Upregulation Of Nitric Oxide Synthase Research Articles

Neuroprotective Effect of Fructus Ligustri Lucidi in Spinal Cord Neuron Injury during H2O2-Mediated Oxidative Stress

Oxidative stress-induced neuronal damage is a significant factor contributing to spinal cord injury. Although previous research has shown that Fructus Ligustri Lucidi (FLL) has neuroprotective benefits in SH-SY5Y, BV2, and PC12 cells, its impact on primary spinal cord neurons, which more accurately reflect the characteristics of central nervous system neurons, remains unexplored. This research investigated how FLL can protect rat primary spinal cord neurons from injury triggered by hydrogen peroxide (H2O2)-mediated oxidative stress. Cell viability, generation of reactive oxygen species (ROS), upregulation of inducible nitric oxide synthase (iNOS), activation of the Nrf2/HO-1 antioxidant pathway, and mitochondrial superoxide were assessed. Rat primary spinal cord neurons were not adversely affected by concentrations of FLL extract up to 100 μg/mL. Furthermore, FLL extract showed a significant protective effect against H2O2-induced neuronal toxicity at 10–100 μg/mL. Fluorescence-activated cell sorting analysis revealed that FLL extract inhibited H2O2-induced ROS generation in a dose-dependent manner. Immunocytochemistry and gene expression analysis confirmed that FLL extract reduced the overexpression of iNOS induced by H2O2 and enhanced the stimulation of the Nrf2/HO-1 pathway, crucial for antioxidant responses. In conclusion, FLL extract demonstrated neuroprotective effects on rat primary spinal cord neurons against the oxidative stress induced by H2O2. FLL extract effectively preserved cell viability, reduced ROS generation, suppressed iNOS overexpression, and activated the Nrf2/HO-1 antioxidant pathway. These results highlight the capacity of FLL extract as a neuroprotective agent against oxidative stress-related spinal neuron damage.

Increased Prolylcarboxypeptidase Expression Can Serve as a Biomarker of Senescence in Culture.

Prolylcarboxypeptidase (PRCP, PCP, Lysosomal Pro-X-carboxypeptidase, Angiotensinase C) controls angiotensin- and kinin-induced cell signaling. Elevation of PRCP appears to be activated in chronic inflammatory diseases [cardiovascular disease (CVD), diabetes] in proportion to severity. Vascular endothelial cell senescence and mitochondrial dysfunction have consistently been shown in models of CVD in aging. Cellular senescence, a driver of age-related dysfunction, can differentially alter the expression of lysosomal enzymes due to lysosomal membrane permeability. There is a lack of data demonstrating the effect of age-related dysfunction on the expression and function of PRCP. To explore the changes in PRCP, the PRCP-dependent prekallikrein (PK) pathway was characterized in early- and late-passage human pulmonary artery endothelial cells (HPAECs). Detailed kinetic analysis of cells treated with high molecular weight kininogen (HK), a precursor of bradykinin (BK), and PK revealed a mechanism by which senescent HPAECs activate the generation of kallikrein upon the assembly of the HK-PK complex on HPAECs in parallel with an upregulation of PRCP and endothelial nitric oxide (NO) synthase (eNOS) and NO formation. The NO production and expression of both PRCP and eNOS increased in early-passage HPAECs and decreased in late-passage HPAECs. Low activity of PRCP in late-passage HPAECs was associated with rapid decreased telomerase reverse transcriptase mRNA levels. We also found that, with an increase in the passage number of HPAECs, reduced PRCP altered the respiration rate. These results indicated that aging dysregulates PRCP protein expression, and further studies will shed light into the complexity of the PRCP-dependent signaling pathway in aging.

Optimal treatment conditions for low-intensity pulsed ultrasound therapy for Alzheimer's disease: applications from mice to humans.

We previously developed a novel therapy with low-intensity pulsed ultrasound (LIPUS) that ameliorates cognitive decline through upregulation of endothelial nitric oxide synthase (eNOS) in mouse models of Alzheimer's disease (AD). In a randomized, double-blind, placebo-controlled pilot trial, we demonstrated that whole-brain LIPUS therapy is safe and tends to suppress the cognitive decline in early AD patients. We herein report the findings of our basic experiments that we performed for the pilot trial in order to apply whole-brain LIPUS therapy to humans, as well. First, we examined the relationship between bone density/thickness and ultrasound transmittance using human temporal bone. Next, based on the results of ultrasound transmittance, we further examined mRNA expression of VEGF, FGF2, and eNOS in response to variable ultrasound frequencies, duty cycles, and sound pressures. There was a significant correlation between bone thickness and transmittance (1.0MHz, P < 0.001), while there was no significant correlation between bone density and transmittance (1.0MHz, P = 0.421). At a frequency of 0.5MHz, the optimum duty cycle was considered to be up to 20%. When the tissue amplitude was in the range of 0.05-0.5MPa, VEGF, FGF2, and eNOS were significantly upregulated by LIPUS. Thus, the conditions necessary for LIPUS therapy for the human brain were identified as sound pressure just below the probe 1.3MPa (tissue amplitude 0.15MPa), duty cycle 5%, and frequency 0.5MHz. We successfully identified the optimal treatment conditions for LIPUS therapy for patients with AD.

