In Vitro Determination of Nitric Oxide Synthase Inhibition, Antioxidant Capacity and Phenolic Content of Various Natural Products (Bee and Herbal Products).

It is obvious that the oxidation process is an undeniable fact and when it comes to aging, one of the first solutions that come to mind is natural products. When it comes to natural products, both plants and bee products play an important, almost combative role against oxidation. For this purpose, natural products of both plant and animal origin were considered together in our study: Linden, green tea, aronia, wild grapes, myrtle, blueberries and basil, honey, pollen and propolis. Total phenolic content values of the extracts ranged between 49.28 and 3859.06 mg gallic acid equivalent/100 g, and propolis, green tea, chestnut flower and aronia samples were found to have the highest values. When looking at the NOS inhibition potential, it was determined that propolis, pollen and aronia samples had the highest percentage inhibition values of 98.11, 92.29, 83.44, respectively. Antioxidant activities of methanolic extracts were investigated using iron(III) reducing/antioxidant capacity (FRAP), 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity test and NOS inhibition tests. The phenolic composition of methanolic extracts was tested using the RP-HPLC-UV (high-performance liquid chromatographic method with ultraviolet) method with 19 phenolic standards.

Folic acid antioxidant supplementation to binge drinking adolescent rats improves hydric-saline balance and blood pressure, but fails to increase renal NO availability and glomerular filtration rate.

Binge drinking (BD) is an especially pro-oxidant pattern of alcohol consumption, particularly widespread in the adolescent population. In the kidneys, it affects the glomerular filtration rate (GFR), leading to high blood pressure. BD exposure also disrupts folic acid (FA) homeostasis and its antioxidant properties. The aim of this study is to test a FA supplementation as an effective therapy against the oxidative, nitrosative, and apoptotic damage as well as the renal function alteration occurred after BD in adolescence. Four groups of adolescent rats were used: control, BD (exposed to intraperitoneal alcohol), control FA-supplemented group and BD FA-supplemented group. Dietary FA content in control groups was 2 ppm, and 8 ppm in supplemented groups. BD provoked an oxidative imbalance in the kidneys by dysregulating antioxidant enzymes and increasing the enzyme NADPH oxidase 4 (NOX4), which led to an increase in caspase-9. BD also altered the renal nitrosative status affecting the expression of the three nitric oxide (NO) synthase (NOS) isoforms, leading to a decrease in NO levels. Functionally, BD produced a hydric-electrolytic imbalance, a low GFR and an increase in blood pressure. FA supplementation to BD adolescent rats improved the oxidative, nitrosative, and apoptotic balance, recovering the hydric-electrolytic equilibrium and blood pressure. However, neither NO levels nor GFR were recovered, showing in this study for the first time that NO availability in the kidneys plays a crucial role in GFR regulation that the antioxidant effects of FA cannot repair.

Nitric Oxide as a Determinant of Human Longevity and Health Span.

The master molecular regulators and mechanisms determining longevity and health span include nitric oxide (NO) and superoxide anion radicals (SOR). L-arginine, the NO synthase (NOS) substrate, can restore a healthy ratio between the dangerous SOR and the protective NO radical to promote healthy aging. Antioxidant supplementation orchestrates protection against oxidative stress and damage-L-arginine and antioxidants such as vitamin C increase NO production and bioavailability. Uncoupling of NO generation with the appearance of SOR can be induced by asymmetric dimethylarginine (ADMA). L-arginine can displace ADMA from the site of NO formation if sufficient amounts of the amino acid are available. Antioxidants such as ascorbic acids can scavenge SOR and increase the bioavailability of NO. The topics of this review are the complex interactions of antioxidant agents with L-arginine, which determine NO bioactivity and protection against age-related degeneration.

Aerobic exercise prevents cardiomyocyte damage caused by oxidative stress in early cardiovascular disease by increasing vascularity while L-arginine supplementation prevents it by increasing activation of the enzyme nitric oxide synthase.

