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Sci Rep
2024 Nov 02;141:26415. doi: 10.1038/s41598-024-77850-4.
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Investigating the potential effect of Holothuria scabra extract on osteogenic differentiation in preosteoblast MC3T3-E1 cells.
Songkoomkrong S
,
Nonkhwao S
,
Duangprom S
,
Saetan J
,
Manochantr S
,
Sobhon P
,
Kornthong N
,
Amonruttanapun P
.
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The present medical treatments of osteoporosis come with adverse effects. It leads to the exploration of natural products as safer alternative medical prevention and treatment. The sea cucumber, Holothuria scabra, has commercial significance in Asian countries with rising awareness of its properties as a functional food. This study aims to investigate the effects of the inner wall (IW) extract isolated from H. scabra on extracellular matrix maturation, mineralization, and osteogenic signaling pathways on MC3T3-E1 preosteoblasts. The IW showed the expression of several growth factors. Molecular docking revealed that H. scabra BMP2/4 binds specifically to mammal BMP2 type I receptor (BMPR-IA). After osteogenic induction, the viability of cells treated with IW extract was assessed and designated with treatment of 0.1, 0.5, 1, and 5 µg/ml of IW extract for 21 consecutive days. On days 14 and 21, treatments with IW extract at 1 and 5 µg/ml showed increased alkaline phosphatase (ALP) activity and calcium deposit levels in a dose-dependent manner compared to the control group. Moreover, the transcriptomic analysis of total RNA of cells treated with 5 µg/ml of IW extract exhibited upregulation of TGF-β, PI3K/Akt, MAPK, Wnt and PTH signaling pathways at days 14. This study suggests that IW extract from H. scabra exhibits the potential to enhance osteogenic differentiation and mineralization of MC3T3-E1 preosteoblasts through TGF-β, PI3K/Akt, MAPK, Wnt and PTH signaling pathways. Further investigation into the molecular mechanisms underlying the effect of IW extract on osteogenesis is crucial to support its application as a naturally derived supplement for prevention or treatment of osteoporosis.
Fig. 1. Schematic diagram illustrated the experimental design of H. scabra IW extract treated with MC3T3-E1 preosteoblasts.
Fig. 2. Gene expression profiles in isolated IW from H. scabra. Digital image of agarose gel electrophoresis showed the expression of H. scabra BMP2A, BMP2/4, BMP5-like, TGF-β2, inhibin, myostatin, activin, and FGF18 mRNA using RT-PCR. The 16s rRNA expression was used as an internal control and no amplification was detected in the no template control (NTC).
Fig. 3. Western blot of the BMP2 in the H. scabra tissues and three dimentional structure of Homsa-BMP2 and Holsc-BMP2/4 with ligand-receptor interaction. (A) Western blot of the BMP2 in the H. scabra tissues. The immunoreacted 45 kDa protein band was only detected in the IW protein. No other immunoreactivity was found in the RN, NR and BW proteins. (B) Amino acid sequences alignment of Homsa-BMP2 (PDB ID: 6omn.2) and Holsc-BMP2/4 from H. scabra. The dark blue highlights indicate 100% sequence identity, while the pale blue highlights represent sequence similarity. Various line colors above and below the sequences highlight the active regions of Homsa-BMP2 (with dark blue and blue lines representing regions of chain A and chain B, respectively) and Holsc-BMP2/4 (with purple and pink lines representing regions of chain A and chain B, respectively). Black and red asterisks (*) indicate the active sites of a crystal structure of human BMP2 and BMPR-IA (PDB ID: 2H62) for chain A and chain B, respectively. Three-dimensional structures of (C) the Homsa-BMP2 (blue (chain A) and cyan (chain B)) and (D) Holsc-BMP2/4 (purple (chain A) and gray (chain B)). (E) 3D-structure of Homsa-BMP2 (blue/cyan) superimposed on Holsc-BMP2/4 (purple/gray). (F) A crystal structure of human BMP2 and BMPR-IA (PDB ID: 2H62) was used as a reference binding template. Structural docking of BMPR-IA binding with Homsa-BMP2 and Holsc-BMP2/4 is illustrated in (G) and (H), respectively. The magnified ribbon diagram highlights the amino acid interactions between the receptor (green) and Homsa-BMP2 (purple) and Holsc-BMP2/4 (pink). Yellow dotted lines represent hydrogen bond interactions.
Fig. 4. The cytotoxic effect of crude IW extract from H. scabra on the proliferation of MC3T3-E1 cells on day 1, day 3 and day 5. The cells were treated with different concentrations (0–100 µg/ml) of IW extract after 1 day, 3 days and 5 days of culture period before subjected to MTT assay. The cell viability of IW-treated groups was shown as the percentage comparing to the control group on each day. Data are expressed as mean ± SEM from n = 5 replications. The statistically significant differences were defined using one-way ANOVA at (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, and (****) p < 0.0001 when the comparison was between the control and osteogenesis-induced IW groups.
Fig. 5. The osteogenic activity of MC3T3-E1 preosteoblasts after 7, 14, and 21 days of incubation with different doses of IW extract of H. scabra. The graph showed the ALP activity calculated from known concentration of ALP and normalized with total protein content (ng/mg protein). Cells were differentiated with indicated increasing concentrations (0.1–5 µg/ml) of IW extract for 7, 14, and 21 days before evaluation of the ALP activity. The statistically significant differences were defined using one-way ANOVA at (**) p < 0.01 and (****) p < 0.0001 when the comparison was between the control and osteogenesis-induced IW groups.
Fig. 6. The bone mineralization of MC3T3-E1 preosteoblasts after 7, 14, and 21 days of differentiation with various doses of IW extract of H. scabra. (A) Digital images showed the mineralized nodules as assessed by ARS staining after incubated with the induction medium containing IW extract at different concentration (0.1, 0.5, 1, and 5 µg/ml) for 7, 14, and 21 days. (B) The quantification analysis of the calcium deposition in the extracellular matrix was evaluated after calcium nodules were stained with ARS. MC3T3-E1 cells were differentiated for 3 weeks with various doses of IW extract and the deposition of calcium was interpreted in terms of ARS concentration (µM). The statistically significant differences were shown at (*) p < 0.05, (**) p < 0.01, and (****) p < 0.0001 when the comparison was between the control and osteogenesis-induced IW groups.
Fig. 7. Identification and functional analysis of differentially expressed genes (DEGs) in control (osteogenic induction medium) compared with treatment (H. scabra IW extract in osteogenic induction medium). (A) The volcano map of DEGs analyzed by DESeq2 (log2 FC ≥ 0.2; Q value ≤ 0.05); the red dots represent upregulated DEGs, the green dots represent downregulated DEGs, and the gray dots represent genes that did not show significant differential expression. (B) Heatmap illustrating the expression of each sample, standardized by log (expression + 1). Each row represents a gene, each column represents a sample, and the colors in the panels indicate the relative expression levels, with blue indicating low expression and red indicating high expression.
Fig. 8. KEGG pathway enrichment identification. (A) KEGG enrichment analyses of the DEGs. (B) GSEA of KEGG.