Detection of nitric oxide-mediated metabolic effects using real-time extracellular flux analysis.

Dendritic cell (DC) activation is marked by key events including: (I) rapid induction and shifting of metabolism favoring glycolysis for generation of biosynthetic metabolic intermediates and (II) large scale changes in gene expression including the upregulation of the antimicrobial enzyme inducible nitric oxide synthase (iNOS) which produces the toxic gas nitric oxide (NO). Historically, acute metabolic reprogramming and NO-mediated effects on cellular metabolism have been studied at specific timepoints during the DC activation process, namely at times before and after NO production. However, no formal method of real time detection of NO-mediated effects on DC metabolism have been fully described. Here, using Real-Time Extracellular Flux Analysis, we experimentally establish the phenomenon of an NO-dependent mitochondrial respiration threshold, which shows how titration of an activating stimulus is inextricably linked to suppression of mitochondrial respiration in an NO-dependent manner. As part of this work, we explore the efficacy of two different iNOS inhibitors in blocking the iNOS reaction kinetically in real time and explore/discuss parameters and considerations for application using Real Time Extracellular Flux Analysis technology. In addition, we show, the temporal relationship between acute metabolic reprogramming and NO-mediated sustained metabolic reprogramming kinetically in single real-time assay. These findings provide a method for detection of NO-mediated metabolic effects in DCs and offer novel insight into the timing of the DC activation process with its associated key metabolic events, revealing a better understanding of the nuances of immune cell biology.

The role of T-type calcium channels in elderly human vascular function: A pilot randomized controlled trial.

Endothelial dysfunction develops with age and may precede cardiovascular disease. Animal data suggest that T-type calcium channels play an important role in endothelial function, but data from humans are lacking. This study included 15 healthy, sedentary, elderly males for a double blinded, randomized controlled trial. For 8weeks, they were given 40mg/day of either efonidipine (L- and T-type calcium channel blocker (CCB)) or nifedipine (L-type CCB). Vascular function was evaluated by graded femoral arterial infusions of acetylcholine (ACh; endothelium-dependent vasodilator) and sodium nitroprusside (endothelium-independent vasodilator) both with and without co-infusion of N-acetylcysteine (NAC; antioxidant). We measured leg blood flow and mean arterial pressure and calculated leg vascular conductance to evaluate the leg vascular responses. Despite no significant change in blood pressure in either group, we observed higher leg blood flow responses (Δ 0.43±0.45l/min, P=0.006) and leg vascular conductance (Δ 5.38±5.67ml/min/mmHg, P=0.005) to intra-arterial ACh after efonidipine, whereas there was no change in the nifedipine group, and no differences between groups. We found no upregulation of endothelial nitric oxide synthase in vastus lateralis muscle biopsies within or between groups. Smooth muscle cell responsiveness was unaltered by efonidipine or nifedipine. Intravenous co-infusion of NAC did not affect endothelium-dependent vasodilatation in either of the CCB groups. These results suggest that 8weeks' inhibition of T- and L-type calcium channels augments endothelium-dependent vasodilatory function in healthy elderly males. Further studies are required to elucidate if T-type calcium channel inhibition can counteract endothelial dysfunction.

Danggui-Shaoyao-San (DSS) ameliorates the progression of osteoarthritis via suppressing the NF-κB signaling pathway: an in vitro and in vivo study combined with bioinformatics analysis.

Osteoarthritis (OA) is a common chronic age-related joint disease characterized primarily by inflammation of synovial membrane and degeneration of articular cartilage. Accumulating evidence has demonstrated that Danggui-Shaoyao-San (DSS) exerts significant anti-inflammatory effects, suggesting that it may play an important role in the treatment of knee osteoarthritis (KOA). In the present study, DSS was prepared and analyzed by high-performance liquid chromatography (HPLC). Bioinformatics analyses were carried out to uncover the functions and possible molecular mechanisms by which DSS against KOA. Furthermore, the protective effects of DSS on lipopolysaccharide (LPS)-induced rat chondrocytes and cartilage degeneration in a rat OA model were investigated in vivo and in vitro. In total, 114 targets of DSS were identified, of which 60 candidate targets were related to KOA. The target enrichment analysis suggested that the NF-κB signaling pathway may be an effective mechanism of DSS. In vitro, we found that DSS significantly inhibited LPS-induced upregulation of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP3), and matrix metalloproteinase-13 (MMP13). Meanwhile, the degradation of collagen II was also reversed by DSS. Mechanistically, DSS dramatically suppressed LPS-induced activation of the nuclear factor kappa B (NF-κB) signaling pathway. In vivo, DSS treatment prevented cartilage degeneration in a rat OA model. DSS could ameliorate the progression of OA through suppressing the NF-κB signaling pathway. Our findings indicate that DSS may be a promising therapeutic approach for the treatment of KOA.

