Nitric Oxide Synthase Research Articles

Whole genome sequencing of two strains of Staphylococcus simulans and their effects on the nitrosomyoglobin formation based on nitric oxide synthase

In order to evaluate the safety and functional characteristics of two potential meat starter cultures, Staphylococcus simulans HZ01 (OM758216) and Staphylococcus simulans HZ02 (OM758217), we performed the whole genome sequences, and then investigated their effects on nitrosomyoglobin formation based on nitric oxide synthase (NOS) catalysis. The results revealed that the two strains did not possess any virulence genes. The gene annotation indicated the gene function is mainly due to translation, ribosomal structure and biogenesis, and amino acid transport and metabolism. Both strains possess nos gene. The phylogenetic tree based on nos gene indicated the two strains formed an independent evolutionary branch. Subsequently, NOS was found to be expressed in both HZ01 and HZ02. NOS activity was higher in HZ01 than in HZ02, whereas NO production in inoculated groups changed considerably with increasing culture time. In the model system, nitrosomyoglobin formed in S. simulans inoculated groups, and the derivatives had absorption peaks at 545 nm and 580 nm. The secondary structure of these derivatives exhibited differences in β-turn proportion and α-helix proportion. Physicochemical properties of fermented sausages inoculated groups indicated that S. simulans HZ01 and HZ02 can significantly improve the color of sausages with no addition of nitrite/nitrate. This result confirmed that the two S. simulans strains can improve the nitrosomyoglobin formation based on the NOS pathway, which contributes to the color of fermented sausages, without the requirement of nitrite or nitrate. Nevertheless, the contribution of the NOS pathway in fermented meat products should be further evaluated.

Investigation of Ginseng-Ophiopogon Injection on Enhancing Physical Function by Pharmacogenomics and Metabolomics Evaluation.

Ginseng-ophiopogon injection (GOI) is a clinically commonly used drug for Qi deficiency syndrome characterized by decreased physical function in China. This study aimed to clarify common pharmacological mechanisms of GOI in enhancing physical function. We performed an integrative strategy of weight-loaded swimming tests in cold water (5.5 °C), hepatic glycogen and superoxide dismutase (SOD) detections, GC-TOF/MS-based metabolomics, multivariate statistical techniques, network pharmacology of known targets and constituents, and KEGG pathway analysis of GOI. Compared with the control group, GOI showed significant increases in the weightloaded swimming time, hepatic levels of glycogen and SOD. Additionally, 34 significantly differential serum metabolites referred to glycolysis, gluconeogenesis and arginine biosynthesis were affected by GOI. The target collection revealed 98 metabolic targets and 50 experimentreported drug targets of ingredients in GOI involved in enhancing physical function. Further, the PPI network analysis revealed that 8 ingredients of GOI, such as ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, and notoginsenoside R1, were well-associated with 48 hub targets, which had good ability in enhancing physical function. Meanwhile, nine hub proteins, such as SOD, mechanistic target of Rapamycin (mTOR), and nitric oxide synthases, were confirmed to be affected by GOI. Finally, 98 enriched KEGG pathways (P<0.01 and FDR<0.001) of GOI were obtained from 48 hub targets of the PPI network. Among them, pathways in cancer, Chagas disease, lipid and atherosclerosis, and PI3K-Akt signaling pathway ranked top four. This study provided an integrative and efficient approach to understanding the molecular mechanism of GOI in enhancing physical function.

Role of Nitric oxide synthase II in the cognitive impairment due to experimental cerebral malaria

The role of nitric oxide (NO) in the pathogenesis of cerebral malaria and its cognitive sequelae remains controversial. Cerebral malaria is still the worst complication of Plasmodium falciparum infection, which is characterized by high rates of morbidity and mortality. Even after recovery from infection due to antimalarial therapy, the development of cognitive impairment in survivors reinforces the need to seek new therapies that demonstrate efficacy in preventing long-lasting sequelae. During disease pathogenesis, reactive oxygen and nitrogen species (RONS) are produced after the established intense inflammatory response. Increased expression of the enzyme inducible nitric oxide synthase (iNOS) seems to contribute to tissue injury and the onset of neurological damage. Elevated levels of NO developed by iNOS can induce the production of highly harmful nitrogen-reactive intermediates such as peroxynitrite. To adress this, we performed biochemical and behavioral studies in C57BL6 mice, aminoguanidine (specific pharmacological inhibitor of the enzyme iNOS) treated and iNOS-/-, infected with Plasmodium berghei ANKA (PbA), with the aim of clarifying the impact of iNOS on the pathogenesis of cerebral malaria. Our findings underscore the effectiveness of both strategies in reducing cerebral malaria and providing protection against the cognitive impairment associated with the disease. Here, the absence or blockade of the iNOS enzyme was effective in reducing the signs of cerebral malaria detected after six days of infection. This was accompanied by a decrease in the production of pro-inflammatory cytokines and reactive oxygen and nitrogen species. In addition, nitrotyrosine (NT-3), a marker of nitrosative stress, was also reduced. Futher, cognitive dysfunction was analyzed fifteen days after infection in animals rescued from infection by chloroquine treatment (25 mg/kg bw). We observed that both interventions on the iNOS enzyme were able to improve memory and learning loss in mice. In summary, our data suggest that the iNOS enzyme has the potential to serve as a therapeutic target to prevent cognitive sequelae of cerebral malaria.

