New insights into micro-algal astaxanthin's effect on deoxynivalenol-induced spleen lymphocytes pyroptosis in Cyprinus carpio: Involving mitophagy and mtROS-NF-κB-dependent NLRP3 inflammasome.

Deoxynivalenol (DON) is one of the most common sources of fungal toxins in fish feed, posing a significant risk to the immune and reproductive systems of fish. Microalgal astaxanthin (MIA), a potent antioxidant derived from microalgae, confers multifarious advantages upon piscine organisms, notably encompassing its anti-inflammatory and antioxidant prowess. Herein, we investigated the potential of MIA in ameliorating the immunotoxicity of DON on carp (Cyprinus carpio L.) based on spleen lymphocytes treated with DON (1.5 ng/ml) and/or MIA (96 µM). Firstly, CCK8 results showed that DON resulted in excessive death of spleen lymphocytes. Secondly, spleen lymphocytes treated with DON had a higher proportion of pyroptosis, and the mRNA and protein levels of pyroptosis (NLRP3, IL-1ß and ASC) in spleen lymphocytes were increased. Thirdly, the relative red fluorescence intensity of JC-1 and DCFH-DA showed decreased mitochondrial membrane potential and increased ROS in spleen lymphocytes treated with DON. Mitochondrial ATP, DNA and NADPH/NADP+ analysis revealed decreased mitochondrial ATP, DNA and NADPH/NADP+ levels in DON-treated lymphocytes, corroborating the association between DON exposure and elevated intracellular ATP, DNA and NADPH/NADP+ in lymphocytes. DON exposure resulted in the downregulation of mitophagy-related genes and proteins (PINK1, Parkin and LC3) in lymphocytes. Notably, these effects were counteracted by treatment with MIA. Furthermore, DON led to the elevated secretion of inflammatory factors (TNF-α, IL-4 and IFN-γ), thereby inducing immune dysfunction in spleen lymphocytes. Encouragingly, MIA treatment effectively mitigated the immunotoxic effects induced by DON, demonstrating its potential in ameliorating pyroptosis, mitochondrial dysfunction and mitophagy impairment via regulating the mtROS-NF-κB axis in lymphocytes. This study sheds light on safeguarding farmed fish against agrobiological threats posed by DON, highlighting the valuable applications of MIA in aquaculture.

Regulatory effect of bacterial melanin on the isoforms of new superoxide-producing associates from rat tissues in rotenone-induced Parkinson's disease.

According to recent research, selective neuronal vulnerability in Parkinson's disease (PD) results from several phenotypic traits, including calcium-dependent, feed-forward control of mitochondrial respiration leading to elevated reactive oxygen species and cytosolic calcium concentration, an extensive axonal arbor, and a reactive neurotransmitter. Therefore, antioxidant therapy is a promising direction in the treatment of PD. In vitro studies have indicated the survival-promoting activity of bacterial melanin (BM) on midbrain dopaminergic neuron cultures. It has been established that BM has a number of protective and anti-inflammatory properties, so there is a high probability of a protective effect of BM in the early stages of PD. In this study, PD was induced through the unilateral intracerebral administration of rotenone followed by bacterial melanin. Tissues (brain, lungs, and small intestine) from the observed groups underwent isolation and purification to extract isoforms of new thermostable superoxide (О2-)-producing associates between NADPH-containing lipoprotein (NLP) and NADPH oxidase-Nox (NLP-Nox). The optical absorption spectral characteristics, specific amounts, stationary concentration of the produced О2-, and the content of NADPH in the observed associates were determined. The optical absorption spectra of the NLP-Nox isoforms in the visible and UV regions in the experimental groups did not differ from those of the control group. However, compared with the control group, the specific content of the total fractions of NLP-Nox isoforms associated with PD groups was higher, especially in the small intestine. These findings suggest that the described changes may represent a novel mechanism for rotenone-induced PD. Furthermore, bacterial melanin demonstrated antioxidant properties and regulated membrane formation in the brain, lung, and small intestine. This regulation occurred by inhibiting the release of new membrane-bound formations (NLP-Nox associates) from these membranes while simultaneously regulating the steady-state concentration of the formed О2-.