Krameria lappacea root extract's anticoccidial properties and coordinated control of CD4 T cells for IL-10 production and antioxidant monitoring.

Recently, the use of botanicals as an alternative to coccidiostats has been an appealing approach for controlling coccidiosis. Therefore, this study was conducted to evaluate the potential role of aqueous methanolic extract (200 mg/kg) of Krameria lappacea (roots) (KLRE) against infection induced by Eimeria papillata. A total of 25 male C57BL/6 mice were divided into five groups (I, II, III, IV, and V). On 1st day of the experiment, all groups except groups I (control) and II (non-infected-treated group with KLRE), were inoculated orally with 103 sporulated E. papillata oocysts. On the day of infection, group IV was treated with KLRE. Group V served as an infected-treated group and was treated with amprolium (coccidiostat). Treatment with extract and coccidiostat was continued for five consecutive days. While not reaching the efficacy level of the reference drug (amprolium), KLRE exhibited notable anticoccidial activity as assessed by key criteria, including oocyst suppression rate, total parasitic stages, and maintenance of nutrient homeostasis. The presence of phenolic and flavonoid compounds in KLRE is thought to be responsible for its positive effects. The Eimeria infection increased the oxidative damage in the jejunum. KLRE treatment significantly increased the activity of catalase and superoxide dismutase. On the contrary, KLRE decreased the level of malondialdehyde and nitric oxide. Moreover, KLRE treatment decreased macrophage infiltration in the mice jejunal tissue, as well as the extent of CD4 T cells and NFkB. E. papillata caused a state of systemic inflammatory response as revealed by the upregulation of inducible nitric oxide synthase (iNOs)-mRNA. Upon treatment with KLRE, the activity of iNOs was reduced from 3.63 to 1.46 fold. Moreover, KLRE was able to downregulate the expression of pro-inflammatory cytokines interferon-γ, nuclear factor kappa B, and interleukin-10 -mRNA by 1.63, 1.64, and 1.38 fold, respectively. Moreover, KLRE showed a significant reduction in the expression of IL-10 protein level from 104.27 ± 8.41 pg/ml to 62.18 ± 3.63 pg/ml. Collectively, K. lappacea is a promising herbal medicine that could ameliorate the oxidative stress and inflammation of jejunum, induced by E. papillata infection in mice.

ALZHEIMER HASTALIĞINDA iNOS İNHİBİTÖRLERİNİN UMUT VERİCİ ROLÜ

Objective: This study aims to explore the role of iNOS inhibitors in Alzheimer's disease (AD), a neurodegenerative disorder affecting millions worldwide. The main symptoms of AD include memory loss, cognitive decline, and behavioral changes. While the exact cause remains uncertain, both genetic and environmental factors are believed to contribute. Recent research has emphasized the significance of nitric oxide (NO) in AD development. Specifically, the upregulation of inducible nitric oxide synthase (iNOS) in AD patients leads to excessive NO production during neuronal inflammation, exacerbating AD and dementia. Therefore, the investigation focuses on the potential of iNOS inhibitors as a novel therapeutic approach for AD treatment. Result and Discussion: In this review, we present the current therapeutic strategies available for Alzheimer's disease (AD) and explore the promising potential of iNOS inhibitors in AD treatment. Specifically, we will focus on their capacity to mitigate NO production and examine their potential neuroprotective effects. Additionally, this review will offer an overview of both natural and synthetic iNOS inhibitors, emphasizing the importance of safety considerations during the development of iNOS inhibitors as therapeutic interventions for AD.

