The Expression Of DUSP1 In Human Normal And Osteoarthritis Fibroblast-like Synoviocytes And Its Protective Role In Osteoarthritis

Osteoarthritis(OA)is the most common form of joint diseases in the world and constitutes a major cause of disability in the aging population.The disease is previously considered as a typical non-inflammatory arthropathy but today it is generally accepted that it is an inflammatory disease.Recent studies have shown that inflammation contributes to the symptoms and the progression of OA.These studies suggest that disease-modifying interventions targeting inflammatory process might be effective for the prevention and treatment of OA.Dual specificity phosphatase1(DUSP1),is also known as mitogen-activated protein kinase phosphatase1(MKP1)that inhibits activity of the MAPKs through dephosphorylating tyrosine and threonine residues in MAPK.Some studies have shown that DUSP1 is an important negative regulator of inflammatory responses,and the induction of DUSP1 gene expression is potentially a novel anti-inflammatory approach.In our study,we investigated the expression of DUSP1 in cultured human normal fibroblast-like synoviocytes and OA FLS and the effect of DUSP1 on the expression of OA related mediators such as MMP-13 and COX2,which occurs through a mechanism involving the inhibition of the p38 MAPK/JNK signaling pathway.We also used dexamethasone to induce the expression of DUSP1 in OA FLS,which could partly demonstrate the anti-inflammatory mechanism of glucocorticoids in OA.The results demonstrated the anti-inflammatory and anti-catabolic actions of DUSP1 on OA FLS,and DUSP1 was a potential target of treatment in OA.Objective1.To culture and identify human normal and osteoarthritis fibroblast-like synoviocytes in vitro.2.To investigate the expression levels of DUSP1 in human normal and osteoarthritis fibroblast-like synoviocytes.3.To verify that dexamethasone could induce the expression of DUSP1 in human osteoarthritis fibroblast-like synoviocytes,and also could inhibit the activiation of p38 MAPK,JNK signaling pathway and the expression of osteoarthritis-related mediators such as MMP-13,COX-2.4.To constitute the lentivirus vector for the overexpression of DUSP1.5.To illuminate that DUSP1 plays a protective role by inhibition of p38 MAPK and JNK signaling pathway.Method1.Human normal and osteoarthritis fibroblast-like synoviocytes were cultured in vitro by enzymatic digestion.Flow cytometry was used to detect the expression of CD55 in FLSs and cell purity.CCK-8 essay was used to compare the proliferation abilities of the normal and osteoarthritis FLSs.2.Fluorogenic quantitative PCR,Western blot and immunofluorescence staining were used to detect the expression of DUSP1 in OA FLSs,normal FLSs and OA FLSs pretreated with dexamethasone.3.Western blot and Fluorogenic quantitative PCR were used to detect the activiation of p38 MAPK and JNK signaling pathway and the expression of osteoarthritis-related mediators such as MMP-13 and COX-2 in above three groups of cells.4.We used a lentivirus vector(GV358)combined with DUSP1 gene to produce LV-DUSP1 by molecular biological technique,and PCR and Western blot were used to identify the lentivirus vector.5.LV-DUSP1 was used to infect the human osteoarthritis FLSs,Then we investigated the infection conditions and observed the infection efficiency.Western blot analysis confirmed DUSP1 overexpression.6.Western blot was used to detect the the changes of the activiation of p38 MAPK and JNK signaling pathway and the expression of osteoarthritis-related mediators.CCK-8 essay was used to detect the changes of the cell proliferation abilities.Results1.The results of flow cytometry showed that the ratio of CD55 positive in cultured cells was 98.3% which accorded with the characteristic of FLSs.2.CCK-8 essay showed that the proliferation rate of osteoarthritis FLSs was significantly higher than normal FLSs.3.The results of fluorogenic quantitative PCR,Western blot and immunofluorescence staining suggested that the expression of DUSP1 was markedly inhibited in human osteoarthritis FLSs and dexamethasone could induce the expression of DUSP1 in human osteoarthritis FLSs.4.Western blot and fluorogenic quantitative PCR showed that dexamethasone could inhibit the activiation of p38 MAPK and JNK signaling pathway and the expression of MMP-13 and COX-2.5.The results of PCR and western blot showed that we successfully constitute the lentivirus vector combined with DUSP1 gene.6.LV-DUSP1 could infect the human FLSs through the observation of fluorescence microscope.7.The results of CCK-8 essay showed that the overexpression of DUSP1 could inhibit the proliferation of human osteoarthritis FLSs.8.Western blot analysis suggested that the overexpression of DUSP1 could inhibit the activiation of p38 and JNK signaling pathway and the expression of MMP-13 and COX-2.Conclusions1.The expression level of DUSP1 in osteoarthritis FLSs was significantly lower than in normal FLSs.2.Dexamethasone could induce the expression of DUSP1 in human osteoarthritis FLSs,inhibit the activiation of p38 MAPK and JNK signaling pathway and the expression of osteoarthritis-related mediators such as MMP-13,COX-2,which suggested that the anti-inflammatory and anti-catabolic role of dexamethasone might partly depend on induction of DUSP1.3.We demonstrated that DUSP1 played a protective role in osteoarthritis by inhibition of p38 MAPK and JNK signaling pathway.4.We demonstrated that the overexpression of DUSP1 could inhibit the proliferation ability of human osteoarthritis FLSs,which showed that DUSP1 might be effective in inhibiting the synovial hyperplasia in osteoarthritis.