Nonthermal biocompatible plasma in stimulating osteogenic differentiation by targeting p38/ FOXO1 and PI3K/AKT pathways in hBMSCs

Abstract

Osteoporosis is manifested by decreased bone density and deterioration of bone architecture, increasing the risk of bone fractures Human bone marrow mesenchymal stem cells (hBMSCs)-based tissue engineering serves as a crucial technique for regenerating lost bone and preventing osteoporosis. Non-thermal biocompatible plasma (NBP) is a potential new therapeutic approach employed in several biomedical applications, including regenerative medicine. NBP affects bone remodeling; however, its role in the regulation of osteogenic differentiation in hBMSCs remains largely unexplored. This study aimed to explore the efficiency of NBP in promoting osteogenic differentiation, and the molecular pathways through which these responses occurred in hBMSCs. We found that NBP facilitated osteogenic differentiation through the upregulation of the bone morphogenic protein signal (BMPs) cascade, which in turn induced the expression of p38 and inhibited the forkhead box protein O1 (FOXO1). To further gain insight into the mechanism through which NBP extensively triggers the initiation of osteogenic differentiation in hBMSCs, PI3K/AKT pathway was also analyzed. Overall, these results highlight that NBP enhances osteogenic differentiation in hBMSCs by the stimulation of the p38/FOXO1 through PI3K/AKT signaling pathways. Therefore, the application of NBP in hBMSCs may offer tremendous therapeutic prospects in the treatment of bone regeneration and osteoporosis prevention.

Keywords: Human bone marrow mesenchymal stem cells; Nonthermal biocompatible plasma; Osteogenic differentiation; PI3K/AKT signaling pathway.

Fig. 1

Schematic structure and dielectric properties of the experiment. A Graphical representation of a micro DBD source. B and C Voltage-current waveform, and D optical emission spectrum (OES) assessment of DBD using N₂ gas. The generation of reactive species E H₂O₂ and F NOx in the culture medium of hBMSCs after 4 and 7 min of plasma treatment. The mean and standard deviation values are used to represent the experimental results. The statistical significance of the data points was assessed using the student’s t-test, and significant differences were indicated by *p < 0.05; **p < 0.01, and ***p < 0.001 vs the untreated control

Fig. 2

Fluorescence images of intracellular ROS and RNS generation in A hBMSCs and B nHDF cell lines after 6 h of NBP treatment at 4 and 7 min, respectively, untreated as control, H₂O₂ (100 µM) as the positive control. ROS and RNS were detected in cells using H₂DCFDA and DAF-FM assay kits (scale bars: 90 μm). The confocal pixel intensity was quantified using the ImageJ software as follows: C hBMSCs and D nHDF cell lines. The hBMSCs were cultivated without an osteogenic inductive medium and data were presented as fold-change normalized to the pixel intensity level of the control. E Flow cytometer histogram H₂DCFDA-stained hBMSCs. The relative intensity of intracellular ROS concentration in hBMSCs was obtained by flow cytometer after 6 h of NBP treatment for 4 and 7 min, untreated as control, with H₂O₂ (100 µM) as the positive control. The mean and standard deviation values are used to represent the experimental results. The statistical significance of the data points was assessed using the student’s t-test, and significant differences were indicated by *p < 0.05; **p < 0.01, and ***p < 0.001 vs the untreated control

Fig. 3

The influence of NBP on osteogenic differentiation of hBMSCs. The cytotoxicity of A hBMSCs and B nHDF cell lines after NBP exposure times of 1, 4, 7, 8, 10, and 20 min, respectively. C Representative images of hBMSCs and nHDF cell morphology were observed under the light microscope. D and F Representative images of Ca deposition in hBMSCs via Alizarin Red Staining at days 4 and 7 under the light microscope. Scale bars, 100 µm. E and G Quantified areas of ARS staining measured by ImageJ software. The mean and standard deviation values are used to represent the experimental results. The statistical significance of the data points was assessed using the student’s t-test, and significant differences were indicated by *p < 0.05; **p < 0.01, and ***p < 0.001 vs the untreated control

Fig. 4

NBP treatment promotes the osteogenic differentiation in hBMSCs. A, B The messenger RNA (mRNA) levels of osteogenic genes, including OCN, COL1A1, OSX, Runx-2, and ALP, were measured using RT-qPCR. Total RNA was extracted from hBMSCs at two-time points: 4 and 7 d following combination treatment of NBP at 4 and 7min treatment time and mineral supplement (0.08 g/ml of ascorbic acid, 0.30611 g/ml of β-glycerol phosphate, 0.051 M dexamethasone). GAPDH was used as the internal control. C, D Immunofluorescence staining was performed to detect OCN and COL1A1 protein expression with the administration of a combination treatment of NBP and mineral supplements. OCN and COL1A1 (green) elevated on day 7 of osteogenic differentiation in response to the combination treatment of NBP at 4 and 7 min with a mineral supplement. The green fluorescence represents the OCN and COL1A1, and DAPI is represented by the blue fluorescence. ImageJ software was used to quantify the fluorescence intensities of OCN and COL1A1. The scale bars used in the imaging were 60 µm. The hBMSCs were cultivated for 1 and 7 d after NBP treatment at 4 and 7min with mineral supplements. E-H the protein expression of BMP-2, BMP-3, BMP-4, p-p38, and FOXO1 was analyzed via western blotting. The quantification of band intensities is represented in the graphs. GAPDH was used for the normalization. The mean and standard deviation values are used to represent the experimental results. The statistical significance of the data points was assessed using the student’s t-test, and significant differences were indicated by *p < 0.05; **p < 0.01, and ***p < 0.001 vs the untreated control

