Mitochondrial responses to intracellular Ca2+ release following infrared stimulation.

Many studies of Ca2+ effects on mitochondrial respiration in intact cells have used electrical and/or chemical stimulation to elevate intracellular [Ca2+], and have reported increases in [NADH] and increased ADP/ATP ratios as dominant controllers of respiration. This study tested a different form of stimulation: brief temperature increases produced by pulses of infrared light (IR, 1,863 nm, 8-10°C for ∼5 s). Fluorescence imaging techniques applied to single PC-12 cells in low µM extracellular [Ca2+] revealed IR stimulation-induced increases in both cytosolic (fluo5F) and mitochondrial (rhod2) [Ca2+]. IR stimulation increased O2 consumption (porphyrin fluorescence), and produced an alkaline shift in mitochondrial matrix pH (Snarf1), indicating activation of the electron transport chain (ETC). The increase in O2 consumption persisted in oligomycin, and began during a decrease in NADH, suggesting that the initial increase in ETC activity was not driven by increased ATP synthase activity or an increased fuel supply to ETC complex I. Imaging with two potentiometric dyes [tetramethyl rhodamine methyl ester (TMRM) and R123] indicated a depolarizing shift in ΔΨm that persisted in high [K+] medium. High-resolution fluorescence imaging disclosed large, reversible mitochondrial depolarizations that were inhibited by cyclosporin A (CSA), consistent with the opening of transient mitochondrial permeability transition pores. IR stimulation also produced a Ca2+-dependent increase in superoxide production (MitoSox) that was not inhibited by CSA, indicating that the increase in superoxide did not require transition pore opening. Thus, the intracellular Ca2+ release that follows pulses of infrared light offers new insights into Ca2+-dependent processes controlling respiration and reactive oxygen species in intact cells.NEW & NOTEWORTHY Pulses of infrared light (IR) provide a novel method for rapidly transferring Ca2+ from the endoplasmic reticulum to mitochondria in intact cells. In PC12 cells the resulting ETC activation was not driven by increased ATP synthase activity or NADH. IR stimulation produced a Ca2+-dependent, reversible depolarization of ΔΨm that was partially blocked by cyclosporin A, and a Ca2+-dependent increase in superoxide that did not require transition pore opening.

Deacetylase sirtuin 2 negatively regulates myeloid-derived suppressor cell functions in allograft rejection.

Although myeloid-derived suppressor cells (MDSCs) are critical for allograft survival, their regulatory mechanism remains unclear. Herein, our results showed that metabolism sensor sirtuin 2 (SIRT2) negatively regulates the functions of MDSCs in inducing allogeneic skin graft rejection. Genetic deletion of SIRT2 in myeloid cells (Sirt2Δmye) increased the number of CD11b+Gr1+ MDSCs in bone marrow, spleens, draining lymph nodes, and allografts, inhibited the production of proinflammatory cytokine tumor necrosis factor ɑ, enhanced the production of anti-inflammatory cytokine interleukin 10, and potentiated the suppressive activation of MDSCs in prolonging allograft skin survival. C-X-C motif chemokine receptor 2 is critical for mediating the recruitment and cytokine production of MDSCs induced by SIRT2. Mechanistically, Sirt2Δmye enhanced NAD+ levels, succinate dehydrogenase subunit A (SDHA) activities, and oxidative phosphorylation (OXPHOS) levels in MDSCs after transplantation. Pharmacologically blocking nicotinamide phosphoribosyltransferase effectively reverses the production of cytokines and suppressive activities of MDSC induced by Sirt2Δmye. Blocking OXPHOS with knockdown of SDHA or pharmacological blocking of SDHA significantly restores Sirt2Δmye-mediated stronger MDSC suppressive activity and inflammatory factor productions. Thus, our findings identify a previously unrecognized interplay between NAD+ and SDH-mediated OXPHOS metabolic pathways in regulating MDSC functions induced by the metabolic sensor SIRT2 in allogeneic transplantation.

Effects of NAD+ precursors on blood pressure, C-reactive protein concentration and carotid intima-media thickness: A meta-analysis of randomized controlled trials.

