A Preliminary Study Of Myostatin Role In The Regulation Of Articular Chondrocytes

Objective: We aim to detect the expression of Myostatin and its receptor Act RIIB in osteoarthritis chondrocytes,and to explore the function of Myostatin in osteoarthritis.Methods: Human articular chondrocytes were isolated from articular cartilage specimens of patients with fracture and with knee osteoarthritis,which were identified by morphological observation,toluidine blue staining and COL2A1 immunofluorescence staining.The expression of Myostatin and its receptor Act RIIB in normal articular chondrocytes and osteoarthritis chondrocytes was detected by q PCR.And cell counting,crystal violet staining and CCK-8 kit were used to detect the effect of Myostatin at different concentration(0,5,10,100 and 500 ng/m L)on chondrocyte proliferation at different time(24h,48 h and 72h).Then the effects of 100 ng/m L Myostatin on the expression of inflammatory factors,matrix metalloproteinase and COL2A1 in chondrocytes were detected by q PCR,Western blot and immunofluorescence staining.At last,the transcriptome of chondrocytes treated with Myostatin was sequenced by high-throughput RNA sequencing technology,and the differentially expressed genes induced by Myostatin were screened.The potential mechanism of Myostatin regulating chondrocyte function was revealed by GO clustering and KEGG signaling pathway analysis.Results:(1)Human articular chondrocytes were identified by morphological observation,toluidine blue staining and COL2A1 immunofluorescence staining.(2)The expression of Myostatin and its receptor Act RIIB were significantly increased in osteoarthritis chondrocytes,suggesting that Myostatin may play an important role in the pathological process of osteoarthritis.(3)100 ng/m L and 500 ng/m L Myostatin significantly inhibited the activity of chondrocytes after 48 hours of Myostatin treatment;after 72 hours of Myostatin treatment,low concentration of Myostatin significantly inhibited the activity of chondrocytes,and the degree of inhibition increased with the increase of Myostatin concentration.(4)Myostatin could promote the expression of COX-2,i NOS,IL-6,promote the expression of MMP3 and MMP13,and inhibit the expression of COL2A1 protein.(5)There were 483 differentially expressed genes in chondrocytes treated with Myostatin,among which 152 genes were up-regulated and 331 genes were down regulated,which indicated that Myostatin could effectively regulate the gene expression of chondrocytes;GO cluster analysis showed that the differentially expressed genes regulated by Myostatin mainly regulated mitotic cell cycle,chromosome segregation,microtubule binding and ATP binding.The results of KEGG signaling pathway showed that the differentially expressed genes regulated by Myostatin were mainly enriched in cell cycle,DNA replication,p53 signaling pathway,FOXO signaling pathway and cell senescence.Among them,28 differentially expressed genes were enriched in cell cycle pathway.After treatment of Myostatin in chondrocytes,27 cell cycle related proteins were down regulated,suggesting that Myostatin inhibits chondrocyte activity by downregulating the expression of cell cycle related proteins.Conclusions:(1)The expression of Myostatin and its receptor Act RIIB were significantly increased in osteoarthritis chondrocytes.(2)Myostatin inhibits the activity of chondrocytes,promotes the expression of inflammatory factors and the degradation of matrix.(3)The differentially expressed genes regulated by Myostatin were mainly enriched in cell cycle,DNA replication,p53 signaling pathway,FOXO signaling pathway and cell senescence.