Activation of NLRP3 inflammasome in lung epithelial cells triggers radiation-induced lung injury.

BACKGROUND: Radiation-induced lung injury (RILI) is the most common and serious complication of chest radiotherapy. However, reported radioprotective agents usually lead to radiation resistance in tumor cells. The key to solving this problem is to distinguish between the response of tumor cells and normal lung epithelial cells to radiation damage. METHODS: RNA-Seq was used to recognize potential target of alleviating the progression of RILI as well as inhibiting tumor growth. The activation of NLRP3 inflammasome in lung epithelial cells was screened by qRT-PCR, western blotting, immunofluorescence, and ELISA. An in vivo model of RILI and in vitro conditioned culture model were constructed to evaluate the effect of NLRP3/interleukin-1ß on fibroblasts activation. ROS, ATP, and (NADP)+/NADP(H) level in lung epithelial cells was detected to explore the mechanism of NLRP3 inflammasome activation. The lung macrophages of the mice were deleted to evaluate the role of lung epithelial cells in RILI. Moreover, primary cells were extracted to validate the results obtained from cell lines. RESULTS: NLRP3 activation in epithelial cells after radiation depends on glycolysis-related reactive oxygen species accumulation. DPYSL4 is activated and acts as a negative regulator of this process. The NLRP3 inflammasome triggers interleukin-1ß secretion, which directly affects fibroblast activation, proliferation, and migration, eventually leading to lung fibrosis. CONCLUSIONS: Our study suggests that NLRP3 inflammasome activation in lung epithelial cells is essential for radiation-induced lung injury. These data strongly indicate that targeting NLRP3 may be effective in reducing radiation-induced lung injury in clinical settings.

Renal protective effects of astragalus root in rat models of chronic kidney disease.

BACKGROUND: Astragalus root is a commonly used herb in traditional Chinese medicine. Although renoprotective effects have been reported in some clinical and experimental studies, the details remain unknown. METHODS: We used 5/6 nephrectomized rats as chronic kidney disease (CKD) models. At 10 weeks, they were divided into four groups, namely, CKD, low-dose astragalus (AR400), high-dose astragalus (AR800), and sham groups. At 14 weeks, they were sacrificed for the evaluation of blood, urine, mRNA expression in the kidney, and renal histopathology. RESULTS: Kidney dysfunction was significantly improved following astragalus administration (creatinine clearance: sham group; 3.8 ± 0.3 mL/min, CKD group; 1.5 ± 0.1 mL/min, AR400 group; 2.5 ± 0.3 mL/min, AR800 group; 2.7 ± 0.1 mL/min). Blood pressure, urinary albumin, and urinary NGAL levels were significantly lower in the astragalus-treated groups than those in the CKD group. Excretion of urinary 8-OHdG, an oxidative stress marker, and intrarenal oxidative stress were lower in the astragalus-treated groups than those in the CKD group. Furthermore, the mRNA expression of NADPH p22 phox, NADPH p47 phox, Nox4, renin, angiotensin II type 1 receptor, and angiotensinogen in the kidney was lower in the astragalus-treated groups compared with the CKD group. CONCLUSION: This study suggests that astragalus root slowed CKD progression, possibly through the suppression of oxidative stress and the renin-angiotensin system.

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.

Choline and citicoline ameliorate oxidative stress in acute kidney injury in rats.