BACKGROUND: There are contradictory effects regarding the effect of NAD+ precursor on blood pressure and inflammation. In order to obtain a better viewpoint from them, this study aimed to comprehensively investigate the effects of NAD+ precursor supplementation on blood pressure, C-reactive protein (CRP) and carotid intima-media thickness (CIMT). METHODS: PubMed/MEDLINE, Web of Science, SCOPUS and Embase databases were searched using standard keywords to identify all controlled trials investigating the effects of NAD+ precursor on blood pressure, CRP and CIMT. Pooled weighted mean difference (WMD) and 95% confidence intervals (95% CI) were achieved by random-effects model analysis for the best estimation of outcomes. RESULTS: Twenty-nine articles (with 8664 participants) were included in this article. Results from meta-analyses of RCTs from random-effects models indicated a significant reduction in systolic (SBP) (weighted mean difference (WMD): -2.54 mmHg, p < .001) and diastolic blood pressure (DBP) (WMD: -2.15 mmHg, p < .001), as well as in CRP (WMD: -.93 mg/L, 95% CI -1.47 to -.40, p < .001) concentrations and CIMT (WMD: -.01 mm, 95% CI -.02 to -.00, p = .005) with the NAD+ precursors supplementation compared with the control group. In addition, a greater effect of supplementation with NAD+ precursors in reducing blood pressure (BP) were observed with the highest dose (≥2 g) and duration of the intervention (>12 weeks), as well as with NA supplementation when compared to NE. CONCLUSIONS: Overall, these findings suggest that NAD+ precursor supplementation might have a beneficial effect on cardiovascular risk factors such as BP, CRP concentration and CIMT.

NAD+-boosting compounds enhance nitric oxide production and prevent oxidative stress in endothelial cells exposed to plasma from patients with COVID-19.

SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), induces vascular endothelial dysfunction, but the mechanisms are unknown. We tested the hypothesis that the "circulating milieu" (plasma) of patients with COVID-19 would cause endothelial cell dysfunction (characterized by lower nitric oxide (NO) production), which would be linked to greater reactive oxygen species (ROS) bioactivity and depletion of the critical metabolic co-substrate, nicotinamide adenine dinucleotide (NAD+). We also investigated if treatment with NAD+-boosting compounds would prevent COVID-19-induced reductions in endothelial cell NO bioavailability and oxidative stress. Human aortic endothelial cells (HAECs) were exposed to plasma from men and women (age 18-85 years) who were hospitalized and tested positive (n = 34; 20 M) or negative (n = 13; 10 M) for COVID-19. HAECs exposed to plasma from patients with COVID-19 also were co-incubated with NAD+ precursors nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN). Acetylcholine-stimulated NO production was 27% lower and ROS bioactivity was 54% higher in HAECs exposed to plasma from patients with COVID-19 (both p < 0.001 vs. control); these responses were independent of age and sex. NAD+ concentrations were 30% lower in HAECs exposed to plasma from patients with COVID-19 (p = 0.001 vs. control). Co-incubation with NR abolished COVID-19-induced reductions in NO production and oxidative stress (both p > 0.05 vs. control). Co-treatment with NMN produced similar results. Our findings suggest the circulating milieu of patients with COVID-19 promotes endothelial cell dysfunction, characterized by lower NO bioavailability, greater ROS bioactivity, and NAD+ depletion. Supplementation with NAD+ precursors may exert a protective effect against COVID-19-evoked endothelial cell dysfunction and oxidative stress.

NMNAT2 is downregulated in glaucomatous RGCs, and RGC-specific gene therapy rescues neurodegeneration and visual function.

The lack of neuroprotective treatments for retinal ganglion cells (RGCs) and optic nerve (ON) is a central challenge for glaucoma management. Emerging evidence suggests that redox factor NAD+ decline is a hallmark of aging and neurodegenerative diseases. Supplementation with NAD+ precursors and overexpression of NMNAT1, the key enzyme in the NAD+ biosynthetic process, have significant neuroprotective effects. We first profile the translatomes of RGCs in naive mice and mice with silicone oil-induced ocular hypertension (SOHU)/glaucoma by RiboTag mRNA sequencing. Intriguingly, only NMNAT2, but not NMNAT1 or NMNAT3, is significantly decreased in SOHU glaucomatous RGCs, which we confirm by in situ hybridization. We next demonstrate that AAV2 intravitreal injection-mediated overexpression of long half-life NMNAT2 mutant driven by RGC-specific mouse γ-synuclein (mSncg) promoter restores decreased NAD+ levels in glaucomatous RGCs and ONs. Moreover, this RGC-specific gene therapy strategy delivers significant neuroprotection of both RGC soma and axon and preservation of visual function in the traumatic ON crush model and the SOHU glaucoma model. Collectively, our studies suggest that the weakening of NMNAT2 expression in glaucomatous RGCs contributes to a deleterious NAD+ decline, and that modulating RGC-intrinsic NMNAT2 levels by AAV2-mSncg vector is a promising gene therapy for glaucomatous neurodegeneration.

