Deficiency of neutral cholesterol ester hydrolase 1 (NCEH1) impairs endothelial function in diet-induced diabetic mice.

BACKGROUND: Neutral cholesterol ester hydrolase 1 (NCEH1) plays a critical role in the regulation of cholesterol ester metabolism. Deficiency of NCHE1 accelerated atherosclerotic lesion formation in mice. Nonetheless, the role of NCEH1 in endothelial dysfunction associated with diabetes has not been explored. The present study sought to investigate whether NCEH1 improved endothelial function in diabetes, and the underlying mechanisms were explored. METHODS: The expression and activity of NCEH1 were determined in obese mice with high-fat diet (HFD) feeding, high glucose (HG)-induced mouse aortae or primary endothelial cells (ECs). Endothelium-dependent relaxation (EDR) in aortae response to acetylcholine (Ach) was measured. RESULTS: Results showed that the expression and activity of NCEH1 were lower in HFD-induced mouse aortae, HG-exposed mouse aortae ex vivo, and HG-incubated primary ECs. HG exposure reduced EDR in mouse aortae, which was exaggerated by endothelial-specific deficiency of NCEH1, whereas NCEH1 overexpression restored the impaired EDR. Similar results were observed in HFD mice. Mechanically, NCEH1 ameliorated the disrupted EDR by dissociating endothelial nitric oxide synthase (eNOS) from caveolin-1 (Cav-1), leading to eNOS activation and nitric oxide (NO) release. Moreover, interaction of NCEH1 with the E3 ubiquitin-protein ligase ZNRF1 led to the degradation of Cav-1 through the ubiquitination pathway. Silencing Cav-1 and upregulating ZNRF1 were sufficient to improve EDR of diabetic aortas, while overexpression of Cav-1 and downregulation of ZNRF1 abolished the effects of NCEH1 on endothelial function in diabetes. Thus, NCEH1 preserves endothelial function through increasing NO bioavailability secondary to the disruption of the Cav-1/eNOS complex in the endothelium of diabetic mice, depending on ZNRF1-induced ubiquitination of Cav-1. CONCLUSIONS: NCEH1 may be a promising candidate for the prevention and treatment of vascular complications of diabetes.

Reversible S-nitrosylation of bZIP67 by peroxiredoxin IIE activity and nitro-fatty acids regulates the plant lipid profile.

Nitric oxide (NO) is a gasotransmitter required in a broad range of mechanisms controlling plant development and stress conditions. However, little is known about the specific role of this signaling molecule during lipid storage in the seeds. Here, we show that NO is accumulated in developing embryos and regulates the fatty acid profile through the stabilization of the basic/leucine zipper transcription factor bZIP67. NO and nitro-linolenic acid target and accumulate bZIP67 to induce the downstream expression of FAD3 desaturase, which is misregulated in a non-nitrosylable version of the protein. Moreover, the post-translational modification of bZIP67 is reversible by the trans-denitrosylation activity of peroxiredoxin IIE and defines a feedback mechanism for bZIP67 redox regulation. These findings provide a molecular framework to control the seed fatty acid profile caused by NO, and evidence of the in vivo functionality of nitro-fatty acids during plant developmental signaling.

Nasal nitric oxide measurement in allergic rhinitis and non-allergic rhinitis: a meta-analysis.

The goal of this meta-analysis was to study nasal nitric oxide (nNO) measurements in allergic rhinitis (AR) and non-allergic rhinitis (non-AR). The protocol was registered with PROSPERO (no: CRD4202124828). Electronic databases from PubMed, Google Scholar, Scopus, Web of Science, and Cochrane were all thoroughly searched and studies were chosen based on the qualifying requirements. The quality of the studies was evaluated by Joanna Briggs Institute evaluation tools, and publication bias using funnel plots. The meta-analysis included 18 studies, whereas the systematic review included 20 studies, totaling 3097 participants (1581 AR, 458 non-AR, and 1058 healthy/control). Patients with AR had significantly greater nNO levels than the control group, although this did not change significantly before or after treatment. AR patients had significantly greater nNO levels than non-AR patients, but there was no significant difference between non-AR patients and healthy controls. Nineteen of the studies were of high quality and the remaining one was of moderate quality. nNO measurement has a promising role in the management of AR and non-AR patients, but more investigations are needed to document clinical benefits.