Exploring the Promising Role of Guggulipid in Rheumatoid Arthritis Management: An In-depth Analysis.

Guggulipid, an oleo-gum resin extracted from the bark of Commiphora wightii of the Burseraceae family, holds a significant place in Ayurvedic medicine due to its historical use in treating various disorders, including inflammation, gout, rheumatism, obesity, and lipid metabolism imbalances. This comprehensive review aims to elucidate the molecular targets of guggulipids and explore their cellular responses. Furthermore, it summarizes the findings from in-vitro, in-vivo, and clinical investigations related to arthritis and various inflammatory conditions. A comprehensive survey encompassing in-vitro, in-vivo, and clinical studies has been conducted to explore the therapeutic capacity of guggulipid in the management of rheumatoid arthritis. Various molecular pathways, such as cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), PI3-kinase/AKT, JAK/STAT, nitric oxide synthase (iNOS), and NFκB signaling pathways, have been targeted to assess the antiarthritic and anti-inflammatory effects of this compound. The research findings reveal that guggulipid demonstrates notable antiarthritic and anti-inflammatory effects by targeting key molecular pathways involved in inflammatory responses. These pathways include COX-2, VEGF, PI3-kinase/AKT, JAK/STAT, iNOS, and NFκB signaling pathways. in-vitro, in-vivo, and clinical studies collectively support the therapeutic potential of guggulipid in managing rheumatoid arthritis and related inflammatory conditions. This review provides a deeper understanding of the therapeutic mechanisms and potential of guggulipid in the management of rheumatoid arthritis. The collective evidence strongly supports the promising role of guggulipid as a therapeutic agent, encouraging further research and development in guggulipid-based treatments for these conditions.

Riboflavin kinase binds and activates inducible nitric oxide synthase to reprogram macrophage polarization

Riboflavin kinase (RFK) is essential in riboflavin metabolism, converting riboflavin to flavin mononucleotide (FMN), which is further processed to flavin adenine dinucleotide (FAD). While RFK enhances macrophage phagocytosis of Listeria monocytogenes, its role in macrophage polarization is not well understood. Our study reveals that RFK deficiency impairs M(IFN-γ) and promotes M(IL-4) polarization, both in vitro and in vivo. Mechanistically, RFK interacts with inducible nitric oxide (NO) synthase (iNOS), which requires FMN and FAD as cofactors for activation, leading to increased NO production that alters energy metabolism by inhibiting the tricarboxylic acid cycle and mitochondrial electron transport chain. Exogenous FAD reverses the metabolic and polarization changes caused by RFK deficiency. Furthermore, bone marrow adoptive transfer from high-riboflavin-fed mice into wild-type tumor-bearing mice reprograms tumor-associated macrophage polarization and inhibits tumor growth. These results suggest that targeting RFK-iNOS or modulating riboflavin metabolism could be potential therapies for macrophage-related immune diseases.

Deletion of AT1a receptors selectively in the proximal tubules of the kidney alters the hypotensive and natriuretic response to atrial natriuretic peptide via NPRA/cGMP/NO signaling.