OBJECTIVES: The purpose of this study is to investigate the effects of cholinergic anti-inflammatory pathway (CAP)-activating drugs, choline and citicoline (Cytidinediphosphate-choline, CDP-choline), on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) parameters and the contribution of NADPH Oxidase4 (NOX4) p22phox. BACKGROUND: Endotoxemia induces a systemic inflammatory response characterized by the production of pro-inflammatory mediators and reactive oxygen species (ROS), which eventually develops acute kidney injury (AKI). NADPH Oxidase4 (NOX4) p22phox pathway contributes to the development of endotoxemia-induced AKI. Inflammatory response can be controlled by CAP. METHODS: Expressions levels of KIM-1, TNF-α, NOX4, p22phox and NFκB in the kidney tissues of rats were analyzed via RT-PCR in experimental groups; 1. Control, 2. LPS (10 mg/kg) + saline, 3. LPS + CDP-choline (375 mg/kg) and 4. LPS + choline (90 mg/kg). Choline and ROS levels in kidney tissues were also measured by a spectrofluorometric assay. RESULTS: LPS-induced elevations of ROS levels were decreased by CDP-choline or choline administration (p < 0.001). LPS-elevated KIM-1, TNFα, NOX4, p22 phox, and NFκB expressions were significantly decreased by choline or CDP-choline treatments (p < 0.001). CONCLUSION: Decreased ROS production in kidney tissues in treatment groups suggests that choline or CDP-choline may have therapeutic potential in endotoxemia-associated AKI via downregulating NOX4 and p22phox expressions (Tab. 1, Fig. 5, Ref. 45). Text in PDF www.elis.sk Keywords: endotoxemia, choline, cytidine diphosphate choline, acute kidney injury, reactive oxygen species.

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.

NADP modulates RNA m6A methylation and adipogenesis via enhancing FTO activity.

Metabolism is often regulated by the transcription and translation of RNA. In turn, it is likely that some metabolites regulate enzymes controlling reversible RNA modification, such as N6-methyladenosine (m6A), to modulate RNA. This hypothesis is at least partially supported by the findings that multiple metabolic diseases are highly associated with fat mass and obesity-associated protein (FTO), an m6A demethylase. However, knowledge about whether and which metabolites directly regulate m6A remains elusive. Here, we show that NADP directly binds FTO, independently increases FTO activity, and promotes RNA m6A demethylation and adipogenesis. We screened a set of metabolites using a fluorescence quenching assay and NADP was identified to remarkably bind FTO. In vitro demethylation assays indicated that NADP enhances FTO activity. Furthermore, NADP regulated mRNA m6A via FTO in vivo, and deletion of FTO blocked NADP-enhanced adipogenesis in 3T3-L1 preadipocytes. These results build a direct link between metabolism and RNA m6A demethylation.

In Vitro ADME and Preclinical Pharmacokinetics of Ulotaront, a TAAR1/5-HT1A Receptor Agonist for the Treatment of Schizophrenia.

PURPOSE: Ulotaront (SEP-363856) is a TAAR1 agonist with 5-HT1A agonist activity currently in clinical development for the treatment of schizophrenia. The objectives of the current study were to characterize the in vitro ADME properties, preclinical PK, and to evaluate the DDI potential of ulotaront and its major metabolite SEP-383103. METHODS: Solubility, permeability, plasma protein binding, CYP inhibition and induction, transporter inhibition and uptake studies were conducted in vitro. Phenotyping studies were conducted using recombinant human CYPs and FMOs, human liver microsomes and human liver homogenates. Preclinical plasma and brain pharmacokinetics were determined after a single intraperitoneal, intravenous, and oral administration of ulotaront. RESULTS: Ulotaront is a compound of high solubility, high permeability, and low binding to plasma proteins. Ulotaront metabolism is mediated via both NADPH-dependent and NADPH-independent pathways, with CYP2D6 as the major metabolizing enzyme. Ulotaront is an inducer of CYP2B6, and an inhibitor of CYP2D6, OCT1 and OCT2, while SEP-383103 is neither a CYP inducer nor a potent inhibitor of CYPs and human transporters. Ulotaront exhibits rapid absorption, greater than 70% bioavailability, approximately 3.5 L/kg volume of distribution, 1.5-4 h half-life, 12-43 ml/min/kg clearance, and good penetration across the blood-brain barrier in preclinical species. CONCLUSIONS: Ulotaront has been designated as a BCS1 compound by US FDA. The ability of ulotaront to penetrate the blood-brain barrier for CNS targeting has been demonstrated in mice and rats. The potential for ulotaront and SEP-383103 to act as perpetrators of CYP and transporter-mediated DDIs is predicted to be remote.

Zinc oxide nanoparticles (ZnO-NPs) exhibit immune toxicity to crucian carp (Carassius carassius) by neutrophil extracellular traps (NETs) release and oxidative stress.