Pullenvalenes A-D: Nitric Oxide-Mediated Transcriptional Activation (NOMETA) Enables Discovery of Triterpene Aminoglycosides from Australian Termite Nest-Derived Fungi.

We report on the use of nitric oxide-mediated transcriptional activation (NOMETA) as an innovative means to detect and access new classes of microbial natural products encoded within silent biosynthetic gene clusters. A small library of termite nest- and mangrove-derived fungi and actinomyces was subjected to cultivation profiling using a miniaturized 24-well format approach (MATRIX) in the presence and absence of nitric oxide, with the resulting metabolomes subjected to comparative chemical analysis using UPLC-DAD and GNPS molecular networking. This strategy prompted study of Talaromyces sp. CMB-TN6F and Coccidiodes sp. CMB-TN39F, leading to discovery of the triterpene glycoside pullenvalenes A-D (1-4), featuring an unprecedented triterpene carbon skeleton and rare 6-O-methyl-N-acetyl-d-glucosaminyl glycoside residues. Structure elucidation of 1-4 was achieved by a combination of detailed spectroscopic analysis, chemical degradation, derivatization and synthesis, and biosynthetic considerations.

Syagrus coronata fixed oil prevents rotenone-induced movement disorders and oxidative stress in Drosophila melanogaster.

One prominent aspect of Parkinson's disease (PD) is the presence of elevated levels of free radicals, including reactive oxygen species (ROS). Syagrus coronata (S. coronata), a palm tree, exhibits antioxidant activity attributed to its phytochemical composition, containing fatty acids, polyphenols, and flavonoids. The aim of this investigation was to examine the potential neuroprotective effects of S. coronata fixed oil against rotenone-induced toxicity using Drosophila melanogaster. Young Drosophila specimens (3-4 d old) were exposed to a diet supplemented with rotenone (50 µM) for 7 d with and without the inclusion of S. coronata fixed oil (0.2 mg/g diet). Data demonstrated that rotenone exposure resulted in significant locomotor impairment and increased mortality rates in flies. Further, rotenone administration reduced total thiol levels but elevated lipid peroxidation, iron (Fe) levels, and nitric oxide (NO) levels while decreasing the reduced capacity of mitochondria. Concomitant administration of S. coronata exhibited a protective effect against rotenone, as evidenced by a return to control levels of Fe, NO, and total thiols, lowered lipid peroxidation levels, reversed locomotor impairment, and enhanced % cell viability. Molecular docking of the oil lipidic components with antioxidant enzymes showed strong binding affinity to superoxide dismutase (SOD) and glutathione peroxidase (GPX1) enzymes. Overall, treatment with S. coronata fixed oil was found to prevent rotenone-induced movement disorders and oxidative stress in Drosophila melanogaster.

Bradykinin attenuates endothelial-mesenchymal transition following cardiac ischemia-reperfusion injury.