In the proximal tubules of the kidney, angiotensin II (ANG II) binds and activates ANG II type 1 (AT1a) receptors to stimulate proximal tubule Na+ reabsorption, whereas atrial natriuretic peptide (ANP) binds and activates natriuretic peptide receptors (NPRA) to inhibit ANG II-induced proximal tubule Na+ reabsorption. These two vasoactive systems play important counteracting roles to control Na+ reabsorption in the proximal tubules and help maintain blood pressure homeostasis. However, how AT1a and NPRA receptors interact in the proximal tubules and whether natriuretic effects of NPRA receptor activation by ANP may be potentiated by deletion of AT1 (AT1a) receptors selectively in the proximal tubules have not been studied previously. The present study used a novel mouse model with proximal tubule-specific knockout of AT1a receptors, PT-Agtr1a-/-, to test the hypothesis that deletion of AT1a receptors selectively in the proximal tubules augments the hypotensive and natriuretic responses to ANP. Basal blood pressure was about 16 ± 3 mmHg lower (P < 0.01), fractional proximal tubule Na+ reabsorption was significantly lower (P < 0.05), whereas 24-h urinary Na+ excretion was significantly higher, in PT-Agtr1a-/- mice than in wild-type mice (P < 0.01). Infusion of ANP via osmotic minipump for 2 wk (0.5 mg/kg/day ip) further significantly decreased blood pressure and increased the natriuretic response in PT-Agtr1a-/- mice by inhibiting proximal tubule Na+ reabsorption compared with wild-type mice (P < 0.01). These augmented hypotensive and natriuretic responses to ANP in PT-Agtr1a-/- mice were associated with increased plasma and kidney cGMP levels (P < 0.01), kidney cortical NPRA and NPRC mRNA expression (P < 0.05), endothelial nitric oxide (NO) synthase (eNOS) and phosphorylated eNOS proteins (P < 0.01), and urinary NO excretion (P < 0.01). Taken together, the results of the present study provide further evidence for important physiological roles of intratubular ANG II/AT1a and ANP/NPRA signaling pathways in the proximal tubules to regulate proximal tubule Na+ reabsorption and maintain blood pressure homeostasis.NEW & NOTEWORTHY This study used a mutant mouse model with proximal tubule-selective deletion of angiotensin II (ANG II) type 1 (AT1a) receptors to study, for the first time, important interactions between ANG II/AT1 (AT1a) receptor/Na+/H+ exchanger 3 and atrial natriuretic peptide (ANP)/natriuretic peptide receptor (NPRA)/cGMP/nitric oxide signaling pathways in the proximal tubules. The results of the present study provide further evidence for important physiological roles of proximal tubule ANG II/AT1a and ANP/NPRA signaling pathways in the regulation of proximal tubule Na+ reabsorption and blood pressure homeostasis.

Neuroprotective and anti-inflammatory effects of the RIPK3 inhibitor GSK872 in an MPTP-induced mouse model of Parkinson’s disease

Parkinson's disease (PD) is a neurodegenerative disorder triggered by the loss of dopaminergic neurons in the substantia nigra (SN). Recent studies have demonstrated that necroptosis is involved in dopaminergic neuronal cell death and the resulting neuroinflammation. During the process of necroptosis, a necrosome complex is formed consisting of the proteins receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL). Although the neuroprotective effects of the RIPK1-specific inhibitor necrostatin-1, as well as RIPK3 and MLKL knockout in mice, have been described, the effects of RIPK3 pharmacological inhibitors have not yet been reported in animal models of PD. In the present study, we investigated the neuroprotective effects of GSK872, a specific RIPK3 inhibitor, in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. GSK872 rescued MPTP-induced motor impairment and inhibited tyrosine hydroxylase-positive dopaminergic cell death in the SN and striatum. Additionally, GSK872 inhibited the MPTP-induced increase in the expression of p-RIPK3 and p-MLKL in both the dopaminergic neurons and microglia, as assessed by biochemical and histological analyses. GSK872 further inhibited microglial activation and the expression of inflammatory mediators including NLRP3, interleukin (IL)-1β, IL-6, tumor necrosis factor-alpha, and inducible nitric oxide synthase in the SN region of MPTP mice. Using in vitro experiments, we validated the effects of GSK872 on necroptosis in SH-SY5Y neuronal and BV2 microglial cells. Overall, our results suggest that GSK872 exerts neuroprotective and anti-inflammatory effects, and may thus have therapeutic potential for PD.

Butyrate-Mediated Modulation of Paraoxonase-1 Alleviates Cardiorenometabolic Abnormalities in a Rat Model of Polycystic Ovarian Syndrome.

