Double‐Pronged NAD Preservation: Delaying Cellular Senescence and Initiating Musculoskeletal Regeneration

Age‐related changes and NAD⁺ deficiency play a significant role in determining the efficacy of cellular therapy (Hope et al. 2025). However, the metabolic alterations in NAD⁺ levels within the musculoskeletal system during the aging process remain incompletely understood. To address this knowledge gap, we performed a single‐cell transcriptomic analysis using data from publicly accessible databases (Kedlian et al. 2024; Zhong et al. 2020). Notably, to evaluate alterations in the musculoskeletal system under aging conditions, four cell populations were identified: myofibers (MF), muscle stem cells (MuSC), chondrocytes (CH), and osteoblasts (OB) (Figure 1a,b and Figure S1). Differential analysis results revealed a decreased proportion of precursor MuSCs within the aging sample, whereas the proportion of adult MFs was increased, indicating a decline in regenerative capacity with advancing age (Figure 1c). Cluster heatmaps revealed that NAD⁺ metabolism‐related genes, including NAMPT, NMNAT1, sirtuins, PARPs, and CD38 (Aksoy et al. 2006), demonstrated significant differences between young and aging samples (Figure 1d and Figure S2). Particularly, the expression of most NAD⁺‐related genes and signaling pathways was downregulated in aging tissues (Figure 1e and Figure S3). Subsequently, we examined the effects of aging on musculoskeletal tri‐lineage differentiation, including chondrogenesis, osteogenesis, and myogenesis, as well as on NAD⁺ metabolism (Figure 1f and Figure S4). Under three different aging models—oxidative stress induced by tert‐butyl hydroperoxide (TBHP), DNA damage due to doxorubicin (DOX), and replicative senescence observed in late‐passage cells—the proportion of β‐galactosidase (β‐gal)‐positive senescent musculoskeletal precursor cells was significantly increased (Figure 1g). Lineage differentiation is an energy‐dependent process with adenosine triphosphate (ATP) serving as its primary energy source (Powell 2018). Results from quantitative analyses supported that aging leads to a reduction in the concentration of cellular ATP, resulting in an energy deficit (Figure 1h). NAD⁺ exists in two interconvertible forms: the reduced form (NADH) and the oxidized form (NAD⁺). The oxidation of NADH to NAD involves the loss of an electron, a process tightly coupled to cellular energy generation (Cahoon and Freitag 2018). For cellular NAD⁺ and NADH metabolism, aging not only reduces the concentration of NAD⁺ but also decreases the NAD⁺/NADH ratio, with an unspecified alteration in NADH levels (Figure 1i,j and Figure S5). Collectively, these findings demonstrate that musculoskeletal aging was correlated with dysregulation in NAD⁺ metabolism and decreased NAD⁺ levels.