AIMS: Endothelial-mesenchymal transition (EndMT) is a crucial pathological process contributing to cardiac fibrosis. Bradykinin has been found to protect the heart against fibrosis. Whether bradykinin regulates EndMT has not been determined. MATERIALS AND METHODS: Rats were subjected to ligation of the left anterior descending coronary artery for 1 h and subsequent reperfusion to induce cardiac ischemia-reperfusion (IR) injury. Bradykinin (0.5 µg/h) was infused by an osmotic pump implanted subcutaneously at the onset of reperfusion. Fourteen days later, the functional, histological, and molecular analyses were performed to investigate the changes in cardiac fibrosis and EndMT. Human coronary artery endothelial cells were utilized to determine the molecular mechanisms in vitro. RESULTS: Bradykinin treatment improved cardiac function and decreased fibrosis following cardiac IR injury, accompanied by ameliorated EndMT and increased nitric oxide (NO) production. In vitro experiments found that bradykinin mitigated transforming growth factor ß1 (TGFß1)-induced EndMT. Significantly, the bradykinin B2 receptor antagonist or endothelial nitric oxide synthase inhibitor abolished the effects of bradykinin on EndMT inhibition, indicating that the bradykinin B2 receptor and NO might mediate the effects of bradykinin on EndMT inhibition. CONCLUSION: Bradykinin plays an essential role in the process of cardiac fibrosis. Bradykinin preserves the cellular signature of endothelial cells, preventing them from EndMT following cardiac IR injury, possibly mediated by bradykinin B2 receptor activation and NO production.

Dilation of Pregnant Rat Uterine Arteries with Phenols from Extra Virgin Olive Oil Is Endothelium-Dependent and Involves Calcium and Potassium Channels.

During pregnancy, uterine vasculature undergoes significant circumferential growth to increase uterine blood flow, vital for the growing feto-placental unit. However, this process is often compromised in conditions like maternal high blood pressure, particularly in preeclampsia (PE), leading to fetal growth impairment. Currently, there is no cure for PE, partly due to the adverse effects of anti-hypertensive drugs on maternal and fetal health. This study aimed to investigate the vasodilator effect of extra virgin olive oil (EVOO) phenols on the reproductive vasculature, potentially benefiting both mother and fetus. Isolated uterine arteries (UAs) from pregnant rats were tested with EVOO phenols in a pressurized myograph. To elucidate the underlying mechanisms, additional experiments were conducted with specific inhibitors: L-NAME/L-NNA (10-4 M) for nitric oxide synthases, ODQ (10-5 M) for guanylate cyclase, Verapamil (10-5 M) for the L-type calcium channel, Ryanodine (10-5 M) + 2-APB (3 × 10-5 M) for ryanodine and the inositol triphosphate receptors, respectively, and Paxilline (10-5 M) for the large-conductance calcium-activated potassium channel. The results indicated that EVOO-phenols activate Ca2+ signaling pathways, generating nitric oxide, inducing vasodilation via cGMP and BKCa2+ signals in smooth muscle cells. This study suggests the potential use of EVOO phenols to prevent utero-placental blood flow restriction, offering a promising avenue for managing PE.

Fractional exhaled nitric oxide in idiopathic pulmonary arterial hypertension and mixed connective tissue disease complicating pulmonary hypertension.

BACKGROUND: Fractional exhaled nitric oxide (FeNO) has been extensively studied in various causes of pulmonary hypertension (PH), but its utility as a noninvasive marker remains highly debated. The objective of our study was to assess FeNO levels in patients with idiopathic pulmonary arterial hypertension (IPAH) and mixed connective tissue disease complicating pulmonary hypertension (MCTD-PH), and to correlate them with respiratory functional data, disease severity, and cardiopulmonary function. METHODS: We collected data from 54 patients diagnosed with IPAH and 78 patients diagnosed with MCTD-PH at the Shanghai Pulmonary Hospital Affiliated to Tongji University. Our data collection included measurements of brain natriuretic peptide (pro-BNP), cardiopulmonary exercise test (CPET), pulmonary function test (PFT), impulse oscillometry (IOS), and FeNO levels. Additionally, we assessed World Health Organization functional class (WHO-FC) of each patient. RESULTS: (1) The fractional exhaled concentration of nitric oxide was notably higher in patients with IPAH compared to those with MCTD-PH. Furthermore, within the IPAH group, FeNO levels were found to be lower in cases of severe IPAH compared to mild IPAH (P = 0.024); (2) In severe pulmonary hypertension as per the WHO-FC classification, FeNO levels in IPAH exhibited negative correlations with FEV1/FVC (Forced Expiratory Velocity at one second /Forced Vital Capacity), MEF50% (Maximum Expiratory Flow at 50%), MEF25%, and MMEF75/25% (Maximum Mid-expiratory Flow between 75% and 25%), while in severe MCTD-PH, FeNO levels were negatively correlated with R20% (Resistance at 20 Hz); (3) ROC (Receiving operator characteristic curve) analysis indicated that the optimal cutoff value of FeNO for diagnosing severe IPAH was 23ppb; (4) While FeNO levels tend to be negatively correlated with peakPETO2(peak end-tidal partial pressure for oxygen) in severe IPAH, in mild IPAH they had a positive correlation to peakO2/Heart rate (HR). An interesting find was observed in cases of severe MCTD-PH, where FeNO levels were negatively correlated with HR and respiratory exchange ratio (RER), while positively correlated with O2/HR throughout the cardiopulmonary exercise test. CONCLUSION: FeNO levels serve as a non-invasive measure of IPAH severity. Although FeNO levels may not assess the severity of MCTD-PH, their significant makes them a valuable tool when assessing severe MCTD-PH.