Nicotinamide prevention in diabetes-induced alterations in the rat liver.

Objective. The study was performed to elucidate whether nicotinamide (NAm) can attenuate the diabetes-induced liver damage by correction of ammonia detoxifying function and disbalance of NAD-dependent processes in diabetic rats. Methods. After four weeks of streptozotocin-induced diabetes, Wistar male rats were treated for two weeks with or without NAm. Urea concentration, arginase, and glutamine synthetase activities, NAD+ levels, and NAD+/NADH ratio were measured in cytosolic liver extracts. Expression of parp-1 gene in the liver was estimated by quantitative polymerase chain reaction and PARP-1 cleavage evaluated by Western blotting. Results. Despite the blood plasma lipid peroxidation products in diabetic rats were increased by 60%, the activity of superoxide dismutase (SOD) was reduced. NAm attenuated the oxidative stress, but did not affect the enzyme activity in diabetic rats. In liver of the diabetic rats, urea concentration and arginase activity were significantly higher than in the controls. The glutamine synthetase activity was decreased. Decline in NAD+ level and cytosolic NAD+/NADH ratio in the liver of diabetic rats was observed. Western blot analysis demonstrated a significant up-regulation of PARP-1 expression accompanied by the enzyme cleavage in the diabetic rat liver. However, no correlation was seen between mRNA expression of parp-1 gene and PARP-1 protein in the liver of diabetic rats. NAm markedly attenuated PARP-1 cleavage induced by diabetes, but did not affect the parp-1 gene expression. Conclusions. NAm counteracts diabetes-induced impairments in the rat liver through improvement of its detoxifying function, partial restoration of oxidative stress, NAD+ level, normalization of redox state of free cytosolic NAD+/NADH-couples, and prevention of PARP-1 cleavage.

Isoamericanin A improves lipopolysaccharide-induced memory impairment in mice through suppression of the nicotinamide adenine dinucleotide phosphateoxidase-dependent nuclear factor kappa B signaling pathway.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Isoamericanin A (ISOA) is a natural lignan possessing great potential for AD treatment. This study investigated the efficacy of ISOA on memory impairments in the mice intrahippocampal injected with lipopolysaccharide (LPS) and the underlying mechanism. Y-maze and Morris Water Maze data suggested that ISOA (5 and 10 mg/kg) ameliorated short- and long-term memory impairments, and attenuated neuronal loss and lactate dehydrogenase activity. ISOA exerted anti-inflammatory effect demonstrating by the reduction of ionized calcium-binding adapter molecule 1 positive cells and suppression of marker protein and pro-inflammation cytokines expressions induced by LPS. ISOA suppressed the nuclear factor kappa B (NF-κB) signaling pathway by inhibiting IκBα phosphorylation and NF-κB p65 phosphorylation and nuclear translocation. ISOA inhibited superoxide and intracellular reactive oxygen species accumulation by reducing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, demonstrating by suppressing NADP+ and NADPH contents, gp91phox expression, and p47phox expression and membrane translocation. These effects were enhanced in combination with NADPH oxidase inhibitor apocynin. The neuroprotective effect of ISOA was further proved in the in vitro models. Overall, our data revealed a novel pharmacological activity of ISOA: ameliorating memory impairment in AD via inhibiting neuroinflammation.

Evaluation of Antibacterial Activity of Thiourea Derivative TD4 against Methicillin-Resistant Staphylococcus aureus via Destroying the NAD+/NADH Homeostasis.

To develop effective agents to combat bacterial infections, a series of thiourea derivatives (TDs) were prepared and their antibacterial activities were evaluated. Our results showed that TD4 exerted the most potent antibacterial activity against a number of Staphylococcus aureus (S. aureus), including the methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis and Enterococcus faecalis strains, with the minimum inhibitory concentration (MIC) at 2-16 µg/mL. It inhibited the MRSA growth curve in a dose-dependent manner and reduced the colony formation unit in 4× MIC within 4 h. Under the transmission electron microscope, TD4 disrupted the integrity of MRSA cell wall. Additionally, it reduced the infective lesion size and the bacterial number in the MRSA-induced infection tissue of mice and possessed a good drug likeness according to the Lipinski rules. Our results indicate that TD4 is a potential lead compound for the development of novel antibacterial agent against the MRSA infection.