Zinc oxide nanoparticles (ZnO-NPs) are widely used in sunscreens, cosmetics, paint, construction materials, and other products. ZnO-NPs released into the environment can harm aquatic creatures and pose a health risk to humans through the food chain. ZnO-NPs are toxic to fish, but there are few reports on its immunotoxicity on crucian carp (Carassius carassius). In this study, ZnO-NPs increased the biochemical indexes of the liver in serum, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In histopathological observation, many inflammatory cells were filled in the liver's central vein stimulated by ZnO-NPs. Furthermore, ZnO-NPs could increase malondialdehyde (MDA) level, lessen superoxide dismutase (SOD) level, and elevate the level of neutrophil extracellular traps (NETs). However, deoxyribonuclease I (DNase I) alleviated all biochemical indexes and histopathological changes. Immunofluorescence in vitro confirmed that NETs were composed of citrullinated histone 3, myeloperoxidase, and neutrophil elastase. ZnO-NPs-increased NETs were dependent on reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase and were also related to partial processes of glycolysis. Our study confirms that ZnO-NPS has a toxic effect on the liver of crucian carp. DNase I can prevent liver damage caused by ZnO-NPs, which provides a new insight into the immunotoxicity of ZnO-NPs to fish.

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.

Taurine alleviates kidney injury in a thioacetamide rat model by mediating Nrf2/HO-1, NQO-1, and MAPK/NF-κB signaling pathways.

This study investigated the molecular mechanisms by which taurine exerts its reno-protective effects in thioacetamide (TAA) - induced kidney injury in rats. Rats received taurine (100 mg/kg daily, intraperitoneally) either from day 1 of TAA injection (250 mg/kg twice weekly for 6 weeks) or after 6 weeks of TAA administration. Taurine treatment, either concomitant or later as a therapy, restored kidney functions, reduced blood urea nitrogen (BUN), creatinine, and malondialdehyde (MDA), increased renal levels of superoxide dismutase (SOD), and reversed the increase of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) caused by TAA. Taurine treatment also led to a significant rise in nuclear factor erythroid 2-related factor 2 (Nrf2), hemoxygenase-1 (HO-1), and NADPH quinone oxidoreductase-1 (NQO-1) levels, with significant suppression of extracellular signal-regulated kinase (ERK) 1/2, nuclear factor kappa B (NF-κB), and tumor necrosis factor α (TNF-α) gene expressions, and interleukin-18 (IL-18) and TNF-α protein levels compared with those in TAA kidney-injured rats. Taurine exhibited reno-protective potential in TAA-induced kidney injury through its antioxidant and anti-inflammatory effects. Taurine antioxidant activity is accredited for its effect on Nrf-2 induction and subsequent activation of HO-1 and NQO-1. In addition, taurine exerts its anti-inflammatory effect via regulating NF-κB transcription and subsequent production of pro-inflammatory mediators via mitogen-activated protein kinase (MAPK) signaling regulation.

Mitoquinone intravitreal injection ameliorates retinal ischemia-reperfusion injury in rats involving SIRT1/Notch1/NADPH axis.

Retinal ischemia-reperfusion injury (RIRI) is an important pathological process of many ocular diseases. Mitoquinone (MitoQ), a mitochondrial targeted antioxidant, is a potential compound for therapeutic development of RIRI. This study observed the effect of MitoQ on RIRI, and further explored its possible molecular mechanism. Temporary increase in intraocular pressure was used to establish rat model of RIRI to observe the effect of MitoQ treatment on retinal function, pathological injury, oxidative stress, inflammation and apoptosis. Immunohistochemistry and Western blot were used to detect expressions of cleaved caspase 3, B cell leukemia/lymphoma 2 associated X (Bax), nicotinamide adenine dinucleotide phosphate oxidase (NOX1), NOX4, cleaved-Notch 1, hairy and enhancer of split 1 (Hes1), and sirtuin 1 (SIRT 1) in retina were detected by immunohistochemistry and Western blot. MitoQ treatment significantly improved retinal function and pathological injury, inhibited the over-production of reactive oxygen species, increased the expression of superoxide dismutase 1 (SOD 1), suppressed the releases of inflammatory cytokines, and inhibited retinal cells apoptosis. MitoQ also down-regulated the expressions of cleaved caspase 3, Bax, NOX 1, NOX 4, cleaved-Notch 1, and Hes 1, increased the expression of SIRT 1 protein and its activity. These effects were significantly reversed by SIRT1 inhibitor EX527. Our data suggests that MitoQ, as a potentially effective drug for improving RIRI, may act through the SIRT1/Notch1/NADPH signal axis.