A novel multifunctional microneedle patch for synergistic photothermal- gas therapy against maxillofacial malignant melanoma and associated skin defects.

Considering the high recrudescence and the long-lasting unhealed large-sized wound that affect the aesthetics and cause dysfunction after resection of maxillofacial malignant skin tumors, a groundbreaking strategy is urgently needed. Photothermal therapy (PTT), which has become a complementary treatment of tumors, however, is powerless in tissue defect regeneration. Therefore, a novel multifunctional sodium nitroprusside and Fe2+ ions loaded microneedles (SNP-Fe@MNs) platform was fabricated by accomplishing desirable NIR-responsive photothermal effect while burst releasing nitric oxide (NO) after the ultraviolet radiation for the ablation of melanoma. Moreover, the steady releasing of NO in the long term by the platform can exert its angiogenic effects via upregulating multiple related pathways to promote tissue regeneration. Thus, the therapeutic dilemma caused by postoperative maxillofacial skin malignancies could be conquered through promoting tumor cell apoptosis via synergistic PTT-gas therapy and subsequent regeneration process in one step. The bio-application of SNP-Fe@MNs could be further popularized based on its ideal bioactivity and appealing features as a strategy for synergistic therapy of other tumors occurred in skin.

Effects of Goose Astrovirus Type 2 Infection on Peripheral Blood Lymphocyte and Macrophage Activity In Vitro.

Goose astrovirus type 2 (GAstV-2) is a novel pathogen causing visceral gout in goslings; it not only causes necrosis of renal epithelial cells but also causes spleen damage, indicating that GAstV-2 induces immunosuppression in goslings. However, to date, the interaction between GAstV-2 and immune cells remains unclear. In this study, peripheral blood lymphocytes and macrophages were isolated from goslings without GAstV-2 infection and then inoculated in vitro with GAstV-2, and the virus localization in the lymphocytes and macrophages, proliferation and apoptosis of lymphocytes, and phagocytic activity, reactive oxygen species (ROS) and nitric oxide (NO) production, and cell polarity in macrophages were determined. The results showed that GAstV-2 was observed in the cytoplasm of CD4 and CD8 T cells and macrophages, indicating that GAstV-2 can infect both lymphocytes and macrophages. GAstV-2 infection reduced the lymphocyte proliferation induced by Concanavalin A and lipopolysaccharide stimulation and increased the lymphocyte apoptosis rate and mRNA expression of Fas, demonstrating that GAstV-2 causes damage to lymphocytes. Moreover, GAstV-2 infection enhanced phagocytic activity and production of ROS and NO and induced a proinflammatory phenotype in macrophages (M1 macrophages), indicating that macrophages play an antiviral role during GAstV-2 infection. In conclusion, these results demonstrate that GAstV-2 infection causes damages to lymphocytes, and host macrophages inhibit GAstV-2 invasion during infection.