Effects of aldo-keto reductase family 1 member A on osteoblast differentiation associated with lactate production in MC3T3-E1 preosteoblastic cells.

Aldo-keto reductase family 1 member A (AKR1A) is an NADPH-dependent aldehyde reductase widely expressed in mammalian tissues. In this study, induced differentiation of MC3T3-E1 preosteoblasts was found to increase AKR1A gene expression concomitantly increased NOx- (nitrite + nitrate), increased glucose uptake, increased [NAD(P)+]/[NAD(P)H] and lactate production but decreased reactive oxygen species (ROS) without changes in endothelial nitric oxide synthase (eNOS) expression in differentiated osteoblasts (OBs). A study using gain- and loss-of-function MC3T3-E1 cells indicated that AKR1A is essential for modulating OB differentiation and gene expression of collagen 1 A1, receptor activator of nuclear factor kappa-B ligand, and osteoprotegerin in OBs. Immunofluorescence microscopy also revealed that changes in AKR1A expression altered extracellular collagen formation in differentiated OBs. Consistently, analyses of alkaline phosphatase activity and calcium deposits of matrix mineralization by Alizarin Red S staining verified that AKR1A is involved in the regulation of OB differentiation and bone matrix formation. In addition, AKR1A gene alterations affected the levels of NOx-, eNOS expression, glucose uptake, [NAD(P)+]/[NAD(P)H] dinucleotide redox couples, lactate production, and ROS in differentiated OBs. Herein, we report that AKR1A-mediated denitrosylation may play a role in the regulation of lactate metabolism as well as redox homeostasis in cells, providing an efficient way to quickly gain energy and to significantly reduce oxidative stress for OB differentiation.

Nicotinamide reverses deficits in puberty-born neurons and cognitive function after maternal separation.

BACKGROUND: Early life stress (ELS) is associated with the development of schizophrenia later in life. The hippocampus develops significantly during childhood and is extremely reactive to stress. In rodent models, ELS can induce neuroinflammation, hippocampal neuronal loss, and schizophrenia-like behavior. While nicotinamide (NAM) can inhibit microglial inflammation, it is unknown whether NAM treatment during adolescence reduces hippocampal neuronal loss and abnormal behaviors induced by ELS. METHODS: Twenty-four hours of maternal separation (MS) of Wistar rat pups on post-natal day (PND)9 was used as an ELS. On PND35, animals received a single intraperitoneal injection of BrdU to label dividing neurons and were given NAM from PND35 to PND65. Behavioral testing was performed. Western blotting and immunofluorescence staining were used to detect nicotinamide adenine dinucleotide (NAD+)/Sirtuin3 (Sirt3)/superoxide dismutase 2 (SOD2) pathway-related proteins. RESULTS: Compared with controls, only MS animals in the adult stage (PND56-65) but not the adolescent stage (PND31-40) exhibited pre-pulse inhibition deficits and cognitive impairments mimicking schizophrenia symptoms. MS decreased the survival and activity of puberty-born neurons and hippocampal NAD+ and Sirt3 expression in adulthood. These observations were related to an increase in acetylated SOD2, microglial activation, and significant increases in pro-inflammatory IL-1ß, TNF-α, and IL-6 expression. All the effects of MS at PND9 were reversed by administering NAM in adolescence (PND35-65). CONCLUSIONS: MS may lead to schizophrenia-like phenotypes and persistent hippocampal abnormalities. NAM may be a safe and effective treatment in adolescence to restore normal hippocampal function and prevent or ameliorate schizophrenia-like behavior.

Exogenous Nicotinamide Adenine Dinucleotide Attenuates Postresuscitation Myocardial and Neurologic Dysfunction in a Rat Model of Cardiac Arrest.

