Nicotinamide adenine dinucleotide (NAD⁺) metabolism and inflammatory diseases are being increasingly linked. Serum NAD⁺ levels were significantly elevated during inflammation. Murine serum NAD⁺ ranges from 0.1 to 0.5 micromoles physiologically, but during inflammation, levels of NAD⁺ in mice could increase up to 10 micromoles ^[1][16]. NAD⁺ has been implicated in the modulation of acute systemic inflammation, as it exerts regulatory control over immune and metabolic pathways in the context of sepsis ^[2][56]. The NAD⁺ salvage pathway is crucial for the inflammatory response to mount an appropriate response in LPS-induced monocytes ^[3][57]. In addition, researchers have found that NAD⁺ may maintain inflammatory states, activated immune systems, and cytokine storms by controlling NF-κB transcriptional activity ^[4][5][6][38,58,59].

NAD⁺ metabolism maintains intestinal homeostasis. Serum NAD⁺ levels increased three-fold in inflammatory bowel disease (IBD) patients compared to healthy people ^[7][18]. Metabolomic analysis of UC patients showed that “nicotinate and nicotinamide metabolism” was the most significant metabolic feature of UC-inflamed tissues, with a decrease in NAD⁺ levels and elevated levels of its metabolites NAM and ADPr. This suggests that NAD⁺ depletion in UC may result from increased NAD⁺ catabolism ^[8][17]. Although NAMPT is an enzyme in the NAD⁺ salvage pathway, proteomic profiles of proteins involved in NAD⁺ metabolism in IBD patients show that it is pro-inflammatory and tumorigenic ^[6][59].

IBD requires a balance between NAD⁺ biosynthesis and consumption to maintain intestinal homeostasis ^[7][18]. However, its pathogenesis is unknown ^[9][6]. Elevated activity of NAD⁺-consuming enzymes in IBD can cause gut inflammation ^[7][18]. In vitro and in vivo experiments have shown that NAD⁺ administration improves inflammation-related intestinal permeability by inhibiting NF-κB ^[10][60]. The gut microbiota provides alternative NAD⁺ synthesis pathways and enhances NAM or NR supplementation ^[11][61]. Therefore, biomedicine could utilize the gut microbiota to treat IBD by modulating NAD⁺ metabolism during intestinal inflammation. It was observed that NMN and NAD⁺ supplementation improved intestinal stem cell function in aged mice via mTOR and SIRT1 ^[12][13][62,63], and NMN prevented intestinal organoids from aging in old mice ^[14][64]. These results suggest that methods that increase NAD⁺ levels or activate sirtuins could protect the gut barrier and prevent IBD from starting or worsening. Nevertheless, it is important to note that augmenting NAD⁺ levels alone is not sufficient for preserving intestinal homeostasis entirely. Experimental evidence has shown that reducing NAD⁺ and SIRT1 levels in the colon of mice, using the olefin receptor agonist norisopodine (which expands epigenetic Treg cells as an aryl hydrocarbon receptor agonist), alleviates DSS-induced colitis ^[15][65]. As a result, drugs targeting the NAD⁺ pathway may help manage IBD.

The NAD⁺ metabolizing enzymes sirtuins, CD38, PARPs, NNMT, and NAMPT are linked to the inflammatory processes in IBD (Table 1).

Mitochondria regulate cell metabolism and viability and maintain cell integrity and function ^[86][133]. Recent research has shown that mitochondria are essential for coordinating innate and adaptive immune responses. Inflammation can begin with mitochondrial dysfunction and ROS production ^[89][218]. Elevated ROS levels in the gut activate inflammatory and cell death pathways ^[90][219]. Therefore, targeting ROS in cells could reduce damage to the gut barrier caused by inflammation.