L-Arginine and chronic exercise reduce oxidative stress. However, it is unclear how they affect cardiomyocytes during cardiovascular disease (CVD) development. The aim of this research was to investigate the possible effects of L-arginine supplementation and aerobic training on systemic oxidative stress and their consequences on cardiomyocytes during cardiometabolic disease onset caused by excess fructose. Wistar rats were allocated into four groups: control (C), fructose (F, 10% fructose in water), fructose training (FT; moderate running, 50-70% of the maximal velocity), and fructose arginine (FA; 880 mg/kg/day). Fructose was given for two weeks and fructose plus treatments for the subsequent eight weeks. Body composition, blood glucose, insulin, lipid profile, lipid peroxidation, nitrite, metalloproteinase-2 (MMP-2) activity, left ventricle histological changes, microRNA-126, -195, and -146, eNOS, p-eNOS, and TNF-α expressions were analyzed. Higher abdominal fat mass, triacylglycerol level, and insulin level were observed in the F group, and both treatments reversed these alterations. Myocardial vascularization was impaired in fructose-fed groups, except in FT. Cardiomyocyte hypertrophy was observed in all fructose-fed groups. TNF-α levels were higher in fructose-fed groups than in the C group, and p-eNOS levels were higher in the FA than in the C and F groups. Lipid peroxidation was higher in the F group than in the FT and C groups. During CVD onset, moderate aerobic exercise reduced lipid peroxidation, and both training and L-arginine prevented metabolic changes caused by excessive fructose. Myocardial vascularization was impaired by fructose, and cardiomyocyte hypertrophy appeared to be influenced by pro-inflammatory and oxidative environments.

The effects of a comparatively higher dose of 1000 mg/kg/d of oral L- or D-arginine on the L-arginine metabolic pathways in male Sprague-Dawley rats.

Oral L-arginine supplements are popular mainly for their nitric oxide mediated vasodilation, but their physiological impact is not fully known. L-arginine is a substrate of several enzymes including arginase, nitric oxide synthase, arginine decarboxylase, and arginine: glycine amidinotransferase (AGAT). We have published a study on the physiological impact of oral L- and D-arginine at 500 mg/kg/day for 4 wks in male Sprague-Dawley rats. We investigated the effects of oral L-arginine and D-arginine at a higher dose of 1000 mg/kg/d for a longer treatment duration of 16 wks in 9-week-old male Sprague-Dawley rats. We measured the expression and activity of L-arginine metabolizing enzymes, and levels of their metabolites in the plasma and various organs. L-arginine did not affect the levels of L-arginine and L-lysine in the plasma and various organs. L-arginine decreased arginase protein expression in the upper small intestine, and arginase activity in the plasma. It also decreased AGAT protein expression in the liver, and creatinine levels in the urine. L-arginine altered arginine decarboxylase protein expression in the upper small intestine and liver, with increased total polyamines plasma levels. Endothelial nitric oxide synthase protein was increased with D-arginine, the presumed metabolically inert isomer, but not L-arginine. In conclusion, oral L-arginine and D-arginine at a higher dose and longer treatment duration significantly altered various enzymes and metabolites in the arginine metabolic pathways, which differed from alterations produced by a lower dose shorter duration treatment published earlier. Further studies with differing doses and duration would allow for a better understanding of oral L-arginine uses, and evidence based safe and effective dose range and duration.

Putrescine supplementation shifts macrophage L-arginine metabolism related-genes reducing Leishmania amazonensis infection.

Leishmania is a protozoan that causes leishmaniasis, a neglected tropical disease with clinical manifestations classified as cutaneous, mucocutaneous, and visceral leishmaniasis. In the infection context, the parasite can modulate macrophage gene expression affecting the microbicidal activity and immune response. The metabolism of L-arginine into polyamines putrescine, spermidine, and spermine reduces nitric oxide (NO) production, favoring Leishmania survival. Here, we investigate the effect of supplementation with L-arginine and polyamines in infection of murine BALB/c macrophages by L. amazonensis and in the transcriptional regulation of genes involved in arginine metabolism and proinflammatory response. We showed a reduction in the percentage of infected macrophages upon putrescine supplementation compared to L-arginine, spermidine, and spermine supplementation. Unexpectedly, deprivation of L-arginine increased nitric oxide synthase (Nos2) gene expression without changes in NO production. Putrescine supplementation increased transcript levels of polyamine metabolism-related genes Arg2, ornithine decarboxylase (Odc1), Spermidine synthase (SpdS), and Spermine synthase (SpmS), but reduced Arg1 in L. amazonensis infected macrophages, while spermidine and spermine promoted opposite effects. Putrescine increased Nos2 expression without leading to NO production, while L-arginine plus spermine led to NO production in uninfected macrophages, suggesting that polyamines can induce NO production. Besides, L-arginine supplementation reduced Il-1b during infection, and L-arginine or L-arginine plus putrescine increased Mcp1 at 24h of infection, suggesting that polyamines availability can interfere with cytokine/chemokine production. Our data showed that putrescine shifts L-arginine-metabolism related-genes on BALB/c macrophages and affects infection by L. amazonensis.