OBJECTIVES: To investigate the therapeutic potential and underlying mechanisms of exogenous nicotinamide adenine dinucleotide+ on postresuscitation myocardial and neurologic dysfunction in a rat model of cardiac arrest. DESIGN: Thirty-eight rats were randomized into three groups: 1) Sham, 2) Control, and 3) NAD. Except for the sham group, untreated ventricular fibrillation for 6 minutes followed by cardiopulmonary resuscitation was performed in the control and NAD groups. Nicotinamide adenine dinucleotide+ (20 mg/kg) was IV administered at the onset of return of spontaneous circulation. SETTING: University-affiliated research laboratory. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: Nicotinamide adenine dinucleotide+. MEASUREMENTS AND MAIN RESULTS: Hemodynamic and myocardial function were measured at baseline and within 4 hours following return of spontaneous circulation. Survival analysis and Neurologic Deficit Score were performed up to 72 hours after return of spontaneous circulation. Adenosine triphosphate (adenosine triphosphate) level was measured in both brain and heart tissue. Mitochondrial respiratory chain function, acetylation level, and expression of Sirtuin3 and NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9 (NDUFA9) in isolated mitochondrial protein from both brain and heart tissue were evaluated at 4 hours following return of spontaneous circulation. The results demonstrated that nicotinamide adenine dinucleotide+ treatment improved mean arterial pressure (at 1 hr following return of spontaneous circulation, 94.69 ± 4.25 mm Hg vs 89.57 ± 7.71 mm Hg; p < 0.05), ejection fraction (at 1 hr following return of spontaneous circulation, 62.67% ± 6.71% vs 52.96% ± 9.37%; p < 0.05), Neurologic Deficit Score (at 24 hr following return of spontaneous circulation, 449.50 ± 82.58 vs 339.50 ± 90.66; p < 0.05), and survival rate compared with that of the control group. The adenosine triphosphate level and complex I respiratory were significantly restored in the NAD group compared with those of the control group. In addition, nicotinamide adenine dinucleotide+ treatment activated the Sirtuin3 pathway, down-regulating acetylated-NDUFA9 in the isolated mitochondria protein. CONCLUSIONS: Exogenous nicotinamide adenine dinucleotide+ treatment attenuated postresuscitation myocardial and neurologic dysfunction. The responsible mechanisms may involve the preservation of mitochondrial complex I respiratory capacity and adenosine triphosphate production, which involves the Sirtuin3-NDUFA9 deacetylation.

Exercise training and NR supplementation to improve muscle mass and fitness in adolescent and young adult hematopoietic cell transplant survivors: a randomized controlled trial {1}.

BACKGROUND: Advances in hematopoietic cell transplantation (HCT) have led to marked improvements in survival. However, adolescents and young adults (AYAs) who undergo HCT are at high risk of developing sarcopenia (loss of skeletal muscle mass) due to the impact of HCT-related exposures on the developing musculoskeletal system. HCT survivors who have sarcopenia also have excess lifetime risk of non-relapse mortality. Therefore, interventions that increase skeletal muscle mass, metabolism, strength, and function are needed to improve health in AYA HCT survivors. Skeletal muscle is highly reliant on mitochondrial energy production, as reflected by oxidative phosphorylation (OXPHOS) capacity. Exercise is one approach to target skeletal muscle mitochondrial OXPHOS, and in turn improve muscle function and strength. Another approach is to use "exercise enhancers", such as nicotinamide riboside (NR), a safe and well-tolerated precursor of nicotinamide adenine dinucleotide (NAD+), a cofactor that in turn impacts muscle energy production. Interventions combining exercise with exercise enhancers like NR hold promise, but have not yet been rigorously tested in AYA HCT survivors. METHODS/DESIGN: We will perform a randomized controlled trial testing 16 weeks of in-home aerobic and resistance exercise and NR in AYA HCT survivors, with a primary outcome of muscle strength via dynamometry and a key secondary outcome of cardiovascular fitness via cardiopulmonary exercise testing. We will also test the effects of these interventions on i) muscle mass via dual energy x-ray absorptiometry; ii) muscle mitochondrial OXPHOS via an innovative non-invasive MRI-based technique, and iii) circulating correlates of NAD+ metabolism via metabolomics. Eighty AYAs (ages 15-30y) will be recruited 6-24 months post-HCT and randomized to 1 of 4 arms: exercise + NR, exercise alone, NR alone, or control. Outcomes will be collected at baseline and after the 16-week intervention. DISCUSSION: We expect that exercise with NR will produce larger changes than exercise alone in key outcomes, and that changes will be mediated by increases in muscle OXPHOS. We will apply the insights gained from this trial to develop individualized, evidence-supported precision initiatives that will reduce chronic disease burden in high-risk cancer survivors. TRIAL REGISTRATION: ClinicalTrials.gov, NCT05194397. Registered January 18, 2022, https://clinicaltrials.gov/ct2/show/NCT05194397 {2a}.