Regulation of endogenous hormone and miRNA in leaves of alfalfa (Medicago sativa L.) seedlings under drought stress by endogenous nitric oxide.

BACKGROUND: Alfalfa (Medicago sativa. L) is one of the best leguminous herbage in China and even in the world, with high nutritional and ecological value. However, one of the drawbacks of alfalfa is its sensitivity to dry conditions, which is a global agricultural problem. The objective of this study was to investigate the regulatory effects of endogenous nitric oxide (NO) on endogenous hormones and related miRNAs in alfalfa seedling leaves under drought stress. The effects of endogenous NO on endogenous hormones such as ABA, GA3, SA, and IAA in alfalfa leaves under drought stress were studied. In addition, high-throughput sequencing technology was used to identify drought-related miRNAs and endogenous NO-responsive miRNAs in alfalfa seedling leaves under drought stress. RESULT: By measuring the contents of four endogenous hormones in alfalfa leaves, it was found that endogenous NO could regulate plant growth and stress resistance by inducing the metabolism levels of IAA, ABA, GA3, and SA in alfalfa, especially ABA and SA in alfalfa. In addition, small RNA sequencing technology and bioinformatics methods were used to analyze endogenous NO-responsive miRNAs under drought stress. It was found that most miRNAs were enriched in biological pathways and molecular functions related to hormones (ABA, ETH, and JA), phenylpropane metabolism, and plant stress tolerance. CONCLUSION: In this study, the analysis of endogenous hormone signals and miRNAs in alfalfa leaves under PEG and PEG + cPTIO conditions provided an important basis for endogenous NO to improve the drought resistance of alfalfa at the physiological and molecular levels. It has important scientific value and practical significance for endogenous NO to improve plant drought resistance.

Nitric oxide-induced lipophagic defects contribute to testosterone deficiency in rats with spinal cord injury.

Introduction: Males with acute spinal cord injury (SCI) frequently exhibit testosterone deficiency and reproductive dysfunction. While such incidence rates are high in chronic patients, the underlying mechanisms remain elusive. Methods and results: Herein, we generated a rat SCI model, which recapitulated complications in human males, including low testosterone levels and spermatogenic disorders. Proteomics analyses showed that the differentially expressed proteins were mostly enriched in lipid metabolism and steroid metabolism and biosynthesis. In SCI rats, we observed that testicular nitric oxide (NO) levels were elevated and lipid droplet-autophagosome co-localization in testicular interstitial cells was decreased. We hypothesized that NO impaired lipophagy in Leydig cells (LCs) to disrupt testosterone biosynthesis and spermatogenesis. As postulated, exogenous NO donor (S-nitroso-N-acetylpenicillamine (SNAP)) treatment markedly raised NO levels and disturbed lipophagy via the AMPK/mTOR/ULK1 pathway, and ultimately impaired testosterone production in mouse LCs. However, such alterations were not fully observed when cells were treated with an endogenous NO donor (L-arginine), suggesting that mouse LCs were devoid of an endogenous NO-production system. Alternatively, activated (M1) macrophages were predominant NO sources, as inducible NO synthase inhibition attenuated lipophagic defects and testosterone insufficiency in LCs in a macrophage-LC co-culture system. In scavenging NO (2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO)) we effectively restored lipophagy and testosterone levels both in vitro and in vivo, and importantly, spermatogenesis in vivo. Autophagy activation by LYN-1604 also promoted lipid degradation and testosterone synthesis. Discussion: In summary, we showed that NO-disrupted-lipophagy caused testosterone deficiency following SCI, and NO clearance or autophagy activation could be effective in preventing reproductive dysfunction in males with SCI.

Platelet rich plasma alleviates endometritis induced by lipopolysaccharide in mice via inhibiting TLR4/NF-κB signaling pathway.