Nicotinic acid adenine dinucleotide phosphate activates two-pore channel TPC1 to mediate lysosomal Ca2+ release in endothelial colony-forming cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca2+ -releasing messenger that increases the intracellular Ca2+ concentration by mobilizing the lysosomal Ca2+ store through two-pore channels 1 (TPC1) and 2 (TPC2). NAADP-induced lysosomal Ca2+ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca2+ pool endowed with TPC1 is also present in human endothelial colony-forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca2+ store and TPC1 in circulating ECFCs by harnessing Ca2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe ß-naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca2+ sequestration by acidic organelles, caused endogenous Ca2+ release in the presence of a replete inositol-1,4,5-trisphosphate (InsP3 )-sensitive endoplasmic reticulum (ER) Ca2+ pool. Likewise, the amount of ER releasable Ca2+ was reduced by disrupting lysosomal Ca2+ content. Liposomal delivery of NAADP induced a transient Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP-induced Ca2+ release also required ER-embedded InsP3 receptors. Finally, NAADP-induced lysosomal Ca2+ release was found to trigger vascular endothelial growth factor-induced intracellular Ca2+ oscillations and proliferation, while it did not contribute to adenosine-5'-trisphosphate-induced Ca2+ signaling. These findings demonstrated that NAADP-induced TPC1-mediated Ca2+ release can selectively be recruited to induce the Ca2+ response to specific cues in circulating ECFCs.

Exogenous NADPH ameliorates myocardial ischemia-reperfusion injury in rats through activating AMPK/mTOR pathway.

Our previous study shows that nicotinamide adenine dinucleotide phosphate (NADPH) plays an important role in protecting against cerebral ischemia injury. In this study we investigated whether NADPH exerted cardioprotection against myocardial ischemia/reperfusion (I/R) injury. To induce myocardial I/R injury, rats were subjected to ligation of the left anterior descending branch of coronary artery for 30 min followed by reperfusion for 2 h. At the onset of reperfusion, NADPH (4, 8, 16 mg· kg-1· d-1, iv) was administered to the rats. We found that NADPH concentrations in plasma and heart were significantly increased at 4 h after intravenous administration. Exogenous NADPH (8-16 mg/kg) significantly decreased myocardial infarct size and reduced serum levels of lactate dehydrogenase (LDH) and cardiac troponin I (cTn-I). Exogenous NADPH significantly decreased the apoptotic rate of cardiomyocytes, and reduced the cleavage of PARP and caspase-3. In addition, exogenous NADPH reduced mitochondrial vacuolation and increased mitochondrial membrane protein COXIV and TOM20, decreased BNIP3L and increased Bcl-2 to protect mitochondrial function. We conducted in vitro experiments in neonatal rat cardiomyocytes (NRCM) subjected to oxygen-glucose deprivation/restoration (OGD/R). Pretreatment with NADPH (60, 500 nM) significantly rescued the cell viability and inhibited OGD/R-induced apoptosis. Pretreatment with NADPH significantly increased the phosphorylation of AMPK and downregulated the phosphorylation of mTOR in OGD/R-treated NRCM. Compound C, an AMPK inhibitor, abolished NADPH-induced AMPK phosphorylation and cardioprotection in OGD/R-treated NRCM. In conclusion, exogenous NADPH exerts cardioprotection against myocardial I/R injury through the activation of AMPK/mTOR pathway and inhibiting mitochondrial damage and cardiomyocyte apoptosis. NADPH may be a potential candidate for the prevention and treatment of myocardial ischemic diseases.

Expression of Ca²âº-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells.