Polycystic ovarian syndrome (PCOS) has been associated with cardiovascular risks and comorbid pathologies, particularly cardiorenal disorder. Short-chain fatty acids (SCFAs), especially butyrate, are essential fatty acids that regulate metabolic health and ameliorate granulosa inflammation in PCOS. However, the effect of butyrate on cardiorenal damage associated with PCOS is unknown. This study investigated the impact of SCFA and butyrate on cardiorenal abnormalities in PCOS rat model and the probable involvement of paraoxonase-1 (PON-1). Eight-week-old female Wistar rats were allotted into three groups, n = 5, namely control (CTL), PCOS (LEZ), and LEZ + BUT. Induction of PCOS with letrozole (1mg/kg) lasted for 21days, while treatment with butyrate (200mg/kg) commenced after the induction and lasted for 6weeks uninterruptedly. PCOS rats showed hyperandrogenism, multiple ovarian cysts, disrupted metabolic indices (fasting insulin and homeostatic model of insulin resistance), and increased (p < 0.05) plasma troponin T, urea, and creatinine, as well as increased cardiac/renal stroma cell-derived factor-1/caspase-6, malondialdehyde/nuclear factor-kappaB, transforming growth factor-β1, and renal ϒ-glutamyl transferase, while a significant decrease (p < 0.05) in systemic nitric oxide/endothelial nitric oxide synthase and cardiac/renal hypoxia-inducible factor-1α and nuclear factor erythroid 2-related factor 2, which were accompanied with a decreased level of PON-1. These systemic and cardiorenal derangements were reversed by butyrate administration. The results demonstrate the therapeutic benefits of SCFAs, butyrate, against cardiorenometabolic disorder in a model of PCOS. This beneficial effect is accompanied by an elevated level of PON-1. The present data possibly provides a preclinical relevance for the management of cardiorenal syndrome in PCOS.

Herbacetin ameliorates lipopolysaccharide-elicited inflammatory response by suppressing NLRP-3/AIM-2 inflammasome activation, PI3K/Akt/MAPKs/NF-κB redox inflammatory signalling, modulating autophagy and macrophage polarization imbalance.

Herbacetin, a flavonol abundant in traditional medicines, is documented as an anti-inflammatory agent. However, information regarding its attributes on lipopolysaccharide (LPS)-induced inflammatory immunopathies has not been delineated yet. The present study aimed to comprehend herbacetin effects on LPS-induced aspects of unwarranted, non-resolving inflammation, particularly via targeting the vicious circle of oxi-inflammatory stress, autophagy-apoptosis, macrophages polarization, impaired inflammasome activation, and inflammatory cascades. In-vitro model of LPS-stimulated RAW 264.7 macrophage was recapitulated to investigate different inflammatory anomalies using enzyme-linked immunosorbent assay, qRT-PCR (Real-Time Quantitative Reverse Transcription PCR), immunoblotting. Concanavalin A challenged splenocytes and in silico studies were performed to measure Tregs population and binding affinity, respectively. Herbacetin administration caused remarkable reduction in nitric oxide, reactive oxygen species, mitochondrial membrane potential hyperpolarization, tumor necrosis factor-α, interferon-γ, interleukin-6, inducible nitric oxide synthase and ratio of M1/M2 markers (inducible nitric oxide synthase/arginase-1/macrophage scavenger receptor-1/mannose receptor C type-1) in in vitro model of persistent inflammation. Suppression of interleukins-5,17 and matrix metalloproteinases-2,3,9,13 and proliferating cell nuclear antigen, signifies its anti-inflammatory attributes. Noticeable decline in monodansylcadaverine-Lysotracker staining, caspase-6, and enhanced p62, B-cell lymphoma-2 expression indicates apoptosis-autophagosome accumulation inhibition and lysosomal destabilization. These were accompanied by reduced NLRP3 activation, caspase-1, AIM-2 expression, and interleukin-1β release. Subsequently, up-regulated activation of TLR-4, NF-κB, PI3K, Akt, ERK1/2, and JNK was decisively thwarted by herbacetin. In silico investigation signified the interaction of herbacetin with these targets. Decreased cytokines and enhanced Tregs conferred its role in extenuating inflammation facilitated by T-cells depletion. Collectively, these findings comprehend attributes of herbacetin as an alternative therapeutic strategy in relieving LPS-associated chronic inflammatory disorders.

Long-term effects of sub-chronic exposure to L-NAME on reproductive system of male rats.

