Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Cultivation and identification of PDLSCs. ( A ) Morphology of PDLSCs. Scale Bar: 200 μm. ( B ) Lipid droplets of PDLSCs after adipogenic induction. Scale bar: 200 μm. ( C ) Mineralized nodules of PDLSCs after osteogenic induction. Scale bar: 500 μm. ( D ) Flow cytometric analyses of PDLSCs. Mesenchymal stem cell markers (CD44, CD105) were positive and hematopoietic stem cell markers (CD34, CD45) were negative in PDLSCs.

Article Snippet: Cells were seeded in 6-well plates at a density of 1×10 5 cells per well and incubated with osteogenic induction medium (MEM with 10% FBS, 50μg/mL ascorbic acid, 0.01μM dexamethasone, and 10mM β-glycerophosphate, Solarbio, Beijing, China) or adipogenic induction medium (MEM with 10% FBS, 500mM isobutyl-methylxanthine, 10 mM insulin, 60mM indomethacin, and 0.5 M hydrocortisone, Solarbio, Beijing, China).

Techniques:

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of AS-IV on proliferation and osteogenic differentiation of PDLSCs. ( A ) CCK-8 analysis of AS-IV effects on PDLSCs on day 1, 3, and 5. ( B and C ) ALP activity ( B ) and extracellular matrix mineralization ( C ) quantification of PDLSCs affected by AS-IV. ( D ) ALP staining. Scale bar: 500 μm. ( E ) Alizarin Red staining. Scale bar: 500 μm. ( F – H ) qRT-PCR analysis of COL-1 (F), ALP(G), and RUNX-2 ( H ) in PDLSCs cultured with AS-IV. ( I – L ) Western blot and semi-quantitative analysis of COL-1 (J), ALP(K), and RUNX-2 ( L ) in PDLSCs cultured with AS-IV. 20 μM AS-IV group had a significant promoting effect. Data are presented as mean ± SD. ** P <0.01, *** P <0.001, **** P <0.0001. Each experiment was repeated five times.

Article Snippet: Cells were seeded in 6-well plates at a density of 1×10 5 cells per well and incubated with osteogenic induction medium (MEM with 10% FBS, 50μg/mL ascorbic acid, 0.01μM dexamethasone, and 10mM β-glycerophosphate, Solarbio, Beijing, China) or adipogenic induction medium (MEM with 10% FBS, 500mM isobutyl-methylxanthine, 10 mM insulin, 60mM indomethacin, and 0.5 M hydrocortisone, Solarbio, Beijing, China).

Techniques: CCK-8 Assay, Activity Assay, Staining, Quantitative RT-PCR, Cell Culture, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of LY294002 on osteogenic differentiation and pathway-associated factor of PDLSCs following AS-IV incubation. ( A and D ) ALP staining( A ) and ALP activity quantification( D ) of PDLSCs affected by LY294002. Scale bar: 500 μm. ( B and E ) Immunofluorescence staining( B ) of COL-1 (green) and DAPI staining (blue) and quantification(E). Scale bar: 50 μm. ( C and F ) Immunofluorescence staining( C ) of eNOS (green) and DAPI staining (blue) and quantification(F). Scale bar: 50 μm. ( G ) NO2- concentration represented the level of NO in cell supernatants. ( H–K ) Western blot and semi-quantitative analysis of COL-1(I), ALP(J), and RUNX-2 ( K ) in PDLSCs cultured with AS-IV and LY294002. ( L–O ) Western blot and semi-quantitative analysis of eNOS(M), iNOS( N ) and p-AKT/AKT ( O ) after addition of AS-IV at different time points. ( P–S ) Western blot and semi-quantitative analysis of eNOS(Q), iNOS( R ) and p-AKT/AKT ( S ) in PDLSCs cultured with AS-IV and LY294002. Data are presented as mean ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. Each experiment was repeated five times.

Article Snippet: Cells were seeded in 6-well plates at a density of 1×10 5 cells per well and incubated with osteogenic induction medium (MEM with 10% FBS, 50μg/mL ascorbic acid, 0.01μM dexamethasone, and 10mM β-glycerophosphate, Solarbio, Beijing, China) or adipogenic induction medium (MEM with 10% FBS, 500mM isobutyl-methylxanthine, 10 mM insulin, 60mM indomethacin, and 0.5 M hydrocortisone, Solarbio, Beijing, China).

Techniques: Incubation, Staining, Activity Assay, Immunofluorescence, Concentration Assay, Western Blot, Cell Culture

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of L-NAME on osteogenic differentiation and pathway-associated factor of PDLSCs following AS-IV incubation. ( A and B ) ALP staining( A ) and ALP activity quantification( B ) of PDLSCs affected by L-NAME. Scale bar: 500 μm. ( C and D ) Immunofluorescence staining( C ) of COL-1 (green) and DAPI staining (blue) and quantification(D). Scale bar: 50 μm. ( E–H ) Western blot and semi-quantitative analysis of COL-1 (F), ALP(G), and RUNX-2 ( H ) in PDLSCs cultured with AS-IV and L-NAME. ( I–K ) Western blot and semi-quantitative analysis of eNOS( J ) and p-AKT/AKT ( K ) in PDLSCs cultured with AS-IV and L-NAME. Data are presented as mean ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. ns: P> 0.05. Each experiment was repeated five times.

Article Snippet: Cells were seeded in 6-well plates at a density of 1×10 5 cells per well and incubated with osteogenic induction medium (MEM with 10% FBS, 50μg/mL ascorbic acid, 0.01μM dexamethasone, and 10mM β-glycerophosphate, Solarbio, Beijing, China) or adipogenic induction medium (MEM with 10% FBS, 500mM isobutyl-methylxanthine, 10 mM insulin, 60mM indomethacin, and 0.5 M hydrocortisone, Solarbio, Beijing, China).

Techniques: Incubation, Staining, Activity Assay, Immunofluorescence, Western Blot, Cell Culture