Nicotinamide mononucleotide attenuates glucocorticoid‑induced osteogenic inhibition by regulating the SIRT1/PGC‑1α signaling pathway

Abstract

Long-term and high-dose glucocorticoid treatment is recognized as an important influencing factor for osteoporosis and osteonecrosis. Nicotinamide mononucleotide (NMN) is an intermediate of NAD+ biosynthesis, and is widely used to replenish the levels of NAD+. However, the potential role of NMN in glucocorticoid‑induced osteogenic inhibition remains to be demonstrated. In the present study, the protective effects of NMN on dexamethasone (Dex)‑induced osteogenic inhibition, and its underlying mechanisms, were investigated. Bone mesenchymal stem cells were treated with Dex, which decreased the levels of the osteogenic markers alkaline phosphatase, Runt‑related transcription factor 2 and osteocalcin. NMN treatment attenuated Dex‑induced osteogenic inhibition and promoted the expression of sirtuin 1 (SIRT1) and peroxisome proliferator‑activated receptor gamma coactivator (PGC)‑1α. SIRT1 knockdown reversed the protective effects of NMN and reduced the expression levels of PGC‑1α. Collectively, the results of the present study reveal that NMN may be a potential therapeutic target for glucocorticoid‑induced osteoporosis.

Keywords: nicotinamide mononucleotide; osteoporosis; sirtuin 1; peroxisome proliferator‑activated receptor gamma coactivator.

Figure 1.

Dex-induced osteogenic inhibition in BMSCs. BMSCs were exposed to Dex (range, 10⁻⁹−10⁻⁶ M) for 7 days. (A) Reverse transcription-quantitative PCR was used to detect Runx2, ALP and OCN mRNA expression in BMSCs. (B) Western blotting was used to determine the Runx2 and ALP protein expression levels in BMSCs. (C) Relative ALP activity. (D) Cell Counting Kit-8 was used to assess the viability of BMSCs. (E) Alizarin red and ALP staining assays were used to measure the osteogenic function of BMSCs treated with 10⁻⁶ M Dex for 7 days. Scale bar, 20 µm. (F) Relative ALP activity of BMSCs treated with 10⁻⁶ M Dex for 7 days. All experiments were performed ≥3 times. ***P<0.001, **P<0.01, *P<0.05 and ^nsP≥0.05 vs. Veh. Dex, dexamethasone; BMSCs, bone mesenchymal stem cells; ALP, alkaline phosphatase; Runx2, Runt-related transcription factor 2; OCN, osteocalcin; Veh, vehicle.

Figure 2.

Role of NMN in Dex-induced osteogenic inhibition of BMSCs. BMSCs were exposed to 10⁻⁵ M Dex and NMN (1, 5 and 10 mM). (A) Reverse transcription-quantitative PCR was used to detect Runx2, ALP and OCN mRNA expression in BMSCs. (B) Western blotting was used to determine the Runx2 and ALP protein expression levels in BMSCs. (C) Alizarin red and ALP staining assays were used to assess the osteogenic function of BMSCs treated with 10⁻⁵ M Dex for 7 days. Scale bar=20 µm. All experiments were performed ≥3 times. P<0.05 was considered to indicate a statistically significant difference. ***P<0.001 and **P<0.01, as indicated. NMN, nicotinamide mononucleotide; Dex, dexamethasone; BMSC, bone mesenchymal stem cells; ALP, alkaline phosphatase; Runx2, Runt-related transcription factor 2; OCN, osteocalcin.

Figure 3.

SIRT1/PGC-1α signaling is involved in the protective effects of NMN on Dex-induced osteogenic inhibition. BMSCs were treated with control, Dex and Dex + NMN for 7 days. (A) Reverse transcription-quantitative PCR was used to detect the SIRT1, PGC-1α, Runx2 and ALP mRNA expression levels in BMSCs. (B) Western blotting was used to determine the SIRT1, PGC-1α, Runx2 and ALP protein expression levels in BMSCs. (C) Relative ALP activity. (D) Immunofluorescence was used to detect SIRT1 protein expression in BMSCs following 2 days of treatment. Scale bar, 5 µm. All experiments were performed ≥3 times. ***P<0.001 and **P<0.01, as indicated. SIRT1, sirtuin 1; PGC, peroxisome proliferator-activated receptor gamma coactivator; NMN, nicotinamide mononucleotide; Dex, dexamethasone; BMSC, bone mesenchymal stem cells; ALP, alkaline phosphatase; Runx2, Runt-related transcription factor 2.

Figure 4.

SIRT1 knockdown reduces the protective effects of NMN on Dex-induced osteogenic inhibition. Si-NC or si-SIRT1 were transfected into BMSCs before Dex and NMN treatment. SIRT1-knockdown was confirmed by (A) RT-qPCR and (B) western blotting. (C) RT-qPCR was used to detect the SIRT1, PGC-1α and Runx2 mRNA expression levels in BMSCs. (D) Western blotting assays were used to determine the SIRT1, PGC-1α, ALP and Runx2 protein expression levels in BMSCs. (E) Alizarin red and ALP staining assays were used to assess the osteogenic ability of BMSCs. Scale bar, 20 µm. All experiments were performed ≥3 times. ***P<0.001, **P<0.01 *P<0.05 and ^nsP≥0.05, as indicated. Si, small interfering; NC, negative control; NMN, nicotinamide mononucleotide; Dex, dexamethasone; BMSC, bone mesenchymal stem cells; SIRT1, sirtuin 1; PGC, peroxisome proliferator-activated receptor gamma coactivator; ALP, alkaline phosphatase; Runx2, runt-related transcription factor 2; RT-qPCR, reverse transcription-quantitative PCR.

Figure 5.

NMN alleviates Dex-induced osteogenesis by regulating the SIRT1/PGC-1α signaling pathway. NMN, nicotinamide mononucleotide; Dex, dexamethasone; SIRT1, sirtuin 1; PGC, peroxisome proliferator-activated receptor gamma coactivator.