Nω-nitro-l-arginine methyl ester (L-NAME) has been utilized as a nitric oxide synthase antagonist for many years in both basic and clinical research settings to assess its therapeutic potential. Though a number of studies have shown the effect of L-NAME on testicular function, the information regarding the reversibility of these effects upon L-NAME withdrawal is limited. In the present study, male rats (68-80days old) divided randomly into three groups received different doses of L-NAME, i.e. 20mg/kg bw (L20) and 10mg/kg bw (L10) in drinking water, and drinking water only (control) through oral gavage daily for three weeks. The rats were monitored for and sacrificed after 60days of L-NAME treatment termination. The animals had a significantly higher (p < 0.01) mean blood pressure compared to control. Aberrant histological changes were observed in the testes of L-NAME-treated rats. A significant reduction (p < 0.05) in the sperm count and an increase in abnormal sperm morphology (p < 0.05) was observed in L-NAME treated rats. Moreover, the spermatogenic cycle was found to be altered in L-NAME treated rats. No change was observed in serum estradiol levels, while serum testosterone levels were significantly increased (p < 0.05) in L10 and L20 animals. The intra-testicular testosterone was increased significantly (p < 0.01) in L20 animals. A significant decrease (p < 0.05) in superoxide dismutase activity was observed in L20 animals. The sub-chronic exposure to L-NAME resulted in higher mean arterial blood pressure and long-term testicular tissue damage, affecting sperm quality and spermatogenesis.

The alarmin, interleukin-33, increases vascular tone via extracellular signal regulated kinase-mediated Ca2+ sensitization and endothelial nitric oxide synthase inhibition.

Alarmins are classified by their release from damaged or ruptured cells. Many alarmins have been found to increase vascular tone and oppose endothelium-dependent dilatation (EDD). Interleukin (IL)-33 plays a prominent role in lung injury and can be released during vascular injury and in chronic studies found to be cardioprotective. Our recent work has implicated IL-33 in acute vascular dysfunction following inhalation of engineered nanomaterials (ENM). However, the mechanisms linking IL-33 to vascular tone have not been interrogated. We therefore aimed to determine whether IL-33 directly influenced microvascular tone and endothelial function. Isolated feed arteries and in vivo arterioles from male and female Sprague-Dawley rats were used to determine direct vascular actions of IL-33. Mesenteric feed arteries and arterioles demonstrated reduced intraluminal diameters when treated with increasing concentrations of recombinant IL-33. IL-33 activated extracellular signal regulated kinase (ERK)1/2 of rat aortic smooth muscle cells but not phosphorylation of myosin light chain kinase. This suggested IL-33may sensitize arterioles to Ca2+-mediated responses. Indeed, IL-33 augmented the myogenic- and phenylephrine-induced vasoconstriction. Additionally, incubation of arterioles with 1ng IL-33 attenuated ACh-mediated EDD. Mechanistically, in human aortic endothelial cells, we demonstrate that IL-33-mediated ERK1/2 activation leads to inhibitory phosphorylation of serine 602 on endothelial nitric oxide synthase. Finally, we demonstrate that IL-33-ERK1/2 contributes to vascular tone following two known inducers of IL-33; ENM inhalation and the rupture endothelial cells. The present study provides novel evidence that IL-33 increases vascular tone via canonical ERK1/2 activation in microvascular smooth muscle and endothelium. Altogether, it is suggested IL-33 plays a critical role in microvascular homeostasis following barrier cell injury. KEY POINTS: Interleukin (IL)-33 causes a concentration-dependent reduction in feed artery diameter. IL-33 acts on vascular smooth muscle cells to augment Ca2+-mediated processes. IL-33 causes inhibitory phosphorylation of endothelial nitric oxide synthase and opposes endothelium-dependent dilatation. Engineered nanomaterial-induced lung injury and endothelial cell rupture in part act through IL-33 to mediate increased vascular tone.

Retinoic acid ameliorates rheumatoid arthritis by attenuating inflammation and modulating macrophage polarization through MKP-1/MAPK signaling pathway.