The second messenger NAADP triggers Ca(2+) release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2(-/-)), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca(2+) responses as assessed by single-cell Ca(2+) imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2(-/-) cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca(2+)-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca(2+) release. High-affinity [(32)P]NAADP binding still occurs in Tpcn1/2(-/-) tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca(2+)-permeable channels indispensable for NAADP signalling.

Protective Effect of Nicotinamide Adenine Dinucleotide Phosphate on Renal Ischemia-Reperfusion Injury.

BACKGROUND/AIMS: Renal ischemia-reperfusion injury (IRI) is a common consequence of acute kidney injury. Nicotinamide adenine dinucleotide phosphate (NADPH), which is derived from the pentose phosphate pathway, is essential for the proper functioning of essential redox and antioxidant defense systems. Previous studies have indicated that NADPH is responsible for protecting the brain from ischemic injury. The goal of this study was to analyze the protective function of NADPH in renal IRI. METHODS: The IRI animal model was generated through a midline laparotomy surgery that clamped both sides of the renal pedicles for 40 min to induce renal ischemia. The in vitro model was generated by removing oxygen and glucose from human kidney epithelial cells (HK-2 cells), followed by reoxygenation to imitate IRI. Renal function and histopathological changes were observed and evaluated. Additionally, malondialdehyde and glutathione levels were determined in renal tissue homogenate as indicators of oxidative stress. ROS production in cells was determined by DHE staining. Protein biomarker expression was evaluated by western blot, apoptosis was analyzed by TUNEL staining, and p65 nuclear translocation was visualized by immunofluorescence. RESULTS: Our data indicated that NADPH safeguarded the kidneys from histological and functional damage, and significantly reduce cell injury along with preventing potential increases in blood urea nitrogen and creatinine levels. Furthermore, we observed that NADPH increased glutathione levels, while reducing levels of malondialdehyde and reactive oxygen species. Additionally, our results suggested that NADPH treatment may alleviate IRI-induced apoptosis and inflammation. CONCLUSION: NADPH treatment may protect against renal IRI and should be further developed as a new treatment for acute kidney injury.

Liposomes as a Putative Tool to Investigate NAADP Signaling in Vasculogenesis.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the newest discovered intracellular second messengers, which is able to release Ca2+ stored within endolysosomal (EL) vesicles. NAADP-induced Ca2+ signals mediate a growing number of cellular functions, ranging from proliferation to muscle contraction and differentiation. Recently, NAADP has recently been shown to regulate angiogenesis by promoting endothelial cell growth. It is, however, still unknown whether NAADP stimulates proliferation also in endothelial progenitor cells, which are mobilized in circulation after an ischemic insult to induce tissue revascularization. Herein, we described a novel approach to prepare NAADP-containing liposomes, which are highly cell membrane permeable and are therefore amenable for stimulating cell activity. Accordingly, NAADP-containing liposomes evoked an increase in intracellular Ca2+ concentration, which was inhibited by NED-19, a selective inhibitor of NAADP-induced Ca2+ release. Furthermore, NAADP-containing liposomes promoted EPC proliferation, a process which was inhibited by NED-19 and BAPTA, a membrane permeable intracellular Ca2+ buffer. Therefore, NAADP-containing liposomes stand out as a promising tool to promote revascularization of hypoxic/ischemic tissues by favoring EPC proliferation. J. Cell. Biochem. 118: 3722-3729, 2017. © 2017 Wiley Periodicals, Inc.

Hepatocyte Nicotinamide Adenine Dinucleotide Phosphate Reduced Oxidase 4 Regulates Stress Signaling, Fibrosis, and Insulin Sensitivity During Development of Steatohepatitis in Mice.