Iron oxide polyaniline-coated nanoparticles modulate tumor microenvironment in breast cancer: an in vitro study on the reprogramming of tumor-associated macrophages

BackgroundBreast cancer is the neoplastic disease with the highest incidence and mortality in the female population worldwide. Treatment remains challenging due to various factors. Therefore, it is of great importance to develop new therapeutic strategies that promote the safe destruction of neoplastic cells without compromising patients' quality of life. Among advances in the treatment of breast cancer, immunotherapy stands out as a promising trend. Recent studies have demonstrated the potential of iron oxide nanoparticles in promoting the reprogramming of M2 macrophages (pro-tumor phenotype) into M1 macrophages (anti-tumor phenotype) within the tumor microenvironment, resulting in potent antitumor effects. In this study, the effect of polyaniline-coated iron oxide nanoparticles (Pani/y-Fe2O3) on macrophage polarization and breast cancer cell death was investigated.MethodsThe non-cytotoxic concentration of nanoparticles was determined using the MTT assay. For in vitro co-culture experiments, breast cancer cell lines MCF -7 and MDA-MB -231 and macrophages THP-1 were co-cultured in a Transwell system and then the effects of Pani/y-Fe2O3 on cell viability, gene expression, cytokine profile, and oxidative stress markers were investigated.ResultsThe results showed that Pani/y-Fe2O3 nanoparticles induced M2-to-M1 macrophage polarization in both cell lines through different pathways. In MCF -7 and THP-1 macrophage co-culture, the study showed a decrease in cytokine levels IL -1β, upregulation of M1-associated genes (IL-12, TNF-α) in macrophages, resulting in increased MCF -7 cell death by apoptosis (caspase 3/7+). In MDA-MB -231 co-cultures, increases in cytokines IL -6, IL -1β, and oxidative stress markers were observed, as well as upregulation of the inducible nitric oxide synthase (iNOS) gene in macrophages, leading to tumor cell death via apoptosis-independent pathways (Sytox+).ConclusionsThese findings highlight the potential of Pani/y-Fe2O3 as a promising therapeutic approach in the context of breast cancer treatment by effectively reprogramming M2 macrophages into an anti-tumor M1 phenotype, Pani/y-Fe2O3 nanoparticles demonstrated the ability to elicit antitumor effects in both MCF-7 and MDA-MB-231 breast cancer cell lines.

Cocoa Shell Extract Reduces Blood Pressure in Aged Hypertensive Rats via the Cardiovascular Upregulation of Endothelial Nitric Oxide Synthase and Nuclear Factor (Erythroid-Derived 2)-like 2 Protein Expression.

Cocoa shell is a by-product of cocoa manufacturing. We obtained an aqueous extract (CSE) rich in polyphenols and methylxanthines with antioxidant and vasodilatory properties. We aimed to evaluate the effects of CSE supplementation in aged hypertensive rats on blood pressure and the mechanism implicated. Eighteen-month-old male and female rats exposed to undernutrition during the fetal period who developed hypertension, with a milder form in females, were used (MUN rats). Systolic blood pressure (SBP; tail-cuff plethysmography) and a blood sample were obtained before (basal) and after CSE supplementation (250 mg/kg; 2 weeks, 5 days/week). Plasma SOD, catalase activity, GSH, carbonyls, and lipid peroxidation were assessed (spectrophotometry). In hearts and aortas from supplemented and non-supplemented age-matched rats, we evaluated the protein expression of SOD-2, catalase, HO-1, UCP-2, total and phosphorylated Nrf2 and e-NOS (Western blot), and aorta media thickness (confocal microscopy). MUN males had higher SBP compared with females, which was reduced via CSE supplementation with a significant difference for group, sex, and interaction effect. After supplementation with plasma, GSH, but not catalase or SOD, was elevated in males and females. Compared with non-supplemented rats, CSE-supplemented males and females exhibited increased aorta e-NOS and Nrf2 protein expression and cardiac phosphorylated-Nrf2, without changes in SOD-2, catalase, HO-1, or UCP-2 in cardiovascular tissues or aorta remodeling. In conclusion, CSE supplementation induces antihypertensive actions related to the upregulation of e-NOS and Nrf2 expression and GSH elevation and a possible direct antioxidant effect of CSE bioactive components. Two weeks of supplementation may be insufficient to increase antioxidant enzyme expression.

Anti-inflammatory Effects of Siponimod in a Mouse Model of Excitotoxicity-Induced Retinal Injury

Glaucoma is a leading cause of permanent blindness worldwide and is characterized by neurodegeneration linked to progressive retinal ganglion cell (RGC) death, axonal damage, and neuroinflammation. Glutamate excitotoxicity mediated through N-methyl-D-aspartate (NMDA) receptors plays a crucial role in glaucomatous RGC loss. Sphingosine 1-phosphate receptors (S1PRs) are important mediators of neurodegeneration and neuroinflammation in the brain and the retina. Siponimod is an immunomodulatory drug for multiple sclerosis and is a selective modulator of S1PR subtypes 1 and 5 and has been shown to have beneficial effects on the central nervous system (CNS) in degenerative conditions. Our previous study showed that mice administered orally with siponimod protected inner retinal structure and function against acute NMDA excitotoxicity. To elucidate the molecular mechanisms behind these protective effects, we investigated the inflammatory pathways affected by siponimod treatment in NMDA excitotoxicity model. NMDA excitotoxicity resulted in the activation of glial cells coupled with upregulation of the inflammatory NF-kB pathway and increased expression of TNFα, IL1-β, and IL-6. Siponimod treatment significantly reduced glial activation and suppressed the pro-inflammatory pathways. Furthermore, NMDA-induced activation of NLRP3 inflammasome and upregulation of neurotoxic inducible nitric oxide synthase (iNOS) were significantly diminished with siponimod treatment. Our data demonstrated that siponimod induces anti-inflammatory effects via suppression of glial activation and inflammatory singling pathways that could protect the retina against acute excitotoxicity conditions. These findings provide insights into the anti-inflammatory effects of siponimod in the CNS and suggest a potential therapeutic strategy for neuroinflammatory conditions.