Macrophages are innate immune cells connected with the development of inflammation. Retinoic acid has previously been proved to have anti-inflammatory and anti-arthritic properties. However, the exact mechanism through which retinoic acid modulates arthritis remains unclear. This study aimed to investigate whether retinoic acid ameliorates rheumatoid arthritis by modulating macrophage polarization. This study used retinoic acid to treat mice with adjuvant arthritis and evaluated anti-inflammatory effects by arthritis score, thermal nociceptive sensitization test, histopathologic examination and immunofluorescence assays. In addition, its specific anti-arthritic mechanism was investigated by flow cytometry, cell transfection and inflammatory signaling pathway assays in RAW264.7 macrophages in vitro. Retinoic acid significantly relieved joint pain and attenuated inflammatory cell infiltration in mice. Furthermore, this treatment modulated peritoneal macrophage polarization, increased levels of arginase 1, as well as decreased inducible nitric oxide synthase expression. In vitro, we verified that retinoic acid promotes macrophage transition from the M1 to M2 type by upregulating mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) expression and inhibiting P38, JNK and ERK phosphorylation in lipopolysaccharide-stimulated RAW264.7 cells. Notably, the therapeutic effects of retinoic acid were inhibited by MKP-1 knockdown. Retinoic acid exerts a significant therapeutic effect on adjuvant arthritis in mice by regulating macrophage polarization through the MKP-1/MAPK pathway, and play an important role in the treatment of rheumatic diseases.

Lactobacillus helveticus HY7801 Improves Premenstrual Syndrome Symptoms by Regulating Sex Hormones and Inflammatory Cytokines in a Mouse Model of Metoclopramide-Induced Hyperprolactinemia

Background/Objectives: Premenstrual syndrome (PMS), a clinical condition that manifests in the form of various physical and psychological symptoms, occurs periodically during the luteal phase of the menstrual cycle and reduces quality of life. Methods: Here, we conducted in vitro and in vivo experiments to investigate the effects of Lactobacillus helveticus HY7801 (HY7801) on PMS symptoms. Results: Data from the in vitro experiments showed that HY7801 inhibits prolactin secretion by estradiol-induced GH3 cells, as well as the secretion of pro-inflammatory cytokines by LPS-induced Raw 264.7 cells. Additionally, the oral administration of HY7801 (109 colony-forming units/kg/day) to mice with metoclopramide-induced hyperprolactinemia reduced uterine tissue mass and endometrial thickness, both of which were increased excessively in the presence of prolactin. HY7801 also regulated the serum levels of follicle-stimulating hormone and prostaglandin E1/E2, as well as recovering the progesterone/estradiol ratio. HY7801 also downregulated the serum levels of prolactin and pro-inflammatory cytokines such as interleukin (Il)-6, tumor necrosis factor-alpha (Tnf), and IL-1β. Finally, HY7801 reduced the expression of genes encoding inflammatory cytokines (i.e., Tnf and Il-6), cyclooxygenase-2 (Cox-2), and inducible nitric oxide synthase (iNOS) in mice with hyperprolactinemia. Conclusion: In summary, HY7801 may be a functional bacterium that alleviates PMS symptoms by modulating hormones and inflammatory markers.

Partial involvement of nitric oxide synthase in increased pilocarpine-induced sweating in exercise-trained men.

The physiological mechanisms involved in augmented cholinergic agonist-induced sweating in exercise-trained individuals remain unclear. This study hypothesizes that nitric oxide synthase (NOS) contributes to augmented pilocarpine-induced sweating in habitually exercise-trained individuals. Endurance-trained and untrained men (n=15 each) iontophoretically received 1% L-NAME, a NOS inhibitor, and saline (control) in the forearm and then administered 0.001% and 1% pilocarpine to evaluate sweat rate. L-NAME administration attenuated pilocarpine-induced sweating by 10% in the exercise-trained (P = 0.004) but not in untrained (P = 0.764) groups independent of pilocarpine concentrations. Results indicate that NOS partially contributes to increased cholinergic sweating in exercise-trained men.

Hot-Melt Extrusion Drug Delivery System-Formulated Haematococcus pluvialis Extracts Regulate Inflammation and Oxidative Stress in Lipopolysaccharide-Stimulated Macrophages

Haematococcus pluvialis contains valuable bioactive compounds, including astaxanthin, proteins, and fatty acids. Astaxanthin is known for its various health benefits, such as preserving the redox balance and reducing inflammation. However, its low stability and poor water solubility present challenges for various applications. Hot-melt extrusion (HME) technology enhances the aqueous solubility of H. pluvialis extracts, increasing the usable astaxanthin content through nanoencapsulation (HME-DDS-applied extracts, ASX-60F and ASX-100F). This study compared the effects of HME-DDS-derived extracts (ASX-60F and ASX-100F) and the non-applied extract (ASX-C) under inflammatory and oxidative stress conditions. In animal models of sepsis, 60F and 100F treatment exhibited higher survival rates and a lower expression of pro-inflammatory biomarkers compared to those treated with C. In lipopolysaccharide-stimulated RAW 264.7 macrophages, nitric oxide (NO) production and the expression of pro-inflammatory mediators such as cyclooxygenase-2 and inducible NO synthase were reduced by 60F or 100F treatments via ERK/p-38 mitogen-activated protein kinase (MAPK) signaling. Moreover, 60F or 100F inhibited reactive oxygen species production regulated by nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling. Collectively, these findings suggest that HME-DDS-derived H. pluvialis extracts exert anti-inflammatory and antioxidant effects by inhibiting MAPK phosphorylation and activating Nrf2/HO-1 expression.

