Int J Stem Cells.  2023 Feb;16(1):78-92. 10.15283/ijsc21238.

Synergistic Effect of Hydrogen and 5-Aza on Myogenic Differentiation through the p38 MAPK Signaling Pathway in Adipose-Derived Mesenchymal Stem Cells

• Fei W
• Pang E
• Hou L
• Dai J
• Liu M
• Wang X
• Xie B
• Wang J

• KMID: 2539623
• DOI: http://doi.org/10.15283/ijsc21238

Abstract

Background and Objectives
This study aims to clarify the systems underlying regulation and regulatory roles of hydrogen combined with 5-Aza in the myogenic differentiation of adipose mesenchymal stem cells (ADSCs).
Methods and Results
In this study, ADSCs acted as an in vitro myogenic differentiating mode. First, the Alamar blue Staining and mitochondrial tracer technique were used to verify whether hydrogen combined with 5-Aza could promote cell proliferation. In addition, this study assessed myogenic differentiating markers (e.g., Myogenin, Mhc and Myod protein expressions) based on the Western blotting assay, analysis on cellular morphological characteristics (e.g., Myotube number, length, diameter and maturation index), RT-PCR (Myod, Myogenin and Mhc mRNA expression) and Immunofluorescence analysis (Desmin, Myosin and β-actin protein expression). Finally, to verify the mechanism of myogenic differentiation of hydrogen-bound 5-Aza, we performed bioinformatics analysis and Western blot to detect the expression of p-P38 protein. Hydrogen combined with 5-Aza significantly enhanced the proliferation and myogenic differentiation of ADSCs in vitro by increasing the number of single-cell mitochondria and upregulating the expression of myogenic biomarkers such as Myod, Mhc and myotube formation. The expressions of p-P38 was up-regulated by hydrogen combined with 5-Aza. The differentiating ability was suppressed when the cells were cultivated in combination with SB203580 (p38 MAPK signal pathway inhibitor).
Conclusions
Hydrogen alleviates the cytotoxicity of 5-Aza and synergistically promotes the myogenic differentiation capacity of adipose stem cells via the p38 MAPK pathway. Thus, the mentioned results present insights into myogenic differentiation and are likely to generate one potential alternative strategy for skeletal muscle related diseases.

Keyword

Hydrogen; 5-Azacitidin (5-Aza); Myogenic differentiation; Adipose-derived mesenchymal stem cells; P38 MAPK signaling pathway

Figure

• 

  Fig. 1 (A) Volcanic map of cells cultured in hydrogen shows differentially expressed genes. (B) KEGG enrichment pathway analysis. (C) Analysis of differential gene expression in MAPK signaling pathway, GSEA enrichment analysis. (D) Enrichment analysis of MAPK signal pathway. (E) Enrichment analysis of p38-MAPK signal pathway. (F) Mitochondrial functional enrichment analysis. (G, H) Identifification of adipose-derived stem cells (ADSCs). (G) Flow cytometry analysis results and expression of cell surface CD markers of ADSCs at passage 3. The x-axis is the fluorescence intensity, and the y-axis is the cell number (H). ADSCs was positive for Alizarin red (a), Oil Red O (b), and Toluidine blue staining (c) after induced differentiation.

• 

  Fig. 2 (A∼C) ADSCs viability evaluation after cultured with different differentiation medium for 1 to 3 days. (A) Live-Dead cell staining analysis and Alamar blue staining on 1 and 3 days. (B) Fluorescence quantitative analysis of cell proliferation. (C) Proportion analysis of Live-Dead cells. (D, E) Cell viability was assessed by MTT. (F) Selection of the optimum concentration of hydrogen. (G, H) The analysis of mitochondrial staining. (G) Fluorescent staining of mitochondria by Mito-tracker. (H) Quantitative analysis of fluorescence intensity of single cell mitochondria. All experiments were performed in triplicate (*p<0.05, **p<0.01).

• 

  Fig. 3 (A) β-actin, Myosin and Desmin expression (Immunofluore-scence staining). Molecular expression levels of (a) β-actin, (b) Myosin and (c) Desmin. (B) Observation and analysis of myotube. Immunofluore-scence staining of Mhc protein (red) in ADSCs on day 7 in myogenic differentiation medium; Quantitative analysis of Myotube number (a), Myotube length (b), Myotube diameter (c), Myotube maturation index (myotubes with≥2nuclei) (d), Com-parison of the number of myotubes between the groups (e), Comparison of myotube length between groups (f), Comparison of myotube diameter between groups (g), and comparison of myotube maturation index bet-ween groups (h). (C) Myod, Mhc and Myogenin expression (RT-PCR). All experiments were performed in triplicate (*p<0.05, **p<0.01).

• 

  Fig. 4 (A) Myod, Mhc, Myogenin and p-P38 and expression (Western blot). (B) Molecular mechanism analysis of myogenic differentiation. (B) Western blot analysis of p38 MAPK pathway related proteins in ADSCs on day 7 after incubation with inductor in inhibitor added myogenic differentiation medium; Quantitative gray level based on the Western blot bands for p-P38, Mhc and Myod proteins expression; All experiments were performed in triplicate (*p<0.05, **p<0.01).

Reference

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