Given the observed NAD⁺ deficiency and impaired energy production in musculoskeletal precursor cells, we aimed to investigate a viable strategy to enhance cellular NAD⁺ metabolism. Building on the concept of simultaneously enhancing NAD⁺ biosynthesis (“increasing income”) and limiting its degradation (“reducing expenditure”), we developed a novel formulation comprising the NAD⁺ precursor NMN and the NAD⁺‐consuming enzyme inhibitor API, hereafter referred to as the “N + A” regimen. The live/dead cell assays demonstrated that treatment with NMN, API, or the combined N + A formulation prevented cell death under aging conditions (Figure S6). Regarding NAD⁺ homeostasis, both NMN and API partially restored the TBHP‐induced decline in NAD⁺ levels; however, the combined N + A formulation demonstrated the most pronounced enhancement of both NAD⁺ levels and the NAD⁺/NADH ratio across all the three types of musculoskeletal precursor cells (Figure 2a and Figure S7). Moreover, the in vivo assay of NAD metabolism in cartilage, bone, and muscle samples derived from differently treated mice indicated that the N + A formulation enhanced the NAD⁺ levels and the NAD⁺/NADH ratio (Figure S8). Owing to the importance of CD38, a NAD‐consuming enzyme, the expression level and activity of CD38 were examined in three cell lines (Aksoy et al. 2006). Our results indicated that the TBHP led to an increase in the expression and activity of CD38, and this phenomenon was mitigated by the treatment with N + A (Figure S9). Both the dose‐gradient and time‐gradient assays indicated that API treatment can effectively decrease the expression level and enzyme activity of CD38 in the three musculoskeletal cells (Figures S10 and S11). Furthermore, to investigate the relationship between apigenin and CD38, we conducted a molecular docking assay and found that CD38 is a potential target of apigenin (Figure S12). Additionally, the SA‐β‐galactosidase staining assay revealed that the NAD⁺ reservoir generated by the N + A formulation effectively reduced the proportion of senescent cells among the musculoskeletal precursor cells (Figure 2b and Figure S13). Notably, senescent cells secrete a wide range of bioactive molecules, collectively termed the senescence‐associated secretory phenotype (SASP), which can inhibit cell proliferation and disrupt normal differentiation processes (Wang et al. 2024). Heatmap analysis demonstrated that treatment with the N + A formulation suppressed SASP components such as the marker of double‐strand breaks γ‐H2ax and pro‐inflammatory chemokine CXCL8 (Figure 2c). Stress factors activate the DNA damage response (DDR) pathway, subsequently initiating either the classical P53‐P21^CIP1‐CDK2 or the P16^INK4A‐CDK4/6 signaling pathways, resulting in cell cycle arrest and cellular senescence (Groelly et al. 2023). Western blot and immunofluorescence staining demonstrated that expression levels of P53, P21, and P16 within the nuclear were significantly reduced following treatment with N + A (Figure 2d,e and Figures S14 and S15). Given that mitochondrial dysfunction is one of the primary triggers of cellular senescence, we further investigated the effects of the N + A formulation on mitochondrial function across the three types of precursor cells (Gallage and Gil 2016). Treatment with N + A formulation restored the impaired mitochondrial membrane potential (MMP) and promoted mitochondrial ATP biosynthesis (Figure 2f and Figure S16). Furthermore, N + A treatment effectively increased both the basal and maximal respiration rates in aged cells and significantly enhanced mitochondrial ATP production (Figure S17). Collectively, these findings demonstrate that treatment with the N + A formulation delayed cellular aging and restored mitochondrial function.

Given the robust anti‐aging effects of the N + A formulation in musculoskeletal precursor cells, we subsequently investigated its potential to restore tri‐lineage differentiation capacity under aging conditions. In the context of chondrogenic differentiation, N + A treatment enhanced the secretion of cartilage extracellular matrix (C‐ECM) such as glycosaminoglycans (GAGs) and covalently bound proteoglycans (aggrecan) (Figure 3a). Additionally, N + A treatment enhanced cartilage‐enriched type II collagen (COLII) synthesis at both transcriptional and translational levels. Mechanistically, our findings demonstrated that N + A treatment upregulated the transcription factor SOX9, thereby activating the transcriptional activity of the COLII‐encoding gene Col2a1 (Figure 3b–d and Figure S18). In terms of osteogenic differentiation, N + A treatment promoted the activity of alkaline phosphatase (ALP)—a biomarker of early‐stage osteogenesis—and the deposition of calcium nodules (an indicator of late‐stage osteogenesis) for ossified extracellular matrix (O‐ECM) synthesis (Figure 3e). The marker proteins of osteogenic differentiation, including osteoprotegerin (OPG, encoded by Tnfrsf11b) and osteocalcin (OCN, encoded by Bglap), were upregulated following treatment with N + A formulation. Additionally, we validated that the transcriptional activation mediated by the transcription factors RUNX family transcription factor 2 (RUNX2) and SP7 regulated the increased osteogenesis (Figure 3f–h and Figure S19). Regarding myogenic differentiation, N + A treatment facilitated both myogenic differentiation and myotube formation, as evidenced by increased myotube width and diameter (Figure 3i). Skeletal muscle is typically classified into two major fiber types: slow‐twitch fibers (which express Myh7‐encoded β‐MyHC) and fast‐twitch fibers (which include Myh2‐encoded MyHC IIa, Myh4‐encoded MyHC IIb, and Myh1‐encoded MyHC IIX). These fiber types exhibit distinct contractile properties and metabolic profiles (Motohashi et al. 2019). In N + A‐treated cells, the expression of myogenic differentiation markers, such as the myosin heavy chain (MHC), was upregulated. Notably, treatment with N + A enhanced the expression of myogenic transcription factors, including myogenic differentiation 1 (MyoD1) and myogenin (MYOG), thereby inducing the transcriptional activation of multiple genes within the MyHC superfamily (Figure 3j–l and Figure S20). Taken together, these findings reveal that N + A effectively promoted the tri‐lineage differentiation of musculoskeletal precursor cells.