Embryonic Lethal Abnormal Visual-Like Protein 1 Aggravates Caerulein-Induced AR42J Cell Injury and Macrophage M1 Polarization to Accelerate Acute Pancreatitis by Upregulating TRAF6.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been found to promote the progression of acute pancreatitis (AP). However, its underlying molecular mechanisms in AP need to be further revealed. Caerulein-induced AR42J cells were used to construct AP cell models. Cell viability and apoptosis were measured by Cell Counting Kit 8 assay and flow cytometry. Levels of inflammatory factors and oxidative stress-related markers were assessed. The medium of AR42J cells was collected for coculturing RAW264.7 cells. Macrophage marker CD86+ cell rates were checked with flow cytometry. The levels of TRAF6, embryonic lethal abnormal visual-like protein 1 (ELAVL1), and inducible nitric oxide synthase (iNOS) were examined by Western blot or quantitative real-time polymerase chain reaction. RNA immunoprecipitation assay was performed to evaluate the interaction between ELAVL1 and TRAF6. TRAF6 mRNA stability was tested using actinomycin D treatment. Caerulein treatment suppressed viability, induced AR42J cell apoptosis, inflammation, oxidative stress, and accelerated macrophage M1 polarization. TRAF6 downregulation could alleviate caerulein-induced AR42J cell injury and macrophage M1 polarization. ELAVL1 interacted with TRAF6 to stabilize its expression. Meanwhile, ELAVL1 knockdown relieved caerulein-induced AR42J cell injury and macrophage M1 polarization, while these effects were abolished by TRAF6 overexpression. TRAF6, stabilized by ELAVL1, promoted caerulein-induced AR42J cell injury and macrophage M1 polarization, suggesting that it might accelerate AP9 progression.

MnNi@PVP Nanoenzyme Based on Regulating Inflammation and Immune Homeostasis for the Therapy of Inflammatory Bowel Disease.

Nanozymes exhibiting remarkable antioxidant capabilities represent an effective therapeutic approach for ulcerative colitis (UC). This study synthesized the MnNi@PVP nanoenzyme through high-temperature pyrolysis and the NaCl template method, which exhibited multienzyme activity comprising glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT). This study investigated the therapeutic effect of MnNi@PVP on colitis caused by sodium dextran sulfate (DSS). The findings indicated that MnNi@PVP notably decreased the disease activity index (DAI) score, which encompasses weight loss, colon shortening, and histopathological changes. MnNi@PVP showed effectiveness in addressing oxidative damage and suppressing the levels of proinflammatory markers, such as tumor necrosis factor (TNF-α), inducible nitric oxide synthase (iNOS), and interleukin (IL)-6, by inactivating the TLR4 pathway and macrophages. In addition, MnNi@PVP demonstrated the ability to repair tight junction proteins (occludin and ZO-1) and restore the intestinal barrier. The transcriptome sequencing demonstrated that MnNi@PVP could regulate inflammatory factor expression pathways and immune processes. Additionally, negatively charged MnNi@PVP can selectively bind to inflamed colonic tissues through electrostatic interactions, endowing it with targeted reparative capabilities at the location of intestinal inflammation. The MnNi@PVP, which possesses the reactive oxygen and nitrogen species (RONS) clearance capability examined in this section, is expected to provide the basis for the targeted repair of intestinal function.

Medicinal Chemistry Insights in Neuronal Nitric Oxide Synthase Inhibitors Containing Nitrogen Heterocyclic Compounds: A Mini Review.