Bioactive fraction from Plumeria obtusa L. attenuates LPS-induced acute lung injury in mice and inflammation in RAW 264.7 macrophages: LC/QToF-MS and molecular docking.

In this study, the anti-inflammatory effects of the methanolic extract (TE) of Plumeria obtusa L. (aerial parts) and its fractions were evaluated in vitro, and active fraction was evaluated in vivo. Among tested extracts, dichloromethane fraction (DCM-F) exhibited the strongest inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) in RAW 264.7 macrophages. The effect of DCM-F on LPS-induced acute lung injury (ALI) in mice was studied. The animals were divided into five groups (n = 7) randomly; Gp I: negative control, GP II: positive control (LPS group), GP III: standard (dexamethasone, 2 mg/kg b.wt), GP IV and V: DCM-F (100 mg/kg), and DEM-F (200 mg/kg), respectively. DCM-F at a dose of 200 mg/kg suppressed the ability of LPS to increase the levels of nitric oxide synthase (iNOS), NO, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), as measured by ELISA. In addition, the expression of cyclooxygenase-2 (COX-2) was reduced (determined by immunohistochemistry) and the level of malondialdehyde (MDA) was decreased while that of catalase was restored to the normal values. Furthermore, the histopathological scores of inflammation induced by LPS were reduced. Twenty-two compounds were tentatively identified in DCM-F using LC/ESI-QToF with iridoids, phenolic derivatives and flavonoids as major constituents. Identified compounds were subjected to two different molecular docking processes against iNOS and prostaglandin E synthase-1 target receptors. Notably, protoplumericin A and 13-O-coumaroyl plumeride were the most promising members compared to the co-crystallized inhibitor in each case. These findings suggested that DCM-F attenuates the LPS-induced ALI in experimental animals through its anti-inflammatory and antioxidant potential.

Supplementation with Nitric Oxide Precursors for Strength Performance: A Review of the Current Literature.

Nitric-oxide-stimulating dietary supplements are widely available and marketed to strength athletes and weightlifters seeking to increase muscle performance and augment training adaptations. These supplements contain ingredients classified as nitric oxide (NO) precursors (i.e., "NO boosters"). Endogenous NO is generated via a nitric oxide synthase (NOS)-dependent pathway and a NOS-independent pathway that rely on precursors including L-arginine and nitrates, with L-citrulline serving as an effective precursor of L-arginine. Nitric oxide plays a critical role in endothelial function, promoting relaxation of vascular smooth muscle and subsequent dilation which may favorably impact blood flow and augment mechanisms contributing to skeletal muscle performance, hypertrophy, and strength adaptations. The aim of this review is to describe the NO production pathways and summarize the current literature on the effects of supplementation with NO precursors for strength and power performance. The information will allow for an informed decision when considering the use of L-arginine, L-citrulline, and nitrates to improve muscular function by increasing NO bioavailability.

The Dark Side of an Essential Amino Acid: L-Arginine in Spinal Cord Injury.

L-arginine is a semi-essential amino acid involved in a variety of physiological processes in the central nervous system (CNS). It is essential in the survival and functionality of neuronal cells. Nonetheless, L-arginine also has a dark side; it potentiates neuroinflammation and nitric oxide (NO) production, leading to secondary damage. Therefore, modulating the L-arginine metabolism is challenging because both detrimental and beneficial effects are dependent on this semi-essential amino acid. After spinal cord injury (SCI), L-arginine plays a crucial role in trauma-induced neuroinflammation and regenerative processes via the two key enzymes: nitric oxide synthase (NOS) and arginase (ARG). Studies on L-arginine metabolism using ARG and NOS inhibitors highlighted the conflicting role of this semi-essential amino acid. Similarly, L-arginine supplementation resulted in both negative and positive outcomes after SCI. However, new data indicate that arginine depletion substantially improves spinal cord regeneration after injury. Here, we review the challenging characteristics of L-arginine metabolism as a therapeutic target after SCI.

Therapeutic effects of a soluble guanylate cyclase activator, BAY 60-2770, on lower urinary tract dysfunction in mice with spinal cord injury.