Nicotinamide mitigates radiation injury in submandibular gland by protecting mitochondrial structure and functions.

BACKGROUND: Radiation damage to salivary gland is inevitable in head and neck cancer patients receiving radiotherapy. Safe and effective treatments for protecting salivary glands from radiation are still unavailable. Mitochondrial damage is a critical mechanism in irradiated salivary gland; however, treatment targeting mitochondria has not received much attention. Nicotinamide is a key component of the mitochondrial metabolism. Here, we investigated the effects and underlying mechanisms of nicotinamide on protecting irradiated submandibular gland. METHODS: Submandibular gland cells and tissues were randomly divided into four groups: control, nicotinamide alone, radiation alone, and radiation with nicotinamide pretreatment. Cell viability was detected by PrestoBlue cell viability reagent. Histopathological alterations were observed with HE staining. Pilocarpine-stimulated saliva was measured from Wharton's duct. Cell apoptosis was determined by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Nicotinamide phosphoribosyl transferase was examined with immunofluorescence. The levels of nicotinamide adenine dinucleotide, mitochondrial membrane potential, and adenosine triphosphate were measured with the relevant kits. The mitochondrial ultrastructure was observed under transmission electron microscopy. RESULTS: Nicotinamide significantly mitigated radiation damage both in vitro and in vivo. Also, nicotinamide improved saliva secretion and reduced radiation-induced apoptosis in irradiated submandibular glands. Moreover, nicotinamide improved nicotinamide phosphoribosyl transferase and the levels of nicotinamide adenine dinucleotide/adenosine triphosphate and mitochondrial membrane potential, all of which were decreased by radiation in submandibular gland cells. Importantly, nicotinamide protected the mitochondrial ultrastructure from radiation. CONCLUSION: These findings demonstrate that nicotinamide alleviates radiation damage in submandibular gland by replenishing nicotinamide adenine dinucleotide and maintaining mitochondrial function and ultrastructure, suggesting that nicotinamide could be used as a prospective radioprotectant for preventing radiation sialadenitis.

NADPH is superior to NADH or edaravone in ameliorating metabolic disturbance and brain injury in ischemic stroke.

Our previous studies confirm that exogenous reduced nicotinamide adenine dinucleotide phosphate (NADPH) exerts a neuroprotective effect in animal models of ischemic stroke, and its primary mechanism is related to anti-oxidative stress and improved energy metabolism. However, it is unknown whether nicotinamide adenine dinucleotide (NADH) also plays a neuroprotective role and whether NADPH is superior to NADH against ischemic stroke? In this study we compared the efficacy of NADH, NADPH, and edaravone in ameliorating brain injury and metabolic stress in ischemic stroke. Transient middle cerebral artery occlusion/reperfusion (t-MCAO/R) mouse model and in vitro oxygen glucose deprivation/reoxygenation (OGD/R) model were established. The mice were intravenously administered the optimal dose of NADPH (7.5 mg/kg), NADH (22.5 mg/kg), or edaravone (3 mg/kg) immediately after reperfusion. We showed that the overall efficacy of NADPH in ameliorating ischemic injury was superior to NADH and edaravone. NADPH had a longer therapeutic time window (within 5 h) after reperfusion than NADH and edaravone (within 2 h) for ischemic stroke. In addition, NADPH and edaravone were better in alleviating the brain atrophy, while NADH and NADPH were better in increasing the long-term survival rate. NADPH showed stronger antioxidant effects than NADH and edaravone; but NADH was the best in terms of maintaining energy metabolism. Taken together, this study demonstrates that NADPH exerts better neuroprotective effects against ischemic stroke than NADH and edaravone.

Nicotinamide adenine dinucleotide attenuates acetaminophen-induced acute liver injury via activation of PARP1, Sirt1, and Nrf2 in mice.