Owing to the excellent anti‐senescent and pro‐differentiation effects observed in the previous in vitro studies, we subsequently evaluated the therapeutic effects of orally administered N + A formulation on musculoskeletal degeneration including cartilage, bone, and muscle, in a naturally aged mouse model. With advancing age, articular cartilage undergoes pronounced surface erosion accompanied by structural disorganization of chondrocytes. Meanwhile, the proportion of SOX9‐ or COLII‐positive chondrocytes was significantly reduced in aging mice. Treatment with N + A reduced the OARSI score, enhanced the hyaline cartilage to calcified cartilage ratio, and reactivated the SOX9‐COLII pathway, demonstrating attenuated degeneration of the cartilage (Figure 4a,b and Figure S21a). Regarding either age‐related OP or senile OP, oral administration of the N + A restored the proportion of trabecular bone area, as demonstrated by H&E staining. Micro‐computed tomography (micro‐CT, μ‐CT) analysis demonstrated that both cancellous bone parameters and cortical bone indices were significantly enhanced following treatment with N + A formulation. Additionally, the expression of the RUNX2‐OCN signaling pathway was upregulated after the treatment with N + A formulation (Figure 4c,d and Figure S21b–e). Furthermore, N + A treatment ameliorated the impaired skeletal muscle morphology, as indicated by increased myofiber area and reduced fibrotic area. The expression levels of various myogenic markers within the skeletal muscle were upregulated in aged mice following treatment with N + A formulation (Figure 4e,f and Figure S21f,g). Furthermore, in vivo immunofluorescence staining revealed that administration of the N + A formulation significantly reduced senescence‐associated biomarkers in cartilage, bone, and muscle tissues. These findings supported the potent and robust anti‐aging effects of N + A formulation on the musculoskeletal system (Figure 4g,h and Figure S22a–c). Ultimately, we comprehensively evaluated the functional parameters of the N + A‐treated aged mice. Following treatment with the N + A formulation, the gross morphology of the muscle was restored, characterized by normalized gait parameters and improved paw grip strength (Figure S23a,b). The open‐field locomotor assay demonstrated that treatment with N + A formulation enhanced motor function by improving multiple aspects of movement vigor including duration, distance, and velocity (Figure 4i and Figure S23c,d). Collectively, these findings reveal that oral administration of N + A prevented musculoskeletal degeneration under aging conditions and promoted functional recovery.