Determining the potent immunostimulation potential arising from the heteropolysaccharide structure of a novel fucoidan, derived from Sargassum Zhangii.

A preliminary study was conducted of the chemical, structural properties and immunomodulatory activities of fucoidan isolated from Sargassum Zhangii (SZ). Sargassum Zhangii fucoidan (SZF) was determined to have a sulfate content of 19.74±0.01% (w/w) and an average molecular weight of 111.28kDa. SZF possessed a backbone structure of (1,4)-α-d-linked-galactose, (3,4)-α-l-fucose, (1,3)-α-d-linked-xylose, β-d-linked-mannose and a terminal (1,4)-α-d-linked-glucose. The main monosaccharide composition was determined as (w/w) 36.10% galactose, 20.13% fucose, 8.86% xylose, 7.36% glucose, 5.62% mannose, and 18.07% uronic acids, respectively. An immunostimulatory assay showed that SZF, compared to commercial fucoidans (Undaria pitnnaifida and Fucus vesiculosus sources), significantly elevated nitric oxide production via up-regulation of cyclooxygenase-2 and inducible nitric oxide synthase at both gene and protein levels. These results suggest that SZ has the potential to be a source of fucoidan with enhanced properties that may act as a useful ingredient for functional foods, nutritional supplements, and immune enhancers.

Glycogen phosphorylase isoenzyme GPbb versus GPmm regulation of ventromedial hypothalamic nucleus glucoregulatory neurotransmitter and counter-regulatory hormone profiles during hypoglycemia: Role of L-lactate and octadecaneuropeptide

Glucose accesses the brain primarily via the astrocyte cell compartment, where it passes through the glycogen shunt before catabolism to the oxidizable fuel L-lactate. Glycogen phosphorylase (GP) isoenzymes GPbb and GPmm impose distinctive control of ventromedial hypothalamic nucleus (VMN) glucose-regulatory neurotransmission during hypoglycemia, but lactate and/or gliotransmitter involvement in those actions is unknown. Lactate or the octadecaneuropeptide receptor antagonist cyclo(1–8)[DLeu5] OP (LV-1075) did not affect gene product down-regulation caused by GPbb or GPmm siRNA, but suppressed non-targeted GP variant expression in a VMN region-specific manner. Hypoglycemic up-regulation of neuronal nitric oxide synthase was enhanced in rostral and caudal VMN by GPbb knockdown, yet attenuated by GPMM siRNA in the middle VMN; lactate or LV-1075 reversed these silencing effects. Hypoglycemic inhibition of glutamate decarboxylase65/67 was magnified by GPbb (middle and caudal VMN) or GPmm (middle VMN) knockdown, responses that were negated by lactate or LV-1075. GPbb or GPmm siRNA enlarged hypoglycemic VMN glycogen profiles in rostral and middle VMN. Lactate and LV-1075 elicited progressive rostral VMN glycogen augmentation in GPbb knockdown rats, but stepwise-diminution of rostral and middle VMN glycogen after GPmm silencing. GPbb, not GPmm, knockdown caused lactate or LV-1075 – reversible amplification of hypoglycemic hyperglucagonemia and hypercorticosteronemia. Results show that lactate and octadecaneuropeptide exert opposing control of GPbb protein in distinct VMN regions, while the latter stimulates GPmm. During hypoglycemia, GPbb and GPmm may respectively diminish (rostral, caudal VMN) or enhance (middle VMN) nitrergic transmission and each oppose GABAergic signaling (middle VMN) by lactate- and octadecaneuropeptide-dependent mechanisms.

Effect of dietary inclusion of atorvastatin, garlic, and dill on growth performance, antioxidant defense, gut, and cardio-pulmonary function, and lipogenesis in broiler chickens.

