 kinase activity and whether mascRNA affected IKK and p38 kinase activities to regulate RIPK1 kinase activity in RAW264.7 cells treated with T5.3.In RAW264.7 cells stimulated by RANKL,the effects of mascRNA on TRAF6 protein and proteins related to NF-κB and MAPK signaling pathways were analyzed by Western blot,and the expressions of osteoclast differentiation related genes c-fos and NFATc1 was detected by RT-PCR and q PCR.The effect of mascRNA on osteoclast differentiation was analyzed.4.CIA model of DBA/1 mice was established,polyethylenimine-loaded mascRNA overexpression plasmid was injected into tail vein,and 5Z-7-oxozeaenol was intraperitoneally injected.Joint index score,X-ray imaging analysis,pathological analysis and cytokine expression detection were used to evaluate the therapeutic effect of mascRNA and mascRNA combined with5Z-7-oxozeaenol on RA mice.Results:1.Compared with wild-type RAW264.7 cells,RAW264.7 cells with overexpression of mascRNA showed more obvious caspase-3 and PARP-1 cleavage under the action of T5,and could be inhibited by Nec-1s.2.Phosphorylation of RIPK1 S166 and p38 MAPK decreased in overexpressed mascRNA cells.3.Compared with wild-type RAW264.7 cells,the expression of TRAF6 and phosphorylated IκB in RAW264.7 cells overexpressed with mascRNA was decreased under RANKL stimulation,and the m RNA levels of c-Fos and NFATc1,which are related to osteoclast differentiation,were decreased.4.X-Ray results showed that,compared with the control group,the degree of joint inflammation and joint destruction was significantly reduced in mice treated with mascRNA and mascRNA+5Z-7-oxozeaenol.HE staining results showed that,compared with the control group,the damage of ankle articular surface and the degree of articular cavity stenosis were reduced in the treatment group,and synovial hyperplasia was not obvious.TRAP staining results showed that the number of osteoclasts in the treatment group was significantly reduced compared with the control group. Conclusions:1.mascRNA may promote RIPK1 kinase activity by inhibiting p38 MAPK,thereby enhancing the sensitivity of RAW264.7 cells to T5-induced apoptosis.2.mascRNA inhibits the differentiation of RAW264.7 cells into osteoclasts by inhibiting TRAF6/NF-κB signaling pathway.3.Animal model proves that mascRNA has both anti-inflammatory and anti-bone erosion therapeutic effects on rheumatoid arthritis.