Many scientific reports over the last two decades have focused on the discovery and development of novel nNOS inhibitors. The structural identity of isoforms, bioavailability, pharmacokinetic, and safety profile issues remain major obstacles in the discovery of more potent and selective nNOS inhibitors. This review aims to provide an in-depth overview of the molecular interaction patterns between nNOS active site and inhibitors containing structurally diverse nitrogen heterocyclic compounds and highlight the structural properties needed to develop selective nNOS inhibitors. Previously published data allowed the usage of the structure-driven approach in the designing of selective nNOS inhibitors, which relies on the specific structural features required to achieve isoform-selectivity towards nNOS. The incorporation of chiral pyrrolidine ring, two aminopyridine heads, or a specific amino tail group, along with the inhibitor's capacity to adopt the curled conformation in the nNOS environment significantly strengthens the molecular interaction between the inhibitor and nNOS residues by forming specific electrostatic interactions and non-bonded contacts that are vital for isoform selectivity. Additional structure-activity relationship investigations are necessary to elucidate more structural characteristics that will ultimately resolve the exact structural basis required for isoform-selective inhibition of nNOS.

The expression of contextual fear conditioning involves the dorsal hippocampus TRPV1 receptor interacting with the NMDA/NO/cGMP signalling pathway.

The dorsal hippocampus (dHIP) is pivotal for learning, memory, and defensive responses. Transient receptor potential vanilloid type 1 (TRPV1) receptors in the dHIP modulate contextual fear conditioning by triggering a cascade involving glutamate release, nitric oxide (NO) formation and cyclic guanosine monophosphate (cGMP) production. The present study investigated the involvement of dHIP TRPV1 receptors and their interaction with the glutamate/NO/cGMP signalling pathway in modulating the expression of contextual fear conditioning (CFC). Male Wistar rats were submitted to an aversive contextual conditioning session and, 48 h later, were re-introduced to the same aversive environment where the freezing response and autonomic activity (evidenced by increased arterial pressure and heart rate and a decrease in tail temperature) were measured. The results demonstrated that the TRPV1 antagonist 6-I-CPS in dHIP reduced the expression of CFC, whereas the agonist capsaicin had the opposite effect. Furthermore, dHIP pre-treatment with an NMDA receptor antagonist (AP7), neuronal NO synthase inhibitor (N-propyl-L-arginine), NO scavenger (c-PTIO) or guanylate cyclase inhibitor (ODQ) attenuated capsaicin-induced increases in CFC. Finally, we observed that re-exposure to the aversive chamber increased dHIP NO levels in conditioned animals compared with a non-conditioned group, which was prevented by the administration of the TRPV1 antagonist, 6-I-CPS. Our study revealed that TRPV1 receptors in the dHIP play a crucial role in modulating contextual fear expression by acting through the NMDA receptor/NO/cGMP signalling pathway, providing important insights into the underlying mechanisms and potential therapeutic avenues associated with these pathways.

Abstract 4135059: Enhanced Expression of iNOS is Associated with Aortic Valve Stenosis in Patients Undergoing Hemodialysis

Background: Patients undergoing hemodialysis (HD) have a greater prevalence of aortic valve calcification and aortic valve stenosis (AS). High prevalence and rapid progression of AS have been reported in patients undergoing HD. However, the precise mechanism of the relationship between HD treatment and rapid development of AS is still unknown. Several findings suggest a pivotal role for oxidative stress in the progression and acceleration of atherosclerosis in patients undergoing HD. A considerable number of human diseases have an inflammatory component, and a key mediator of immune activation and inflammation is inducible nitric oxide synthase (iNOS). Overexpressed iNOS has been implicated in numerous pathogenetic processes, including coronary plaque destabilization. To elucidate the role of iNOS in the aortic valve of patients with AS, we studied its presence in aortic valve specimens from patients with AS undergoing HD. Methods: We enrolled 32 patients undergoing HD and 42 patients not undergoing HD who had severe AS and underwent AVR. Valve samples were analyzed immunohistochemically with antibodies against macrophages, microvessels, iNOS and 4-hydroxy-2-nonenal (4-HNE), an index of lipid peroxidation. Immunoreactivity was quantified with computer-aided polarimetry. Double immunostaining was also performed to identify cell types that stained positive for iNOS. Results: Quantitative analysis demonstrated that macrophage-, iNOS-, and 4-HNE-positive areas, as well as the number of microvessels, were significantly higher in patients with AS undergoing HD (macrophages, P &lt;0.001; microvessels, P &lt;0.05; 4-HNE, P &lt;0.05; iNOS , P &lt;0.001). iNOS-positive areas were positively correlated with 4-HNE-positive areas (R=0.71, P &lt;0.0001). Double immunostaining for both iNOS and macrophages identified iNOS-positive cells as macrophages. Conclusions: These findings indicate that accumulation of iNOS in macrophages may increase oxidative stress and contribute to the rapid progression of AS in patients undergoing HD.