BACKGROUND: Endometritis is an inflammatory reaction of the lining of uterus, leading to the occurrence of infertility. Platelet rich plasma (PRP) has been proven to exhibit extremely effective for the treatment of endometrium-associated infertility, but the mechanism of its prevention for endometritis remains unclear. OBJECTIVE: The present study aimed to investigate the protective effect of PRP against endometritis induced by lipopolysaccharide (LPS) and elucidate the mechanism underlying these effects. METHODS: Mouse model of endometritis was established by intrauterine perfusion of LPS. PRP intrauterine infusion was administered at 24 h after LPS induction. After another 24 h, the uterine tissues were harvested to observe histopathological changes, production of proinflammatory cytokines, variation of the Toll-like receptor 4/nuclear factor κB (TLR4/NF-κB) signaling pathways, and validated the anti-inflammatory effect of PRP. The myeloperoxidase (MPO) activity and concentration of nitric oxide (NO) were determined using assay kit. Proinflammatory chemokines (tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6)) were measured by ELISA and Real-Time PCR. The activity of TLR4/NF-κB pathway in uterine tissues was measured by Western blotting. RESULTS: Hematoxylin-eosin staining (H&E) appeared that PRP remarkably relieved the impairment of uterine tissues. Detection of MPO activity and concentration of NO revealed that PRP treatment distinctly mitigated infiltration of inflammatory cells in mice with endometritis induced by LPS. PRP treatment significantly affected the expression of TNF-α, IL-1ß, and IL-6. PRP was also found to suppress LPS-induced activation of TLR4/NF-κB pathway. CONCLUSION: PRP effectively alleviates LPS-induced endometritis via restraining the signal pathway of TLR4/NF-κB. These findings provide a solid foundation for PRP as a potential therapeutic agent for endometritis.

Anti-Inflammatory Effect of Chamaecyparis obtusa (Siebold & Zucc.) Endl. Leaf Essential Oil.

Chamaecyparis obtusa (Siebold & Zucc.) Endl. (C. obtusa) belongs to the Cupressaceae family and is native to East Asian regions. Essential oils extracted from the leaves, bark, branches, and roots of C. obtusa have both aesthetic and medicinal properties and are thus widely used. However, detailed analyses of the active ingredients of C. obtusa extract are lacking. In this study, the sabinene content in the hydro-distillation of C. obtusa leaf essential oil (COD) was analyzed using GC-MS, and the anti-inflammatory effect of COD was compared with that of pure sabinene. Cell viability was evaluated by MTT assay, and nitric oxide (NO) production was measured using Griess reagent. Relative mRNA and protein levels were analyzed using RT-qPCR and western blot, and secreted cytokines were analyzed using a cytokine array kit. The results showed that both COD and sabinene inhibited the expression of inducible nitric oxide synthase (iNOS) and the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 in lipopolysaccharide (LPS)-induced RAW 264.7 cells. COD and sabinene also reduced the production of pro-inflammatory cytokines interleukin (IL)-1ß, IL-6, IL-27, IL-1 receptor antagonist (IL-1ra), and granulocyte-macrophage colony-stimulating factor (GM-CSF). The anti-inflammatory mechanisms of COD and sabinene partially overlap, as COD was shown to inhibit MAPKs and the JAK/STAT axis, and sabinene inhibited MAPKs, thereby preventing LPS-induced macrophage activation.

Effects of Photobiomodulation Therapy on the Expression of Hypoxic Inducible Factor, Vascular Endothelial Growth Factor, and Its Specific Receptor: A Randomized Control Trial in Patients with Diabetic Foot Ulcer.

