Kartogenin Promotes Chondrogenic Differentiation Of Mouse Bone Marrow Mesenchymal Stem Cells By Regulating SOX9

Objective: To investigate the role of small molecule Kartogenin in promoting the chondrogenic differentiation of mouse bone marrow mesenchymal stem cells(BMSCs)through SOX9,and the mechanism of inhibiting cartilage dedifferentiation through SOX9 at later stages.Methods: BMSCs were extracted by whole bone marrow method.The cell phenotypes were identified by flow cytometry.CCK-8 and alixin blue were used to determine the effects of different concentrations of KGN on chondrogenic differentiation and proliferation of BMSCs.Three groups of blank control group(DMSO group),positive control group(TGF-?3 group)and KGN group were used to interfere with chondrogenic differentiation of BMSCs.Quantitative real-time quantitative PCR was used to determine the expression of SOX9 and downstream chondrogenic and cartilage hypertrophy-related genes in each group.Western-blot and immunofluorescence were used to determine the expression of SOX9 and downstream cartilage and cartilage hypertrophy-related proteins.Results: 1.Extraction of cell surface molecules was identified as CD34-,CD45-,CD44+,CD105+ consistent with the surface features of mesenchymal stem cells.2.KGN had no obvious effect on the activity of BMSCs in 0-10?M range.3.KGN-induced chondrogenesis of MSCs is concentration-dependent.The higher the KGN intensity is,the stronger the chondrogenesis is,and 10 ?M is the optimal chondrocyte-inducing concentration.4.The PCR results showed that: 3 days after chondrogenesis induction: SOX9 and downstream Col-2,Agg gene expression in TGF-?3 group,KGN group were significantly higher than the blank control group(P<0.05),TGF-?3 group The RUNX2 gene expression in KGN group was significantly lower than that in DMSO group,and the difference was statistically significant(P<0.05).At 28 days after chondrogenesis,the expression of SOX9 and Col-2 in KGN group was higher than that in DMSO and TGF-?3 groups.The expression of RUNX2 and COL-X genes in TGF-?3 group was higher than that in other two groups.The expression of RUNX2 in KGN group was lower than that in DMSO group(P<0.05).5.Western-blot results showed that after 28 days of culture,cartilage-marked Col-2 protein appeared in TGF-?3 group and KGN group,but SOX9 protein expression in KGN group was higher than that in TGF-?3 group and DMSO group.The expression of RUNX2 protein in KGN group was lower than that in DMSO group and TGF-?3 group.The expression of Col-X protein was highest in TGF-?3 group.The expression of Col-X protein in KGN group was lower than that in TGF-?3 group and DMSO group.6.Immunofluorescence results showed that after 28 days of chondrogenic culture,KGN group had higher SOX9 expression than TGF-?3 group and DMSO group,and KGN group had the lowest RUNX2 protein expression.Conclusion: 1.KGN promotes chondrogenesis of MSCs in a concentration-dependent manner,with 10 ?M being the optimal chondrocyte-inducing concentration.2.KGN promotes chondrogenic differentiation of BMSCs by up-regulating SOX9 expression and activating downstream Col-2,Agg,etc.3.The effect of KGN on inhibiting the late differentiation of cartilage may be through the up-regulation of SOX9 and the inhibition of downstream Col-X and RUNX2.4.Compared with the existing chondrogenic factor TGF-?3,KGN culture does not easily lead to dedifferentiation of cartilage in the late cartilage.