To investigate the mechanisms underlying N + A formulation‐mediated anti‐aging effects in senile musculoskeletal tissues, given the established role of sirtuin 3 (SIRT3) in regulating the NAD⁺ cycling pathway, as well as maintaining mitochondrial redox balance and energy production, we generated a mitochondrial SIRT3 global knockout mouse model (Sirt3^−/−) using CRISPR‐Cas9 technology (Figure 5a,b). The wild‐type (WT) and SIRT3‐deficient aged mice were orally administered either saline or the N + A formulation. The NAD⁺/NADH quantification assay demonstrated that the N + A formulation significantly elevated both the NAD⁺ levels and the NAD⁺/NADH ratio in the peripheral blood of the SIRT3‐deficient aged mice (Figure 5c). In the context of cartilage protection, SIRT3 knockout diminished the beneficial effects of N + A treatment on articular cartilage integrity and C‐ECM marker expression. Additionally, the senescence‐related proteins P53, P21, and P16 associated with DNA damage were upregulated in the articular cartilage of N + A‐treated mice in the absence of SIRT3 (Figure 5d–f and Figure S24a–c). Regarding bone mass recovery, the impaired clearance of senescent skeletal stem cells caused by SIRT3 deletion in vivo partially attenuated the beneficial effects of N + A on both cortical and cancellous bone parameters (Figure 5g–i and Figure S25a–d). The improvement in the structure of the skeletal muscle induced following treatment with N + A formulation was attenuated in SIRT3‐deficient aged mice, accompanied by exacerbated muscular senescence (Figure 5j–l and Figure S26a–c). In contrast, in aged mice with SIRT3 knockout, the protective effects of N + A formulation on motor function recovery were partially attenuated, as evidenced by declines in both muscle strength and physical activity (Figure S27a–d). Moreover, assays of the acetylation level of mitochondrial proteins demonstrated that N + A decreased the acetylation level of mitochondrial proteins, which may be partially attributed to the enhanced activity of the mitochondrial deacetylase SIRT3 (Figure S28). Taken together, these results show that treatment with N + A formulation delayed musculoskeletal aging and functional impairment, partially through the SIRT3 signaling pathway.

Recent research establishes that gut microbiota dysbiosis and ecological imbalances are crucial hallmarks of aging, yet they are underexplored mechanisms (López‐Otín et al. 2023). Given the oral route of administration, the intestine is expected to be the primary organ to initially respond to N + A treatment. Therefore, we investigated age‐related pathological changes in both the ileum and colon of aging mice following treatment with the N + A formulation. Histological staining results revealed that N + A treatment increased the number of goblet cells in the intestine, suggesting an enhanced restorative capacity of the gut. Following N + A treatment, the proportion of cells positive for the tight junction protein ZO‐1 (Tjp1) and mucin 2 (Muc2) was significantly increased, indicating a potential restoration of intestinal mucosal barrier integrity (Figure S29). Based on these findings, we subsequently assessed the fecal microbiota composition in aging and N + A‐treated aging mice using 16S rRNA sequencing. The results revealed that N + A treatment significantly enhanced microbial alpha diversity, as evidenced by the increased Shannon, Chao1, and Simpson indices (Figure S30). Additionally, there was a significant difference in microbial beta diversity between the two groups (Figure S31). Subsequently, the composition of the bacterial community was characterized across multiple taxonomic levels including genus, family, order, class, and phylum (Figures S32 and S33). Notably, following treatment with the N + A regimen, at the genus level, the relative abundances of Alistipes, Bacteroides, Rikenellaceae_RC9_gut_group, and Prevotellaceae_UCG‐001 were decreased, whereas those of Ruminococcus, Oscillibacter, Turicibacter, Coriobacteriaceae_UCG‐002, UCG‐009, and [Ruminococcus]_torques_group were significantly increased (Figure 6a and Figure S34). