Fig 1: AST-IV stimulates osteogenic differentiation of BMSCs via GSK3ß/ß-catenin signalling. (A) Western blotting was used to determine the expression levels of p-S9-GSK3ß, total GSK3ß and ß-catenin in untreated BMSCs and BMSCs treated with AST-IV (0, 10, 20 and 40 µM) and (B) linear association was determined. (C) Western blotting was used to detect and (D) quantify the total GSK3ß and ß-catenin protein changes in BMSCs overexpressing GSK3ß or treated with AST-IV (40 µM). ß-actin was used as an internal control. (E) ALP activity of GSK3ß-overexpressing BMSCs following treatment with AST-IV on day 7. (F) Alizarin red S staining of mineralization conditions in GSK3ß-overexpressing BMSCs with AST-IV incubation on day 21 are (G) expressed as a ratio of the Control (magnification, ×200). Scale bar, 100 µm. (H) Expression levels of Runx2, OCN, OPN and OSX mRNA following GSK3ß overexpression in BMSCs. (I) Runx2, OCN, OPN and OSX protein expression levels were detected via western blotting, with ß-actin acting as the endogenous control and (J) quantified. n=3; *P<0.05, **P<0.01 and ***P<0.001. AST-IV, astragaloside-IV; BMSCs, bone marrow-derived mesenchymal stem cells; GSK3ß, glycogen synthase kinase 3ß; ALP, alkaline phosphatase.
Fig 2: Runx2, OCN, OPN and OSX expression levels in BMSCs following AST-IV incubation. Expression levels of (A) Runx2, (B) OCN, (C) OPN and (D) OSX mRNA in AST-IV-incubated BMSCs (day 7) were detected via quantitative PCR. (E) Runx2, OCN, OPN and OSX protein expression levels were detected by western blotting, with ß-actin acting as the endogenous control and (F) linear associations were determined. n=3. BMSCs, bone marrow-derived mesenchymal stem cells; AST-IV, astragaloside-IV.
Fig 3: CGFe increases RUNX and OSX protein expression. (A) Western blot analysis of RUNX2 and OSX following treatment with various culture conditions for 14 days. GAPDH was used to monitor equal protein sample loading. (B) Quantitative analysis of RUNX2 and OSX protein expression. RUNX2 was upregulated in the CGFe group and downregulated in the CGFe+TNF-a and TNF-a groups. The protein level of OSX was upregulated in the CGFe+TNF-a group, but not significantly upregulated compared with the control group. **P<0.01; NS, CGFe+TNF-a group vs. control group; ##P<0.01, CGFe+TNF-a group vs. control group. CGFe, concentrated growth factor exudate; TNF-a, tumor necrosis factor-a; RUNX2, runt-related transcription factor 2; OCN, osteocalcin; OSX, Osterix.
Fig 4: AST-IV-mediated osteogenic differentiation is dependent on NGF expression levels. (A) Expression levels of NGF mRNA in BMSCs following incubation with 0, 10, 20 or 40 µM AST-IV. (B) NGF protein expression levels and (C) linear association between BMSCs incubated with 0, 10, 20 or 40 µM AST-IV. (D) Determination and (E) quantification of NGF protein expression levels in BMSCs following treatment with AST-IV and anti-NGF antibody or anti-NGF-H0 (negative control). (F) Western blotting and (G) relative expression levels of p-S9-GSK3ß and total GSK3ß in BMSCs following treatment with AST-IV, anti-NGF antibody and anti-NGF-H0. ß-catenin was used as a control. (H) Western blotting and (I) relative protein expression levels of p-S9-GSK3ß and total GSK3ß in GI-treated BMSCs. ß-catenin was used as the control. (J) Runx2, OCN, OPN and OSX mRNA levels in AST-IV-cultured BMSCs following GI and anti-NGF treatment. Runx2, OCN, OPN and OSX protein levels were (K) detected and (L) quantified in AST-IV-cultured BMSCs following GI and anti-NGF treatment. n=3; *P<0.05, **P<0.01 and ***P<0.001. AST-IV, astragaloside-IV; NGF, neuronal growth factor; BMSCs, bone marrow-derived mesenchymal stem cells; GSK3ß, glycogen synthase kinase 3ß; GI, GSK3ß inhibitor.
Fig 5: CGFe increases the expression of osteogenic-associated genes. Following osteogenic induction for 7 or 14 days, the expression of (A) RUNX2, (B) OSX and (C) OCN was determined by reverse transcription-quantitative polymerase chain reaction. The expression of RUNX2 and OSX in the TNF-a groups was lower than that of the control group. On days 4, 7 and 14, the expression of RUNX2 and OSX in the CGFe groups was higher than the control group. OCN gene expression was not significantly different between the experimental groups on day 4. By day 7, OCN expression in the CGFe group began to surpass the control group. After 14 days of culture, OCN expression in the TNF-a was lower than that of the control group. **P<0.01. CGFe, concentrated growth factor exudate; TNF-a, tumor necrosis factor-a; RUNX2, runt-related transcription factor 2; OCN, osteocalcin; OSX, Osterix.
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