The aim of this study was to investigate the protective effect of nicotinamide adenine dinucleotide (NAD+) against acute liver injury (ALI) induced by acetaminophen (APAP) overdose in mice. First, serum transaminases were used to assess the protective effect of NAD+, and the data revealed that NAD+ mitigated the APAP-induced ALI in a dose-dependent manner. Then, we performed hematoxylin-eosin staining of liver tissues and found that NAD+ alleviated the abnormalities of histopathology. Meanwhile, increase in the malondialdehyde content and decrease in glutathione, superoxide dismutase (SOD), and glutathione peroxidase were identified in the APAP group, which were partially prevented by the NAD+ pretreatment. Moreover, compared with the mice treated with APAP only, the expression of poly ADP-ribose polymerase 1 (PARP1), Sirtuin1 (Sirt1), SOD2, nuclear factor erythroid 2-related factor 2 (Nrf2), and hemoxygenase-1 was upregulated, while Kelch-like ECH-associated protein 1 and histone H2AX phosphorylated on Ser-139 were downregulated by NAD+ in NAD+ + APAP group. Conversely, NAD+ could not correct the elevated expression of phospho-Jun N-terminal kinase and phospho-extracellular signal-regulated kinase induced by APAP. Taken together, these findings suggest that NAD+ confers an anti-ALI effect to enhance the expression of PARP1 and Sirt1, and to simultaneously stimulate the Nrf2 anti-oxidant signaling pathway.

Two Laminaria japonica Fermentation Broths Alleviate Oxidative Stress and Inflammatory Response Caused by UVB Damage: Photoprotective and Reparative Effects.

UVB radiation can induce oxidative stress and inflammatory response in human epidermal cells. We establish a UVB-induced damage model of human immortalized epidermal keratinocytes (HaCaT) to explore the protective and reparative effects of Laminaria japonica on UVB-damaged epidermal inflammation after fermentation by white Ganoderma lucidum (Curtis) P. Karst and Saccharomyces cerevisiae. Compared with unfermented Laminaria japonica, fermented Laminaria japonica possesses stronger in vitro free radical scavenging ability. Laminaria japonica white Ganoderma lucidum fermentation broth (LJ-G) and Laminaria japonica rice wine yeast fermentation broth (LJ-Y) can more effectively remove excess reactive oxygen species (ROS) in cells and increase the content of the intracellular antioxidant enzymes heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO-1). In addition, fermented Laminaria japonica effectively reduces the content of pro-inflammatory factors ILs, TNF-α and MMP-9 secreted by cells. The molecular research results show that fermented Laminaria japonica activates the Nrf2 signaling pathway, increases the synthesis of antioxidant enzymes, inhibits the gene expression levels of pro-inflammatory factors, and alleviates cellular oxidative stress and inflammatory response caused by UVB radiation. Based on the above results, we conclude that fermented Laminaria japonica has stronger antioxidant and anti-inflammatory activity than unfermented Laminaria japonica, possesses good safety, and can be developed and used as a functional inflammation reliever. Fermented Laminaria japonica polysaccharide has a more slender morphological structure and more rockulose, with better moisturizing and rheological properties.

The effect of pesticides on the NADH-supported mitochondrial respiration of permeabilized potato mitochondria.

Pesticides can seriously affect the respiratory chain of the mitochondria of many crops, reducing the intensity of plant growth and its yield. Studying the effect of pesticides on the bioenergetic parameters of intact plant mitochondria is a promising approach for assessing their toxicity. In this study, we investigated the effect of some pesticides on isolated potato mitochondria, which used exogenous NADH as a substrate for respiration. We showed that succinate is the most preferred substrate for phosphorylating respiration of intact potato tubers mitochondria. Potato mitochondria poorly oxidize exogenous NADH, despite of the presence of external NADH dehydrogenases. Permeabilization of the mitochondrial membrane with alamethicin increased the availability of exogenous NADH to complex I. However, the pathway of electrons through complex I to complex IV makes intact potato mitochondria susceptible to a number of pesticides such as difenoconazole, fenazaquin, pyridaben and tolfenpyrad, which strongly inhibit the rate of mitochondrial respiration. However, these pesticides only slightly inhibited the rate of oxygen consumption during succinate-supported respiration. Dithianon, the inhibitor of Complex II, is the only pesticide which significantly increased the respiratory rate of NADH-supported respiration of permeabilized mitochondria of potato. Thus, it can be assumed that the alternative NADH dehydrogenases for electron flow represent a factor responsible for plant resistance to xenobiotics, such as mitochondria-targeted pesticides.

Enhanced tolerance of Cupriavidus necator NCIMB 11599 to lignocellulosic derived inhibitors by inserting NAD salvage pathway genes.