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that treatment with N + A downregulated the MAPK signaling pathway and ferroptosis; additionally, it inhibited the degradation of GAGs (Figure 6b and Figure S35). Based on these findings, we proposed that the gut microbiota might contribute to the musculoskeletal protective effects mediated by the N + A formulation. To validate this hypothesis, we performed microbiota depletion experiments combined with FMT, whereby gut microbiota were transferred from three donor groups (aged mice, aged mice that had received N + A treatment, and young mice) to recipient aged mice. Compared to the control group, FMT from aged mice exacerbated senescence in cartilage (Figure 6c–e and Figure S36a–c), bone (Figure 6f–h and Figure S37a–d), and muscle tissues (Figure 6i–k and Figure S38a–c), thereby inducing profoundly severe musculoskeletal degeneration. Conversely, FMT from N + A‐treated aged mice reversed senescence and restored regenerative functions among the three tissues, with the effect closely resembling that of FMT from young mice (Figure S39a–d). NMN supplementation ameliorated the disruptions of intestinal homeostasis through the restoration of deoxycholic acid for the prevention of intestinal infections (Fang et al. 2023). Apigenin directly binds to FGR, which contributes to the enhanced phagocytosis of bacteria by macrophages, thereby enhancing the antibacterial ability associated with aging (Gu et al. 2025). Moreover, it is reported that gut microbiota mediates the deamidation of orally administered NMN and its conversion to nicotinic acid (NA) for NAD generation through the Preiss‐Handler pathway (Yaku et al. 2025). Recent views have demonstrated that NAD precursors not only sustain an increase in systemic NAD⁺ levels but also serve as a potent modulator of gut health (Christen et al. 2026). Collectively, these findings reveal that FMT from N + A formulation‐treated mice ameliorated cartilage, bone, and muscle senescent phenotype in aged mice.

Gut microbiota plays a significant role in the regulation of intestinal homeostasis by producing a wide range of metabolites. These metabolites can penetrate the intestinal mucosa and, in some cases, traverse the intestinal barrier to reach distant organs via the circulatory system (Ezzine et al. 2022). To elucidate key metabolites associated with N + A treatment, untargeted metabolomic profiling was conducted on stool samples collected from both groups. Following administration of the N + A formulation in aged mice, a total of 292 metabolites were downregulated, whereas 403 were upregulated (Figure 7a,b and Figures S40 and S41). The KEGG enrichment analysis revealed that the sphingolipid signaling pathway and sphingolipid metabolism were significantly influenced by the N + A administration. Notably, the sphingolipid metabolism pathway exhibited differential characteristics as revealed by the metabolomic sequencing results (Figure 7c). After screening sphingolipid‐related metabolites from the differential metabolite analysis, two metabolites—phosphoethanolamine (PE) and phytosphingosine (PHS)—were identified as potential candidates. Given the relatively higher abundance and the potential negative effects of PE on senescence (Tighanimine et al. 2024), the PHS was selected as the therapeutic option for degenerative musculoskeletal disorders (Figure 7d). Combined therapeutic strategy of PHS and gamma‐radiation may enhance the sensitivity of cancer cells to radiotherapy (Park et al. 2005). Similarly, supplementation with Flavonifractor plautii ‐biosynthesized PHS alleviated metabolic disorders by enhancing PHS levels along the gut–hepatic axis (Li et al. 2025). The co‐analysis of microbes and metabolites demonstrated a strong positive correlation between Coriobacteriaceae_UCG‐002 (Cintado et al. 2025) and Ruminococcus (Vereecke and Elewaut 2017) with PHS (correlation coefficient > 0.5 and p value < 0.01), indicating that these intestinal microbiotas may serve as potential contributors to PHS (Figure S42). Ultimately, we evaluated the effects of orally administered PHS on mitigating age‐related musculoskeletal impairments. Treatment with PHS significantly alleviated cartilage degeneration (Figure 7e–g and Figure S43a–c), bone loss (Figure 7h–j and Figure S44a–d), and muscle atrophy (Figure 7k–m and Figure S45a–c), accompanied by the suppression of the P53‐P21 and P16 signaling pathways. Besides architectural improvements, treatment with PHS significantly enhanced muscle strength and mobility performance, especially in aged mice (Figure S46a–c). Taken together, these findings indicate that intestinal metabolite PHS may serve as a promising candidate for the rejuvenation of musculoskeletal degeneration.

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.