In the present study, we evaluated the antihyperlipidemic and antioxidant effects of garlic and dill in comparison with atorvastatin to combat lipogenesis in broiler chickens. A total of 400 1-day-old chicks (Ross 308 strain) were randomly distributed into four experimental diets. Dietary treatments included a control diet, the control diet plus atorvastatin at 20mg/kg, the control diet plus garlic dry powder (GDP) at 7.5g/kg, and the control diet plus dill dry powder (DDP) at 7.5g/kg. Chicks were maintained on experimental diets for 42days under the recommended environmental conditions set out by the strain management manual. The results showed that weight gain, feed conversion ratio (FCR), and duodenal, jejunal, and ileal dimensions of villi (height, width, and the surface absorptive area) were improved by in-feed atorvastatin, GDP, or DDP when compared to the control (P < 0.05). The inclusion of atorvastatin or phytobiotic products increased circulatory levels of nitric oxide (NO) but decreased circulatory levels of malondialdehyde (MDA), triacylglycerol (TAG), and low-density lipoproteins cholesterol (LDL), with concomitant reductions in the T, R, and S waves amplitudes in the Lead 2 electrocardiogram (ECG) (P < 0.05). Dietary supplements caused an up-regulation of inducible nitric oxide synthase (iNOS), superoxide dismutase 1 (SOD1), and glutathione peroxidase (GPX) but reduced the expression of key hepatic lipogenic enzymes (fatty acid synthase (FAS) and hydroxy-methylglutaryl-CoA reductase (HMGCR) (P < 0.05). In conclusion, feed supplementation with atorvastatin, GDP, or DDP suppressed lipogenesis, enhanced antioxidant response, and improved gut and cardio-pulmonary function in broiler chicks subjected to hypobaric hypoxia.

UDP-glucose Regulates Dendritic Cell Mitochondrial Respiration via a Nitric Oxide-dependent Mechanism

Abstract Dendritic cells (DCs) are key innate immune cells specialized in fine tuning an immune response. Activation of DCs through Toll-Like Receptors (TLRs) is accompanied by a metabolic switch to increased use of glycolytic metabolism fueled by carbohydrates glucose and glycogen. Our lab has shown that DCs require glucose and glycogen to perform their immune effector functions including: production of cytokines and inflammatory mediators, antigen presentation to T cells, and initiation of the T cell-mediated immune response. Recent studies in inflammatory macrophages showed a secondary metabolite in the glycogen synthetic pathway, uridine diphosphate glucose (UDP-glucose), is upregulated through TLR4 stimulation. UDP-glucose exits the cell and signals in an autocrine manner through its specific purinergic receptor P2Y 14. Additionally, P2Y 14R signaling modulates the expression of proinflammatory pathways including upregulation of inducible nitric oxide synthase (iNOS). Activated bone marrow-derived DCs (BMDCs) upregulate iNOS and produce nitric oxide (NO), which has pleiotropic effects, including inhibiting mitochondrial respiration. Therefore, we hypothesized that the UDP-glucose/P2Y 14R/iNOS axis regulates BMDC metabolism, which has further ramifications on DC cell survival and proinflammatory phenotypes. To investigate the impact of UDP-glucose as an autocrine signaling molecule, we characterized proinflammatory gene transcript and protein levels, as well as the functional outcomes of increased iNOS protein in the cell. These studies showed that DCs use the UDP-glucose/P2Y 14R/iNOS axis to bolster inflammatory phenotypes including inhibition of mitochondrial respiration leading to exacerbated cell death. Supported by grants from NIH (R01 AI158710-01, 1R21AI135385-01A1) and UVM (COBRE VCIID Funding Support and Pilot Project)

MP20-18 EXOGENOUS NITRIC OXIDE, S-NITROSYLATES THE CSF1 RECEPTOR TO SENSITIZE THE CASTRATION-RESISTANT PROSTATE CANCER CELLS TO CSF1R BLOCKADE