BACKGROUND & AIMS: Reactive oxidative species (ROS) are believed to be involved in the progression of nonalcoholic steatohepatitis (NASH). However, little is known about the sources of ROS in hepatocytes or their role in disease progression. We studied the effects of nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (NOX4) in liver tissues from patients with NASH and mice with steatohepatitis. METHODS: Liver biopsy samples were obtained from 5 patients with NASH, as well as 4 patients with simple steatosis and 5 patients without steatosis (controls) from the University of California, Davis Cancer Center Biorepository. Mice with hepatocyte-specific deletion of NOX4 (NOX4(hepKO)) and NOX4(floxp+/+) C57BL/6 mice (controls) were given fast-food diets (supplemented with high-fructose corn syrup) or choline-deficient l-amino acid defined diets to induce steatohepatitis, or control diets, for 20 weeks. A separate group of mice were given the NOX4 inhibitor (GKT137831). Liver tissues were collected and immunoblot analyses were performed determine levels of NOX4, markers of inflammation and fibrosis, double-stranded RNA-activated protein kinase, and phospho-eIF-2α kinase-mediated stress signaling pathways. We performed hyperinsulinemic-euglycemic clamp studies and immunoprecipitation analyses to determine the oxidation and phosphatase activity of PP1C. RESULTS: Levels of NOX4 were increased in patients with NASH compared with controls. Hepatocyte-specific deletion of NOX4 reduced oxidative stress, lipid peroxidation, and liver fibrosis in mice with diet-induced steatohepatitis. A small molecule inhibitor of NOX4 reduced liver inflammation and fibrosis and increased insulin sensitivity in mice with diet-induced steatohepatitis. In primary hepatocytes, NOX4 reduced the activity of the phosphatase PP1C, prolonging activation of double-stranded RNA-activated protein kinase and phosphorylation of extracellular signal-regulated kinase-mediated stress signaling. Mice with hepatocyte-specific deletion of NOX4 and mice given GKT137831 had increased insulin sensitivity. CONCLUSIONS: NOX4 regulates oxidative stress in the liver and its levels are increased in patients with NASH and mice with diet-induced steatohepatitis. Inhibitors of NOX4 reduce liver inflammation and fibrosis and increase insulin sensitivity, and might be developed for treatment of NASH.

Effect of bafilomycin and NAADP on membrane-associated ATPases and respiration of isolated mitochondria of the murine Nemeth-Kellner lymphoma.

The goal of the study was to estimate the effect of a selective V-type H+ -ATPase inhibitor bafilomycin A1 and nicotinic acid adenine dinucleotide phosphate (NAADP) on energetic processes in NK/Ly cell by directly measuring the respiration of isolated mitochondria and ATPase activities. NAADP (7 µM) increased the activity of Na+ /K+ -ATPase in the postmitochondrial fraction of NK/Ly cells, but lower concentration of NAADP decreased it (0.1 and 1 µM). The increase the activity of plasma membrane Ca2+ ATPase (PMCA) under NAADP application (1 and 7 µM) was observed. However, NAADP (1 µM) decreased activities of sarcoendoplasmic reticulum Ca2+ -ATPase (SERCA) and basal Mg2+ -ATPase. Bafilomycin A1 (1 µM) increased the activity of Na+ /K+ -ATPase and potentiated the effect of NAADP (1 µM) on this pump. At the same time, bafilomycin A1 (1 µM) completely prevented all effects of NAADP (1 µM) on activities of PMCA, SERCA, and basal Mg2+ -ATPase, confirming that these effects are dependent on acidic stores. Bafilomycin A1 or NAADP decreased respiratory and oxidative phosphorylation rates in NK/Ly mitochondria when α-ketoglutarate was used as substrate in contrast to succinate. Thus, α-ketoglutarate oxidation is more sensitive to bafilomycin A1 and NAADP influences compared with succinate oxidation. However, bafilomycin A1 + NAADP and any of these compounds separately lead to full uncoupling of mitochondria after ADP addition irrespectively to substrate used. Bafilomycin A1 affects isolated tumor mitochondria more effectively in combination with NAADP. Bafilomycin and NAADP alter some membrane-associated ATPases and inhibit respiration in mitochondria of the Nemeth-Kellner lymphoma. SIGNIFICANCE OF RESEARCH PARAGRAPH: Bafilomycin A1 potentiates the effect of NAADP by inhibiting the mitochondrial energetic process in lymphoma cells and activity of Na+ /K+ -ATPase. The obtained data show promising possibility to use bafilomycin A1 and NAADP as chemotherapeutic agents for lymphoma cells treatment. This is important because lymphomas are seventh most common form of cancer. Today the lymphoma mortality is 15% to 30%, whereas the effectiveness of malignant neoplasms treatment is less than 50%.