Background: Impaired angiogenesis is a significant factor contributing to delayed healing in diabetic foot ulcers (DFUs) due to inadequate oxygenation. Objective: This study aimed to investigate the impact of photobiomodulation (PBM) using a Ga-As laser on the release of serum hypoxia-inducible factor 1-α (HIF-1α), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2, and nitric oxide (NO) in diabetic patients with DFUs. Materials and methods: In this double-blind RCT, a total of 30 patients with grade II DFUs were enrolled. The patients were randomly divided into two groups: the PBM (n = 15) and the placebo (n = 15). In the PBM group, a Ga-As laser (904 nm, 2 J/cm2, 90 W) was given for 3 days/week for 4 weeks (11 sessions). In the placebo group, the power was turned off. Both groups received similar standard wound care. Before and after interventions, the levels of serum HIF-1α, VEGF, NO, and sVEGFR-2 were measured. In addition, the percentage decrease in the wound surface area (%DWSA) was measured. Results: Following the intervention, the results revealed that the PBM group had significantly lower levels of VEGF than the placebo group (p = 0.005). The %DWSA was significantly higher in the PBM group compared to the placebo group (p = 0.003). Moreover, VEGF showed a significant negative correlation with %DWSA (p < 0.001). Conclusions: The observed decrease in serum levels of VEGF and an increase in %DWSA, compared to the placebo group, suggests that PBM effectively improves angiogenesis. Furthermore, the significant correlation found between VEGF levels and %DWSA emphasizes the importance of evaluating wound surface in patients as a dependable indicator of enhanced wound angiogenesis. Clinical Trial Registration: NCT02452086.

Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events.

KEY MESSAGE: Pepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration. Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO-), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), L-cysteine (L-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.

Improved synthesis, molecular modeling and anti-inflammatory activity of new fluorinated dihydrofurano-naphthoquinone compounds.

A series of new fluorinated dihydrofurano-napthoquinone compounds were sucessfully synthesized in good yields using microwave-assisted multi-component reactions of 2-hydroxy-1,4-naphthoquinone, fluorinated aromatic aldehydes, and pyridinium bromide. The products were fully characterized using spectroscopic techniques and evaluated for their anti-inflammatory activity using lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Among 12 new compounds, compounds 8b, 8d, and 8e showed high potent NO inhibitory activity in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells with IC50 values ranging from 1.54 to 3.92 µM. The levels of pro-inflammatory cytokines IL-1ß and IL-6 in LPS-stimulated RAW264.7 macrophages were remarkably decreased after the application of 8b, 8d, 8e and 8k. Molecular docking simulations revealed structure-activity relationships of 8b, 8d, and 8e toward NO synthase, cyclooxygenase (COX-2 over COX-1), and prostaglandin E synthase-1 (mPGES-1). Further physicochemical and pharmacokinetic computations also demonstrated the drug-like characteristics of synthesized compounds. These findings demonstrated the importance of fluorinated dihydrofurano-napthoquinone moieties in the development of potential anti-inflammatory agents.

L-arginine mitigates bleomycin-induced pulmonary fibrosis in rats through regulation of HO-1/PPAR-γ/ß-catenin axis.

Pulmonary fibrosis is a chronic and progressively deteriorating lung condition that can be replicated in laboratory animals by administering bleomycin, a chemotherapeutic antibiotic known for its lung fibrosis-inducing side effects. L-arginine, a semi-essential amino acid, is recognized for its diverse biological functions, including its potential to counteract fibrosis. This study aimed to evaluate the antifibrotic properties of L-arginine on bleomycin-induced pulmonary fibrosis in rats. The administration of a single intratracheal dose of bleomycin resulted in visible and microscopic damage to lung tissues, an uptick in oxidative stress markers, and an elevation in inflammatory, apoptotic, and fibrotic indicators. A seven-day treatment with L-arginine post-bleomycin exposure markedly improved the gross and histological architecture of the lungs, prevented the rise of malondialdehyde and carbonyl content, and enhanced total antioxidant capacity alongside the activities of antioxidant enzymes. Also, L-arginine attenuated the expression of the pro-fibrotic factors, transforming growth factor-ß and lactate dehydrogenase in bronchoalveolar lavage fluid. In the lung tissue, L-arginine reduced collagen deposition, hydroxyproline concentration, and mucus production, along with decreasing expression of α-smooth muscle actin, tumor necrosis factor-α, caspase-3, matrix metalloproteinase-9, and ß-catenin. Moreover, it boosted levels of nitric oxide and upregulated the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), heme oxygenase-1 (HO-1), and E-cadherin and downregulating the expression of ß-catenin. These findings suggest that L-arginine has preventive activities against bleomycin-induced pulmonary fibrosis. This effect can be attributed to the increased production of nitric oxide, which modulates the HO-1/PPAR-γ/ß-catenin axis.