Polyhydroxybutyrate (PHB) is a bio-based, biodegradable and biocompatible plastic that has the potential to replace petroleum-based plastics. Lignocellulosic biomass is a promising feedstock for industrial fermentation to produce bioproducts such as polyhydroxybutyrate (PHB). However, the pretreatment processes of lignocellulosic biomass lead to the generation of toxic byproducts, such as furfural, 5-HMF, vanillin, and acetate, which affect microbial growth and productivity. In this study, to reduce furfural toxicity during PHB production from lignocellulosic hydrolysates, we genetically engineered Cupriavidus necator NCIMB 11599, by inserting the nicotine amide salvage pathway genes pncB and nadE to increase the NAD(P)H pool. We found that the expression of pncB was the most effective in improving tolerance to inhibitors, cell growth, PHB production and sugar consumption rate. In addition, the engineered strain harboring pncB showed higher PHB production using lignocellulosic hydrolysates than the wild-type strain. Therefore, the application of NAD salvage pathway genes improves the tolerance of Cupriavidus necator to lignocellulosic-derived inhibitors and should be used to optimize PHB production.

Synthesis of a Rare Water-Soluble Silver(II)/Porphyrin and Its Multifunctional Therapeutic Effect on Methicillin-Resistant Staphylococcus aureus.

Porphyrin derivatives are popular photodynamic therapy (PDT) agents; however, their typical insolubility in water has made it challenging to separate cells of organisms in a liquid water environment. Herein, a novel water-soluble 5,10,15,20-tetrakis(4-methoxyphenyl-3-sulfonatophenyl) porphyrin (TMPPS) was synthesized with 95% yield by modifying the traditional sulfonation route. The reaction of TMPPS with AgNO3 afforded AgTMPPS an unusual Ag(II) oxidation state (97% yield). The free base and Ag(II) complex were characterized by matrix-assisted laser desorption ionization-mass spectroscopy, and 1H nuclear magnetic resonance, Fourier-transform infrared, UV-vis, fluorescence, and X-ray photolectron spectroscopies. Upon 460 nm laser irradiation, AgTMPPS generated a large amount of 1O2, whereas no ⦁OH was detected. Antibacterial experiments on methicillin-resistant Staphylococcus aureus (MRSA) revealed that the combined action of AgⅡ ions and PDT could endow AgTMPPS with a 100% bactericidal ratio for highly concentrated MRSA (108 CFU/mL) at a very low dosage (4 µM) under laser irradiation at 360 J/cm2. Another PDT response was demonstrated by photocatalytically oxidizing 1,4-dihydronicotinamide adenine dinucleotide to NAD+ with AgTMPPS. The structural features of the TMPPS and AgTMPPS molecules were investigated by density functional theory quantum chemical calculations to demonstrate the efficient chemical and photodynamical effects of AgTMPPS for non-invasive antibacterial therapy.

Selective Growth Suppressive Effect of Pravastatin on Senescent Human Lung Fibroblasts.

Various chemical reagents containing inhibitors of mitochondrial activity, antioxidants, nuclear factor-kappa B (NF-kB) inhibitor, mammalian target of rapamycin (mTOR) inhibitor and other clinical therapeutics were screened in order to identify those that selectively decrease the viability of senescent human lung fibroblasts. Cell viability was measured using the CCK-8 assay. The results showed that pravastatin, a drug for hyperlipidemia, decreased the viability of senescent cells but not non-senescent cells. The effect of pravastatin on senescent cells is thought to be due to the inhibition of cell proliferation, rather than cell death. The effect of pravastatin was further investigated using the glucose metabolism assay, which showed that glucose consumption was inhibited both in non-senescent and senescent cells and intracellular nicotinamide adenine dinucleotide (NAD) was decreased in senescent cells. Changes to the mRNA expression levels of senescence-associated genes in response to pravastatin treatment were quantified by real-time-qPCR. There were no significant changes in the relative mRNA expression levels of IL-1ß, p16, p21, and p53 in pravastatin-treated non-senescent cells, whereas the expression of IL-1ß and p16 were increased by pravastatin only in senescent cells. The results of this study suggest that pravastatin does not induce senolysis, but rather selectively inhibits the proliferation of senescent cells and that cellular senescence is enhanced by decreasing intracellular NAD and promoting IL-1ß production.