You have accessJournal of UrologyCME1 Apr 2023MP20-18 EXOGENOUS NITRIC OXIDE, S-NITROSYLATES THE CSF1 RECEPTOR TO SENSITIZE THE CASTRATION-RESISTANT PROSTATE CANCER CELLS TO CSF1R BLOCKADE Fakiha Firdaus, Derek Van Booven, Rehana Qureshi, Ranjith Ramasamy, and Himanshu Arora Fakiha FirdausFakiha Firdaus More articles by this author , Derek Van BoovenDerek Van Booven More articles by this author , Rehana QureshiRehana Qureshi More articles by this author , Ranjith RamasamyRanjith Ramasamy More articles by this author , and Himanshu AroraHimanshu Arora More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.18AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Sustained oxidative stress in castration-resistant prostate cancer (CRPC) cells potentiates the overall tumor microenvironment (TME). Targeting the TME using colony-stimulating factor 1 receptor (CSF1R) inhibition is a promising therapy for CRPC. However, the therapeutic response to sustained CSF1R inhibition (CSF1Ri) is limited as a monotherapy. We hypothesized that one of the underlying causes for the reduced efficacy of CSF1Ri and increased oxidation in CRPC is the upregulation and uncoupling of endothelial nitric oxide synthase (NOS3). METHODS: Patient biopsies belonging to different Gleason grades were used to determine the levels of NOS3, CSF1, and GSNOR activity. A CSF1-receptor inhibitor, GW2580, was tested in-vitro and in-vivo, singularly and in combination with S-nitrosoglutathione (GSNO), an active NO donor. In-vivo dosage of 40mg/kg/day was used for GW2580, and 10mg/kg/day for GSNO was considered. Markers were checked for their expression in tumor grafts and human specimens using immunohistochemistry. Moreover, we evaluated the percentage of different immune cells using immune phenotyping in tumor cells exposed to different treatment conditions. RESULTS: We show that in high-grade PCa human specimens, NOS3 abundance positively correlates with CSF1-CSF1R signaling and remains uncoupled. The uncoupling diminishes NOS3 generation of sufficient nitric oxide (NO) required for S-nitrosylation of CSF1R at specific cysteine sites (Cys 224, Cys 278, and Cys 830). Exogenous S-nitrosothiol administration (with S-nitrosoglutathione (GSNO)) induces S-nitrosylation of CSF1R and rescues the excess oxidation in tumor regions, in turn suppressing the tumor-promoting cytokines, which are ineffectively suppressed by CSF1R blockade. Together these results suggest that the NO administration could act as an effective combinatorial partner with the CSF1R blockade against CRPC. In this context, we further show that exogenous NO treatment with GSNOR successfully augments the anti-tumor ability of CSF1Ri to reduce the overall tumor burden effectively, decreases the intratumoral percentage of anti-inflammatory macrophages, myeloid-derived progenitor cells and increases the percentage of pro-inflammatory macrophages, cytotoxic T lymphocytes, and effector T cells, respectively. CONCLUSIONS: Together, these findings support the concept that the NO-CSF1Ri combination can act as a therapeutic agent that restores control over TME, which in turn could improve the outcomes of PCa patients. Source of Funding: AUA Research Scholar Award to HAAmerican Cancer Society Grant to R.R. © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e282 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Fakiha Firdaus More articles by this author Derek Van Booven More articles by this author Rehana Qureshi More articles by this author Ranjith Ramasamy More articles by this author Himanshu Arora More articles by this author Expand All Advertisement PDF downloadLoading ...

Co-administration of alcohol and combination antiretroviral therapy (cART) in male Sprague Dawley rats: A study on testicular morphology, oxidative and cytokines perturbations.

Alcohol consumption alongside combination antiretroviral therapy (cART) has attracted research interest, especially because of increasing male infertility. This study investigated the combined effects of alcohol and cART on testicular morphology, biomarkers of oxidative stress, inflammation, and apoptosis. Rats, weighing 330-370 g, were divided into four groups of six animals each; control, alcohol treated (A), cART, and alcohol plus cART treated (A+cART). Following 90 days treatment period, animals were euthanized, testis extracted, and routinely processed for histology and immunohistochemical analysis. Significantly decreased epithelial area fraction, increased luminal and connective tissue area fractions, and reduction of epithelial height and spermatocyte number, were recorded in the treated groups compared to control. Extensive seminiferous epithelial lesions including widened intercellular space, karyolysis, and sloughing of germinal epithelium were recorded in all the treated groups. Furthermore, upregulation of inducible nitric oxide synthase and 8-hydroxydeoxyguanosine, interleukin-6, and caspase 3 recorded in treated animals, was more significant in A+cART group. Also, the levels of interleukin-1β and tumor necrosis factor-α were more elevated in A and cART treated groups than in A+cART, while MDA was significantly elevated in cART and A+cART treated groups compared to control group. Altogether, the results indicate testicular toxicity of the treatments. It is concluded that consuming alcohol or cART induces oxidative stress, inflammation, and apoptosis in testis of rats, which lead to testicular structural and functional derangements, which are exacerbated when alcohol and cART are consumed concurrently. The result will invaluably assist clinicians in management of reproductive dysfunctions in male HIV/AIDS-alcoholic patients on cART.

Low Dose of 5-Aminolevulinic Acid Hydrochloride Alleviates the Damage in Cardiomyocytes Induced by Lenvatinib via PI3K/AKT Signaling Pathway.