The process of NAD⁺ metabolism is intricately intertwined with mitochondrial function. NAD⁺ serves as a critical intermediate in cellular metabolism and acts as an enzymatic cofactor in redox reactions, including glycolysis, the tricarboxylic acid (TCA) cycle, and fatty acid oxidation (FAO) ^[9][6]. These reactions produce NADH, an electron donor from the nicotinamide of NAD⁺ that synthesizes ATP via mitochondrial oxidative phosphorylation ^[91][9]. Mitochondrial function and energy substitution depend on the NAD⁺/NADH ratio, regulated by mitochondrial electron flux ^[92][36]. Reduced NAD⁺ levels impair filamentous cell activity, epigenetic chromatin structure ^[93][220], mitochondrial metabolism, oxidative stress, and ATP production, thereby promoting inflammation and cellular damage ^[94][221]. Cameron et al. found that LPS induction activated mitochondrial ROS production, leading to DNA damage, PARP activation, and NAD⁺ depletion in macrophages ^[64][113].

Mitochondrial dysfunction is linked to defects in NAD⁺ metabolism. NAD⁺ supplementation improves mitochondrial performance and reduces mitochondrial damage and ROS production ^[95][96][97][46,177,222]. The NAD⁺ precursor NAM restores the NAD⁺/NADH balance and reduces IFB-γ production and Th1 differentiation in vitro ^[98][223]. Providing NAD⁺ precursors and targeting NAD⁺ biosynthesis/degradation enzymes could reverse mitochondrial dysfunction. This suggests that NAD⁺ metabolism plays a role in regulating mitochondrial function ^[9][99][6,224]. Minhas et al. found that macrophages synthesize NAD⁺ via the kynurenine pathway. The authors also reported that genetic ablation (in Ido^−/− and Qprt^−/− mice) or pharmacological disruption (1-methyl-L-tryptophan and phthalic acid) reduced intracellular NAD⁺ concentrations, impairing mitochondrial respiration and increasing glycolysis in vitro ^[100][225]. These metabolic changes increase CD86 and CD64 expression, decrease CD206 and CD23 expression, and impair phagocytosis ^[100][225]. The exogenous NAD⁺ precursor NMN restored mitochondrial respiratory parameters and pro-inflammatory markers ^[101][19]. NAD⁺ levels also regulate mitochondrial metabolism via sirtuins ^[16][66]. Low NAD⁺ levels decrease SIRT1 and SIRT3 activity, decrease vital mitochondrial activity, alter mitochondrial morphology, and hyperacetylate mitochondrial proteins ^{[52][102][103][104]}[102,168,226,227]. Hyperactivated PARP1 reduces mitophagy due to SIRT1 impairment ^[96][177].

Mitochondrial dysfunction has been linked to NAD⁺ deficiency. NADH, the reduced form of NAD⁺, is oxidized back to NAD⁺ in complex I of the mitochondrial electron transport chain (ETC) and provides metabolic energy ^[9][6]. In addition, mitochondrial dysfunction decreases NAD⁺/NADH ratio and impairs SIRT3 activity ^[104][227]. The NAD⁺/NADH ratio is imbalanced in CD4+ T cells lacking mitochondrial transcription factor A (Tfam) that controls mitochondrial DNA expression. To compensate for mitochondrial dysfunction, CD4+ T cells lacking Tfam switch to glycolysis, decrease NAD⁺, increase the pro-inflammatory Th1 phenotype, secrete IFB-Γ and TNF-α, and inhibit IL-10 ^[105][228]. Linezolid, a ribosome-targeting antibiotic, affects the mitosome function and cell electron transport chain of Th17 cells. Mitochondrial respiration impairs NAD⁺ regeneration, lowering the NAD⁺/NADH ratio and decreasing Th17 effector function ^[106][229].

Understanding the relationship between NAD⁺ and mitochondria could help explain the pathophysiology of IBD. Inflammatory tissues have higher levels of NAM and ADR and lower levels of NAD⁺. Mitochondrial status and NAD⁺ metabolism are interdependent, and changes in the organism affect inflammation. Mitochondrial dysfunction is a major cause of IBD pathogenesis ^{[107][108][109]}[230,231,232]. The intestinal mucosa of IBD is characterized by hypoxia and increased oxidative stress implicated in various genes involved in mitochondrial function, such as CUL2, LACC1, and NADPH oxidase ^{[110][111][112][113][114]}[233,234,235,236,237]. A recent metabolic analysis showed NAD⁺ metabolic dysregulation and altered mitochondrial status in UC patients. The NAD⁺/NAM ratio decreased in patients with active UC, distinguishing the degree of inflammation from UC. UC alters mitochondria, resulting in a lower mitochondrial density and number in colon cells ^[8][17]. These findings suggest a link between mitochondrial dysfunction and inflammation in UC and NAD⁺ metabolism.

