Evaluation of the osteogenic effect of apigenin on human mesenchymal stem cells by inhibiting inflammation through modulation of NF-κB/IκBα

Abstract

Background and purpose: Apigenin has stimulatory effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) as well as anti-inflammatory properties. This study investigated the osteogenic differentiation of hMSCs in inflammatory conditions treated with apigenin focusing on nuclear factor kappa-light-chain-enhancer of activated B (NF-кB), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammatory pathways.

Experimental approach: Along with osteogenic differentiation of the hMSCs, they became inflamed with lipopolysaccharide (LPS)/palmitic acid (PA) and treated with apigenin. Alizarin red staining, alkaline phosphatase (ALP) activity, and Runt-related transcription factor 2 (RUNX2) gene expression were used to determine the degree of differentiation. Also, gene expression of NLRP3 was performed along with protein expression of interleukin 1-beta (IL-1β), NF-кB, and IκBα.

Findings / results: Apigenin was shown to be effective in neutralizing the inhibitory impact of LPS/PA on osteogenesis. Apigenin increased MSC osteogenic capacity by inhibiting NLRP3 expression and the activity of caspase-1. It was also associated with a considerable decrease in the protein expression of NF-κB and IκBα, as well as IL-1β, in these cells.

Conclusion and implications: The effects of apigenin on osteogenesis under inflammatory conditions were cautiously observed.

Keywords: Apigenin; Inflammation; Mesenchymal stem cells; NF-кB; NLRP3; RUNX2.

Fig. 1

Confirmation of hMSCs by their ability to three lineage differentiation (A1-A3) and (B) flow cytometric characterization of surface CD markers. (A) The hMSCs differentiation potency into (A1) adipocytes by oil red O staining, (A2) osteocytes by alizarin red, and (A3) chondrocytes by alcian blue staining were observed. The white line below the images indicates a magnification of 1000 µm for (A1 and A2) and 100 µm for (A3). (B) Flow cytometric examination of human-derived mesenchymal stem cells CD markers. As a result, while CD73, CD90, and CD105 were found in a high percentage of cells, only 2.41 percent of cells were positive for CD34, and 2.06 percent for CD45

Fig. 2

Apigenin treatment prevented the inflammatory changes of human mesenchymal stem cells caused by LPS/PA. Cells from three donors (n = 3) were used in duplicate in the experiment. NLRP3 gene expression and interleukin-1β protein levels, as well as caspase-1 activity, have been demonstrated in mesenchymal stem cells cultured in the presence of LPS/PA and treated with apigenin. Data represent the means ± SD.*P < 0.05 and ***P < 0.001 indicate significant differences compared to the control group; ^###P < 0.001 versus 1 LPS/PA group. LPS, Lipopolysaccharide; PA, palmitic acid; Api, apigenin; NLRP3, leucine-rich repeat and pyrin domain containing 3.

Fig. 3

Inflammatory and osteogenic changes of LPS/PA-exposed hMSCs treated with apigenin. The test was performed for three independent experiments (n = 3) and in duplicate. (A) microscopic evaluation of hMSCs in the osteogenesis process for 21 days. Osteogenic differentiation of unstained hMSCs (top row) and alizarin red stained hMSCs (bottom row) is observed.; (B) represent the quantitative analysis of changes in osteogenesis by alizarin red staining, the activity of ALP, and gene expression of RUNX2. Moreover, it represents gene expression of NLRP3 and protein level of IL-1β as well as activity of caspase 1 in cultured hMSCs in the osteogenic medium in the presence of LPS/PA and treated with apigenin. All the groups were cultured in an osteogenic medium for 21 days and then treated with two concentrations of apigenin (25 µM and 50 µM) for 14 days after exposure to LPS/PA. OSX was considered the control group. Data represent the means ± SD. ***P< 0.001 Indicates significant differences in comparison with the control group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 versus OSX + LPS/PA. LPS, Lipopolysaccharide; PA, palmitic acid; hMSCs, human mesenchymal stem cells; ALP, alkaline phosphatase; RUNX2, Runt-related transcription factor 2; NLRP3, leucine-rich repeat and pyrin domain containing 3; IL-1β, interleukin-1 beta; Api, apigenin, OSX, osterix.

Fig. 4

(A)Western blot density change of p-NF-kB/NF-kB and p-IkBa/IkBa in hMSCs cultured in osteogenic medium and exposed to LPS/PA and apigenin. This experiment was performed in the cells of three donors (n = 3) and each experiment was performed in duplicate. It represents the changes in the protein expression of (B) NF-κB and (C) IkBain cultured hMSCs in the osteogenic medium in the presence of LPS/PA and treated with apigenin. All the groups were cultured in an osteogenic medium for 21 days and treated with apigenin at 25 and 50 µM for 14 days after exposure to LPS/PA. Data represent the means ± SD. **P < 0.01 and ***P < 0.001 indicate significant differences compared to OSX group; ^###P < 0.001 versus1 OSX + LPS/PA. LPS, Lipopolysaccharide; PA, palmitic acid; hMSCs, human mesenchymal stem cells; Api, apigenin, OSX, osterix; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B.