A 2-week treatment with 5-azacytidine improved the hypercontractility state in prostate from obese mice: Role of the nitric oxide-cyclic guanosine monophosphate signalling pathway.

Benign prostatic hyperplasia (BPH) is characterised by increases in prostate volume and contraction. Downregulation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway contributes to prostate dysfunctions. Previous studies in cancer cells or vessels have shown that the epigenetic mechanisms control the gene and protein expression of the enzymes involved in the production of NO and cGMP. This study is aimed to evaluate the effect of a 2-week treatment of 5-azacytidine (5-AZA), a DNA-methyltransferase inhibitor, in the prostate function of mice fed with a high-fat diet. Functional, histological, biochemical and molecular assays were carried out. Obese mice presented greater prostate weight, α-actin expression and contractile response induced by the α-1adrenoceptors agonist. The relaxation induced by the NO-donor and the protein expression of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) were significantly decreased in the prostate of obese mice. The treatment with 5-AZA reverted the higher expression of α-actin, reduced the hypercontractility state of the prostate and increased the expression of eNOS and sGC and intraprostatic levels of cGMP. When prostates from obese mice treated with 5-AZA were incubated in vitro with inhibitors of the NOS or sGC, the inhibitory effect of 5-AZA was reverted, therefore, showing the involvement of NO and cGMP. In conclusion, our study paves the way to develop or repurpose therapies that recover the expression of eNOS and sGC and, hence, to improve prostate function in BPH.

Protein phosphatase 4 mediates palmitic acid-induced endothelial dysfunction by decreasing eNOS phosphorylation at serine 633 in HUVECs.

Plasma saturated free fatty acid (FFA)-induced endothelial dysfunction (ED) contributes to the pathogenesis of atherosclerosis and cardiovascular diseases. However, the mechanism underlying saturated FFA-induced ED remains unclear. This study demonstrated that palmitic acid (PA) induced ED by activating the NADPH oxidase (NOX)/ROS signaling pathway to activate protein phosphatase 4 (PP4) and protein phosphatase 2A (PP2A), thereby reducing endothelial nitric oxide synthase (eNOS) phosphorylation at Ser633 and Ser1177, respectively. Okadaic acid (OA) and fostriecin (FST), which are inhibitors of PP2A, inhibited the PA-induced decreases in eNOS phosphorylation at Ser633 and Ser1177. The antioxidants N-acetylcysteine (NAC) and apocynin (APO) or knockdown of gp91phox or p67phox (NOX subunits) restored PA-mediated downregulation of PP4R2 protein expression and eNOS Ser633 phosphorylation. Knockdown of the PP4 catalytic subunit (PP4c) specifically increased eNOS Ser633 phosphorylation, while silencing the PP2A catalytic subunit (PP2Ac) restored only eNOS Ser1177 phosphorylation. Furthermore, PA dramatically decreased the protein expression of the PP4 regulatory subunit R2 (PP4R2) but not the other regulatory subunits. PP4R2 overexpression increased eNOS Ser633 phosphorylation, nitric oxide (NO) production, cell migration and tube formation but did not change eNOS Ser1177 phosphorylation levels. Coimmunoprecipitation (Co-IP) suggested that PP4R2 and PP4c interacted with the PP4R3α and eNOS proteins. In summary, PA decreases PP4R2 protein expression through the Nox/ROS pathway to activate PP4, which contributes to ED by dephosphorylating eNOS at Ser633. The results of this study suggest that PP4 is a novel therapeutic target for ED and ED-associated vascular diseases.