Lenvatinib is an oral tyrosine kinase inhibitor (TKI), and has been applied in the clinical trials for the treatment of hepatocellular carcinoma (HCC). The function of 5-aminolevulinic acid hydrochloride (ALA) treatment in protecting cardiomyocytes under lenvatinib stimulation was investigated. H9c2 cells were treated with 2 mg/mL lenvatinib for 48 h and 1 mM ALA in the lenvatinib with low dose 5-aminolevulinic acid treatment group (LL) group, 10 mM ALA in the lenvatinib with high-dose 5-aminolevulinic acid treatment group (LH) group and cells without treatment were used as an internal control. C57/BL mice were treated with 10 mg/kg lenvatinib and 200 mg/kg ALA in the LL group and 400 mg/kg ALA in the LH group by gavage once per day for 4 weeks. The proliferation ability of cells was detected using the methyl thiazolyl tetrazolium (MTT) assay. Target gene expression was calculated through real-time quantitative PCR (qPCR), and target protein expression was calculated through Western blotting analysis. The concentrations of cardiovascular protective factors were detected using enzyme linked immunosorbent assay (ELISA). In these experiments, 10 mM ALA significantly increased the viability rate of cardiomyocytes (105.4 ± 8.0%) compared with the single lenvatinib treatment group (73.2 ± 6.5%). We also noticed that activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathways were activated after low-dose ALA treatment. 5-ALA treatment led to the downregulation of intercellular cell adhesion molecule-1 (ICAM-1) (0.81- and 0.71-fold), vascular cell adhesion molecule (VCAM) (0.63- and 0.66-fold), angiotensin I (ANGI) (0.88- and 0.66-fold), ANGII (0.66- and 0.48-fold) and upregulation of endothelial nitric oxide synthases (eNOS) (1.25- and 1.89-fold) compared with non 5-ALA treatment group. With more experiments on animal models, low-dose of ALA treatment might be a therapeutic strategy to alleviate the damage to cardiomyocytes induced by lenvatinib.

Chemical Characterization and Immunomodulatory Activity of Fucoidan from Sargassum hemiphyllum.

Fucoidan is a sulfated algal polyanionic polysaccharide that possesses many biological activities. In this paper, a fucoidan (SHF) polysaccharide was extracted from Sargassum hemiphyllum collected in the South China Sea. The SHF, with a molecular weight of 1166.48 kDa (44.06%, w/w), consisted of glucose (32.68%, w/w), galactose (24.81%, w/w), fucose (20.75%, w/w), xylose (6.98%, w/w), mannose (2.76%, w/w), other neutral monosaccharides, and three uronic acids, including glucuronic acid (5.39%, w/w), mannuronic acid (1.76%, w/w), and guronuronic acid (1.76%, w/w). The SHF exhibited excellent immunostimulatory activity. An immunostimulating assay showed that SHF could significantly increase NO secretion in macrophage RAW 264.7 cells via upregulation of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) levels based on both gene expression and protein abundance. These results suggest that SHF isolated from Sargassum hemiphyllum has great potential to act as a health-boosting ingredient in the pharmaceutical and functional-food fields.

Chloroform Fraction of Prasiola japonica Ethanolic Extract Alleviates UPM 1648a-Induced Lung Injury by Suppressing NF-κB Signaling

Prasiola japonica is an edible alga, and the ethanol extract of P. japonica (Pj-EE) possesses various biological activities. Interestingly, in a recent study, we observed the potent anti-inflammatory activity of the chloroform fraction of Pj-EE (Pj-EE-CF). Thus, to extend the application of Pj-EE-CF, we further studied its effects on lung injury. To establish an experimental model of lung injury, we nasally administered urban particulate matter UPM 1648a (50 mg/kg) to mice. In addition, BEAS-2B cells were treated with 300 μg/mL of UPM 1648a for in vitro analysis. Intranasal administration of UPM 1648a increased lung injury score, macrophage infiltration, and upregulation of the inflammatory enzyme inducible nitric oxide synthase (iNOS) in lung tissues. On the other hand, oral administration of Pj-EE-CF (25, 50, and 100 mg/kg) alleviated these pathological features as assessed by lung wet/dry ratio, lung injury score, bronchoalveolar lavage fluid (BALF) protein amount in the lung tissues up to 70%, 95%, and 99%, respectively. In addition, Pj-EE-CF down-regulated the release of inflammatory cytokines, interleukins (ILs), tumor necrosis factor (TNF)-α, and interferon (IFN)-γ elevated by UPM 1648a in the lung tissues and lung BALF up to 95%. According to Western blot and luciferase assay, Pj-EE-CF (100 mg/kg in vivo or 50 and 100 μg/mL in vitro) significantly reduced the nuclear factor-κB (NF-κB) signal activated by UPM 1648a. Finally, UPM 1648a increased cellular reactive oxygen species (ROS) levels in BEAS-2B cells, while Pj-EE-CF reduced them. These results suggest that Pj-EE-CF alleviates UPM 1648a-induced lung damage via anti-inflammatory and antioxidant activities and by suppressing NF-κB signaling. In conclusion, these observations imply that Pj-EE-CF could be a practical component of food supplements to mitigate air pollution-derived lung damage.