Int J Stem Cells.  2022 Aug;15(3):334-345. 10.15283/ijsc22044.

Modulation of Osteogenic Differentiation of Adipose-Derived Stromal Cells by Co-Treatment with 3, 4’-Dihydroxyflavone, U0126, and N-Acetyl Cysteine

• Song K
• Yang GM
• Han J
• Gil M
• Dayem AA
• Kim K
• Lim KM
• Kang GH
• Kim S
• Jang SB
• Vellingiri B
• Cho SG

• KMID: 2532407
• DOI: http://doi.org/10.15283/ijsc22044

Abstract

Background and Objectives
Flavonoids form the largest group of plant phenols and have various biological and pharma-cological activities. In this study, we investigated the effect of a flavonoid, 3, 4’-dihydroxyflavone (3, 4’-DHF) on osteogenic differentiation of equine adipose-derived stromal cells (eADSCs).
Methods and Results
Treatment of 3, 4’-DHF led to increased osteogenic differentiation of eADSCs by increasing phosphorylation of ERK and modulating Reactive Oxygen Species (ROS) generation. Although PD98059, an ERK inhibitor, suppressed osteogenic differentiation, another ERK inhibitor, U0126, apparently increased osteogenic differentiation of the 3, 4’-DHF-treated eADSCs, which may indicate that the effect of U0126 on bone morphogenetic protein signaling is involved in the regulation of 3, 4’-DHF in osteogenic differentiation of eADSCs. We revealed that 3, 4’-DHF could induce osteogenic differentiation of eADSCs by suppressing ROS generation and co-treatment of 3, 4’-DHF, U0126, and/or N-acetyl cysteine (NAC) resulted in the additive enhancement of osteogenic differentiation of eADSCs.
Conclusions
Our results showed that co-treatment of 3, 4’-DHF, U0126, and/or NAC cumulatively regulated osteo-genesis in eADSCs, suggesting that 3, 4’-DHF, a flavonoid, can provide a novel approach to the treatment of osteoporosis and can provide potential therapeutic applications in therapeutics and regenerative medicine for human and companion animals.

Keyword

3, 4’-dihydroxyflavone; Osteogenesis; Equine adipose-derived stromal cells; Regenerative medicine

Figure

• 

  Fig. 1 3, 4-’dihydroxyflavone (3, 4’-DHF) enhanced osteogenesis in equine Adipose-Derived Stromal Cells (eADSCs). (A, B) Osteogenic differentiation marker staining of 3, 4’-DHF-treated eADSCs with osteogenic differentiation at day 14 (A) Alkaline phosphatase (ALP) and (B) Alizarin red s. (C) Calcium content ration of 3, 4’-DHF-treated eADSCs with osteogenic differentiation. (D) qRT-PCR analysis of osteogenesis markers (Osteocalcin (OCN), Osteopontin (OPN), RUNX2, and ALP) in eADSCs and 3, 4’-DHF eADSCs. Error bars represent±SD from at least three independent experiments (*p<0.05).

• 

  Fig. 2 3, 4’-DHF induced ERK activation during osteogenic differentiation. (A) Western blot analysis of ERK and AKT phosphorylation during osteogenic differentiation in the presence or absence of 3, 4’-DHF. (B) Expression level of phosphorylated ERK in the presence or absence of PD98059 or 3, 4’-DHF. (C) qRT-PCR analysis of osteogenic differentiation markers ALP and OPN in eADSCs with or without 3, 4’-DHF-treatment in the presence or absence of PD98059. Each experiment was repeated in triplicate and data are presented as means±standard deviation (p<0.05, denoted by*).

• 

  Fig. 3 Treatment with the ERK inhibitor U0126, led to an increase in osteogenesis in eADSCs and 3, 4’-DHF eADSCs via BMP signaling. (A) Western blot analysis of phosphorylated ERK in eADSCs and 3, 4’-DHF eADSCs in the presence or absence of U0126. (B) qRT-PCR of osteogenesis marker gene expression in eADSCs and 3, 4’-DHF eADSCs treated with U0126. (C) qRT-PCR analysis of BMP2 and BMP4 gene expression in the presence or absence of U0126 and PD98059 in eADSCs and 3, 4’-DHF eADSCs. Error bars represent±SD from at least three independent experiments (*p<0.05).

• 

  Fig. 4 3, 4’-DHF regulates osteogenic differentiation by modulation of Reactive Oxygen Species (ROS) signaling. (A) Intracellular ROS level according to 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence by flow cytometry. (B) The intensity of H2DCFDA fluorescence in N-acetyl cysteine (NAC) treated eADSCs and 3, 4’-DHF eADSCs. (C) Expression of osteogenesis marker genes in NAC treated or untreated eADSCs and 3, 4’-DHF eADSCs according to qRT-PCR analysis. (D) Expression of ROS-related genes in eADSCs and 3, 4ʹ-DHF eADSCs in the presence or absence of NAC treatment. Error bars represent±SD from the mean of three independent experiments (*p<0.05).

• 

  Fig. 5 Co-treatment with 3, 4’-DHF, U0126, and NAC regulated osteogenic differentiation. (A) ROS levels were determined by measuring H2DCFDA fluorescence using a flow cytometer, with or without U0126 treatment in eADSCs or 3, 4’-DHF-treated eADSCs during osteogenic differentiation. (B) Intracellular ROS levels were measured following treatment with U0126 or NAC in eADSCs and 3, 4’-DHF eADSCs. (C) qRT-PCR analysis of osteogenic differentiation marker gene expressions in eADSCs and 3, 4’-DHF eADSCs, in the presence or absence with U0126 or NAC. Each experiment was repeated in triplicate and data are presented as means±SD (*p<0.05).

• 

  Fig. 6 Schematic representation of the 3, 4’-DHF, U0126, and NAC modulation of osteogenesis in eADSCs.

Reference

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