The intestinal epithelium forms a selective barrier that blocks toxicants and microbes from the lumen but allows nutrient absorption ^[115][238]. The intestinal epithelial barrier relies on the tight junction (TJ), a circumferential protein complex at opposing apical/basolateral cell junctions ^[116][239]. The occludin and claudin transmembrane protein families form the TJ ^[117][240] and prevent paracellular transport ^[118][241]. Inflammatory diseases, such as IBD, cholestasis, hemorrhagic shock, and sepsis, damage the intestinal epithelial barrier ^[119][242].

Extracellular NAD⁺ prevented activation, induced nitric oxide synthase, increased NO production, and improved epithelial permeability in inflammatory epithelial cells ^[120][243]. NAD⁺ improved intestinal mucosal permeability in LPS-induced CACO-2 cells ^[10][60], indicating that NAD⁺ can reduce the structural and functional changes in pro-inflammatory intestinal epithelial cells. Another study found that the overexpression of SIRT1 inhibited LPS-induced pro-inflammatory cytokines (IL-6, IL-8, and TNF-α), impaired the intestinal epithelial barrier, and reduced the inflammatory response and intestinal epithelial barrier dysfunction ^[121][244]. Quinone oxidoreductase 1 (NQO1) reduces quinone metabolites using NADH as an electron donor ^[122][123][245,246], regulating NAD and NADH in various cellular systems. Quinone oxidoreductase, also known as the antioxidant flavocyanin ^[124][247], clears ROS. NQO1 promotes the barrier function of the intestinal epithelium in mice by regulating the transcription of tight junction molecules. A lack of NQO1 can exacerbate colon inflammation ^[125][248]. As mentioned above, the intestinal cells have NAD⁺ receptors. These receptors could be drug targets to treat intestinal epithelium in an inflammatory environment.

Adult stem cells use glycolysis as an energy source to avoid oxidative stress pathways during mitochondrial respiration ^[126][249]. However, mitochondrial defects are a common cause of adult stem cell senescence, as oxidative respiration is essential for their function in old age ^[127][250]. Early aging mediated by DNA repair defects degrades NAD⁺ through PARP and the loss of mitochondrial homeostasis, reducing MuSC numbers and self-renewal ^[96][177]. The activation of NAD⁺ and SIRT1 can repair mitochondrial defects in aging stem cells and DNA repair-deficient cells. Reduced SIRT3 or SIRT7 activity in hematopoietic stem cells impairs the regenerative capacity of aged mouse hematopoietic stem cells (HSCs) ^[128][129][251,252]. Muscle stem cells (MuSC) have lower NAD⁺ levels and SIRT1 activity with age, contributing to the decline in NAD^{+ [130]}[214]. NR, an NAD⁺ precursor, improves muscle, neural, and melanocyte stem cell function in aged mice, rejuvenating MuSCs and extending lifespan ^[131][253].

The intestinal epithelium is rapidly renewed by the ISC. Early ISC aging research focused on the intestinal epithelium of fruit flies ^[132][254]. Drosophila gut stem cells proliferate with age due to environmental changes or tissue damage. Mammalian ISCs are mainly Lgr5-expressing cells at crypt bases ^[133][134][255,256]. Recent studies have reported a decline in ISC function in mammals with advancing age, thereby highlighting the impact of aging on ISC dynamics. Interestingly, it has been observed that the modulation of Wnt signaling pathways can ameliorate the impaired ISC function commonly observed in older individuals ^[135][136][257,258]. Paneth cells support ISCs, regardless of age. In contrast, ISCs cells in mice become less active with age ^[137][259]. NAD⁺ supplementation with precursor NR can repair age-related ISC deficiencies and restore ISC quantity and vitality ^[12][62]. Compared to young mice, NR treatment reduced the sensitivity of aged mice to DSS, suggesting that NR can repair the damage in the gut of old mice by restoring the ISC pool ^[12][62]. Therefore, increasing NAD⁺ levels can activate ISCs in the intestine, speeding up intestinal barrier repair and promoting the recovery of IBD mucosa.