The Coordinated Function And Mechanism Study Of SIRT1 In BMP2-induced Chondrogenic Differentiation And Cartilage Homeostasis Maintaining

Objective:To investigate the effects of silent information regulator 2related enzyme 1?SIRT1?on bone morphogenetic protein 2?BMP2?inducing chondrogenic differentiation of stem cells and cartilage maintenance under oxidative stress.Methods:The adenovirus Ad-SIRT1 and Ad-BMP2 were constructed and the expression of SIRT1 and BMP2 was confirmed by Western blot and Real-time PCR.To explore whether SIRT1 can promote chondrogenic differentiation of stem cells induced by BMP2,it was divided into Ad-GFP group,Ad-SIRT1 group,Ad-BMP2 group and Ad-BMP2+Ad-SIRT1 group.Western blot was used to detect the expression of Sox9,Col2A1,Aggrecan,Real-time PCR was used to detect the expression of Sox9 and Col2A1,and Alcian blue staining was used to detect the extracellular matrix of cartilage in each group at different times.In addition,the subcutaneous transplantation of stem cells was also carried out in nude mice,which were divided into Ad-GFP group,Ad-SIRT1 group,Ad-BMP2 group and Ad-BMP2+Ad-SIRT1 group.The subcutaneous bone tissues obtained at different time points were stained with H&E,Alcian blue and Masson staining to observe the cartilage maturity of each group.The effects of SIRT1 on chondrogenic differentiation and cartilage homeostasis under oxidative stress were further studied.The experiment was divided into three groups:Ad-BMP2,Ad-BMP2+H₂O₂ and Ad-BMP2+Ad-SIRT1+H₂O₂.Flow cytometry and Western blot were used to detect the apoptosis of cells in each treatment group under the stimulation of H₂O₂.Western blot was used to detect the expression of Sox9,Col2A1,Aggrecan cartilage differentiation markers in each treatment group.Meanwhile,the expression of MMP2,NF-?B and its acetylation level were detected.The expression of Col2A1 and Aggrecan in each group was detected by immunofluorescence.Results:Western blot and Real-time PCR showed that the group of Ad-SIRT1 and Ad-BMP2 were highly expressed at mRNA and protein levels.Western blot and Real-time PCR showed that both Ad-BMP2 and Ad-BMP2+Ad-SIRT1 could promote the expression of Sox9,Col2A1 and Aggrecan,and the expression of Sox9,Col2A1 and Aggrecan in Ad-BMP2+Ad-sirt1 group was significantly higher than that in Ad-BMP2 group,Alcian blue staining also showed that the staining of Ad-BMP2+Ad-SIRT1group was deeper than that of Ad-BMP2 group,suggesting that Ad-BMP2+Ad-SIRT1 group secreted more cartilage extracellular matrix.Furthermore,the subcutaneous transplantation of stem cells in nude mice showed that the cartilage of Ad-BMP2+Ad-SIRT1 group was more mature than that of Ad-BMP2 group.Western blot and flow cytometry showed that the apoptosis of Ad-BMP2+Ad-SIRT1+H₂O₂ group was significantly lower than that of Ad-BMP2+H2O2 group,which confirmed that SIRT1 could inhibit oxidative stress-induced apoptosis.Western blot and Real-time PCR showed that the chondrogenic differentiation markers Sox9,Col2A1 and Aggrecan could be induced in both Ad-BMP2 and Ad-BMP2+Ad-SIRT1groups,but the expression of chondrogenic differentiation markers was more obvious in Ad-BMP2+Ad-SIRT1 group.The results of Alcian blue staining showed that cells in the Ad-BMP2+Ad-SIRT1 group secreted more extracellular matrix than those in the Ad-BMP2 group.Subcutaneous transplantation experiments were performed in nude mice also showed that SIRT1 can promote BMP2 to induce stem cell chondrogenic differentiation.Cellular immunofluorescence and Western blot results showed taht the expression of Sox9,Col2A1 and Aggrecan in the Ad-BMP2+Ad-SIRT1+H₂O₂ group was significantly higher than that in the Ad-BMP2+H₂O₂ group,while the expression of MMP2 and Ac-p65 were significantly lower.It was confirmed that SIRT1 can significantly inhibit the extracellular matrix decomposition of cartilage and promote the maintenance of cartilage homeostasis in the process of chondrogenic differentiation induced by BMP2.Conclusion:SIRT1 can promote the chondrogenic differentiation of BMP2 induced stem cells,and SIRT1 can promote the maintenance of cartilage homeostasis during the chondrogenic differentiation of BMP2induced stem cells.