Fig. 5

Inhibition of p38 pathways reversed the enhancing effects of NBP on osteogenic differentiation of hBMSCs. A The relative mRNA expression of OCN, COL1A1, OSX, Runx-2, and ALP in hBMSCs, they were cultivated in culture media for 2 d with or without SB203580 (10 μm) or NBP (4 min) and quantified via qRT-PCR. GAPDH was used as the internal control. B Immunofluorescence staining of OCN and COL1A1 and, C its relative pixel intensities on day 2, scale bars: 100 μm. D Results of western blot analysis of phosphorylated p38, and BMP-2. E Represents the quantification of band intensity on day 2 were performed. GAPDH was used for the normalization. The mean and standard deviation values are used to represent the experimental results. The statistical significance of the data points was assessed using the student’s t-test, and significant differences were indicated by *p < 0.05; **p < 0.01 compared with NC, compared with NBP (4 min)

Fig. 6

NBP treatment promoted hBMSCs osteogenic differentiation through the activation of PI3K/AKT pathways. A, B The relative mRNA expression of hmTOR, PIK3CA, PIK3R1 and PIK3R2 is upregulated in hBMSCs quantified via qRT-PCR. Total RNA was extracted from hBMSCs at two-different points: combination treatment of NBP at 4 and 7min treatment time and mineral supplement (0.08 g/ml of ascorbic acid, 0.30611 g/ml of β-glycerol phosphate, 0.051 M dexamethasone) and without mineral supplements following NBP at 4 and 7min treatment time. GAPDH was used as the internal control. C, D Western blot analysis was performed to detect PI3K, p-AKT, and PTEN protein expression with the administration of NBP treatment at 4 and 7min on day 2. D The quantification of band intensities is represented in the graphs. GAPDH was used for the normalization. E, F Representative images of Ca deposition in hBMSCs via Alizarin Red Staining at day 2 under the light microscope. Scale bars, 60 µm. F Quantified areas of ARS staining measured by ImageJ software. The mean and standard deviation values are used to represent the experimental results. The statistical significance of the data points was assessed using the student’s t-test, and significant differences were indicated by *p < 0.05; **p < 0.01, and ***p < 0.001 vs the untreated control

Fig. 7

LY294002 reversed the stimulating effect of NBP and blocked the PI3K/AKT pathways. A The amount of gene levels of hmTOR, PIK3CA, PIKR1, and PIK3R2 in hBMSCs, were cultivated in culture media for 2 d with or without LY294002 (10 μm) or NBP (4 min). hBMSCs were cultured with or without NBP treatment at 4 min or LY294002 (10 μm); B Results of western blot analysis of PI3K, phosphorylated AKT, and PTEN. C Represents the quantification of band intensity on day 2 were performed. GAPDH was used for the normalization. D Representative images of Ca deposition in hBMSCs via Alizarin Red Staining on day 2 under the light microscope. Scale bars, 100 µm. E Quantified areas of ARS staining measured by ImageJ software. The mean and standard deviation values are used to represent the experimental results. The statistical significance of the data points was assessed using the student’s t-test, and significant differences were indicated by *p < 0.05; **p < 0.01 compared with NC, compared with NBP (4 min)

Fig. 8

The comparison of inhibitory effects on p38 and PI3K/AKT signal translocation by the PI3K/AKT and p38 inhibitors. A The relative mRNA expression of OCN, COL1A1, OSX, Runx-2, and ALP in hBMSCs was measured by qRT-PCR. Cells were cultured in complete media for 2 d with or without LY294002 (10 μm) or NBP (4 min). B Results of western blot analysis of phosphorylated p-38. C Representing the quantification of band intensity on day 2 were performed. GAPDH was used for the normalization. D–F hBMSCs were cultured with or without NBP treatment at 4 min or SB205380 (10 μm); D Results of western blot analysis of PI3K and PTEN. E Represents the quantification of band intensity on day 2 were performed. GAPDH was used for the normalization. F The relative mRNA expression of hmTOR, PIK3CA, PIKR1, and PIK3R2 in hBMSCs, were cultivated in culture media for 2 d with or without SB205380 (10 μm) or NBP (4 min). The mean and standard deviation values are used to represent the experimental results. The statistical significance of the data points was assessed using the student’s t-test, and significant differences were indicated by *p < 0.05; **p < 0.01 compared with NC, compared with NBP (4 min)

Scheme 1

Graphic illustration of a possible mechanism for the manner in which NBP induced osteogenic differentiation in hBMSCs. It was discovered that NBP causes the production of ROS, which drives PI3K/AKT signaling pathways and stimulates osteogenic genes and proteins linked to osteogenic differentiation