Pro-Osteogenic and Anti-Inflammatory Synergistic Effect of Orthosilicic Acid, Vitamin K2, Curcumin, Polydatin and Quercetin Combination in Young and Senescent Bone Marrow-Derived Mesenchymal Stromal Cells

Abstract

During aging, bone marrow mesenchymal stromal cells (MSCs)-the precursors of osteoblasts-undergo cellular senescence, losing their osteogenic potential and acquiring a pro-inflammatory secretory phenotype. These dysfunctions cause bone loss and lead to osteoporosis. Prevention and intervention at an early stage of bone loss are important, and naturally active compounds could represent a valid help in addition to diet. Here, we tested the hypothesis that the combination of two pro-osteogenic factors, namely orthosilicic acid (OA) and vitamin K2 (VK2), and three other anti-inflammatory compounds, namely curcumin (CUR), polydatin (PD) and quercetin (QCT)-that mirror the nutraceutical BlastiMin Complex^® (Mivell, Italy)-would be effective in promoting MSC osteogenesis, even of replicative senescent cells (sMSCs), and inhibiting their pro-inflammatory phenotype in vitro. Results showed that when used at non-cytotoxic doses, (i) the association of OA and VK2 promoted MSC differentiation into osteoblasts, even when cultured without other pro-differentiating factors; and (ii) CUR, PD and QCT exerted an anti-inflammatory effect on sMSCs, and also synergized with OA and VK2 in promoting the expression of the pivotal osteogenic marker ALP in these cells. Overall, these data suggest a potential role of using a combination of all of these natural compounds as a supplement to prevent or control the progression of age-related osteoporosis.

Keywords: inflammaging; mesenchymal stromal cells; natural compounds; osteogenesis; osteoporosis; senescence.

Figure 1

In vitro characterization of replicative senescent MSCs (sMSCs). (A) Characterization of sMSC by cumulative population doubling curve and (SA)-β-Gal activity. (B) Telomere length in bone marrow mesenchymal stromal cells, calculated according to 2^−ΔΔCt method using a single-copy reference DNA for normalization. (C) p16^ink4a and p21 mRNA expression. Data are reported as relative expression according to 2^−ΔΔCt method using IPO8 as housekeeping. (D) Representative Western blot analysis of p16^ink4a, p21 and PCNA protein expression and densitometric analysis using β-Actin as loading control. The results are expressed as mean ±SD from three independent biological replicates. Paired t test, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. yMSC.

Figure 2

Dose-response curve of yMSC and sMSC to orthosilicic acid, vitamin K2, curcumin, polydatin, quercetin and their combinations. (A) yMSC and (B) sMSC were treated with different concentrations of orthosilicic acid (from 50 μM to 200 μM), vitamin K2 (from 0.05 μM to 10 μM), their combination (OA+VK2, 75 μM OA and 0.1 μM VK2) or with PEG-60 hydrogenated castor oil (vitamin K2 solvent) alone as a control for 24 h or 7 days. (C) sMSC were treated with different concentrations of curcumin (from 0.6 μM to 40 μM), polydatin (from 6.25 μM to 100 μM), quercetin (from 0.125 μM to 2 μM), their combination (C+P+Q, 1 μM CUR, 10 μM PD, 0.5 μM QCT) or with DMSO alone as a control for the indicated times. (D) sMSC were treated for 7 days with the combination of OA, VK2, CUR, PD and QCT (MIX) or with DMSO alone as a control. The MTT assay was used to assess cell viability upon treatment. The results are presented as a percentage of cell viability normalized to the viability of PEG-60 hydrogenated castor oil-treated (Ctr) for VK2 or DMSO-treated cells (Ctr)—for CUR, PD and QCT—and presented as mean value ±SD from three independent biological replicates.

Figure 3

Orthosilicic acid and Vitamin K2 have an osteogenic effect on yMSC. yMSC were treated with OA (75 μM), VK2 (0.1 μM) and their combination (OA+VK2) and RUNX2, ALP, Col1α1 and OCN mRNA expression analyzed after 7 and 14 days of treatment (A,B). (C) miR-98 expression analysis in 7 days-treated yMSC. Data are reported as relative expression according to 2^−ΔΔCt method, using IPO8 or RNU44 as housekeeping. (D) ALP activity of 7 days treated with yMSC. Data are reported as fold change vs untreated yMSC. (E) Representative Western blot analysis showing RUNX2, Col1α1 and OCN protein expression, with Gapdh or β-actin as loading controls. The bands were quantified by ImageJ. All data are reported as fold change vs untreated yMSC. The results are expressed as mean ± SD from three independent biological replicates. Paired t test, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated yMSC.

Figure 4

Curcumin, polydatin and quercetin exert an anti-inflammatory effect on sMSC. (A) IL-1β, IL-6, IL-8, MCP-1 mRNA and miR-21 and miR-146a expression of sMSC treated with CUR, PD and QCT for 24h. Data are reported as relative expression according to 2^−ΔΔCt method, using IPO8 and RNU44 as housekeeping. (B) Concentration (pg/mL) of IL-8 and MCP-1 in the conditioned medium of untreated and treated sMSC. Histograms represent the mean of three independent experiments ± SD. (C) Representative Western blot analysis showing P-p38 and P-NF-κB protein expression, using β-actin as loading control. The bands were quantified by ImageJ. The results are expressed as mean ± SD from three independent biological replicates. Paired t test, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. untreated yMSC, * p < 0.05, ** p < 0.01, *** p <0.001 vs. untreated sMSC.

Figure 5

Anti-inflammatory and pro-osteogenic effect of OA+VK2, C+P+Q and their combination (MIX) in sMSC. (A) IL-1β, MCP-1, IL-6, IL-8, ALP, COL1α1 mRNA and miR-98, miR-21, miR-146a expression in sMSC treated with OA+VK2, C+P+Q and MIX for 7 days and yMSC (not differentiated). Data are reported as relative expression according to 2^−ΔΔCt and using IPO8 and RNU44 as housekeeping. (B) Concentration (pg/mL) of IL-8 and MCP-1 in conditioned media of yMSC, untreated and treated sMSC. (C) ALP activity in sMSC treated for 7 days with OA+VK2, C+P+Q and MIX. (D) Representative Western blot analysis of P-p38 and P-NF-κB protein expression with β-actin as loading control. The bands were quantified by ImageJ. The results are expressed as mean ± SD from three independent biological replicates. Paired t test, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. untreated yMSC, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated sMSC.

Figure 6

Schematic representation of orthosilicic acid (OA), vitamin K2 (VK2), curcumin (CUR), polydatin (PD) and quercetin (QCT) effects on MSC and sMSC. OA and VK2 combination up-regulated RUNX2, ALP and COL1α1 (upper part of the figure). CUR, PD and QCT combination inhibited SASP, which plays a role in low bone degeneration in aging. The association of the five compounds (OA, VK2, CUR, PD and QCT) and increased ALP and COL1α1 expression and inhibited SASP in sMSC. Overall, it can be suggested that reducing inflammation and promoting MSC (both young and senescent) differentiation by using these natural compounds might be a valid strategy to prevent bone loss during aging. This figure was created using the Servier Medical Art Commons Attribution 3.0 Unported Licence (http://smart.servier.com (accessed on 21 April 2023)).