We aimed to evaluate the effects of a soluble guanylate cyclase (sGC) activator, BAY 60-2770, on neurogenic lower urinary tract dysfunction in mice with spinal cord injury (SCI). Mice were divided into the following three groups: spinal cord intact (group A), SCI + vehicle (group B), and SCI + BAY 60-2770 (group C). SCI mice underwent Th8-Th9 spinal cord transection and treatment with BAY 60-2770 (10 mg/kg/day) once daily for 2-4 wk after SCI. We evaluated urodynamic parameters using awake cystometry and external urethral sphincter electromyograms (EMG); mRNA levels of mechanosensory channels, nitric oxide (NO)-, ischemia-, and inflammation-related markers in L6-S1 dorsal root ganglia, the urethra, and bladder tissues; and protein levels of cGMP in the urethra at 4 wk after SCI. With awake cystometry, nonvoiding contractions, postvoid residual, and bladder capacity were significantly larger in group B than in group C. Voiding efficiency (VE) was significantly higher in group C than in group B. In external urethral sphincter EMGs, the duration of notch-like reductions in intravesical pressure and reduced EMG activity time were significantly longer in group C than in group B. mRNA expression levels of transient receptor potential ankyrin 1, transient receptor potential vanilloid 1, acid-sensing ion channel (ASIC)1, ASIC2, ASIC3, and Piezo2 in the dorsal root ganglia, and hypoxia-inducible factor-1α, VEGF, and transforming growth factor-ß1 in the bladder were significantly higher in group B than in groups A and C. mRNA levels of neuronal NO synthase, endothelial NO synthase, and sGCα1 and protein levels of cGMP in the urethra were significantly lower in group B than in groups A and C. sGC modulation might be useful for the treatment of SCI-related neurogenic lower urinary tract dysfunction.NEW & NOTEWORTHY This is the first report to evaluate the effects of a soluble guanylate cyclase activator, BAY 60-2770, on neurogenic lower urinary tract dysfunction in mice with spinal cord injury.

Pleurotus albidus (Agaricomycetes) Antioxidant Action Induces Vasodilation in Aorta Arteries: The Influence of the NADPH/NOS Balance.

The main objective of this work was to evaluate whether Pleurotus albidus extract exerts influences on aorta artery tone by its antioxidant properties. The hearts and aortic arteries of male Wistar rats were removed for use in biochemical analysis and vascular reactivity. Both tissues were exposed to P. albidus extract at different concentrations for 30 min and were then exposed to a free radical generation system for 30 min. The extract reduced lipid peroxidation levels and increased catalase and glutathione peroxidase activity in cardiac tissue. In the aorta, P. albidus extract demonstrated a direct vasodilatory effect, which was associated with a reduction in nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity and an increase in sulfhydryl levels and nitric oxide synthase (NOS) activity. Our findings suggest that P. albidus extract has regulatory potential on aorta arteries, regulating the balance of NOX/NOS enzymes and then influencing vessel tone. Further studies are needed to determine the protective mechanisms of the extract.

Hypochlorous acid facilitates inducible nitric oxide synthase subunit dissociation: The link between heme destruction, disturbance of the zinc-tetrathiolate center, and the prevention by melatonin.

Inducible nitric oxide synthase (iNOS) is a zinc-containing hemoprotein composed of two identical subunits, each containing a reductase and an oxygenase domain. The reductase domain contains binding sites for NADPH, FAD, FMN, and tightly bound calmodulin and the oxygenase domain contains binding sites for heme, tetrahydrobiopterin (H4B), and l-arginine. The enzyme converts l-arginine into nitric oxide (NO) and citrulline in the presence of O2. It has previously been demonstrated that myeloperoxidase (MPO), which catalyzes formation of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride (Cl-), is enhanced in inflammatory diseases and could be a potent scavenger of NO. Using absorbance spectroscopy and gel filtration chromatography, we investigated the role of increasing concentrations of HOCl in mediating iNOS heme destruction and subsequent subunit dissociation and unfolding. The results showed that dimer iNOS dissociation between 15 and 100 µM HOCl was accompanied by loss of heme content and NO synthesis activity. The dissociated subunits-maintained cytochrome c and ferricyanide reductase activities. There was partial unfolding of the subunits at 300 µM HOCl and above, and the subunit unfolding transition was accompanied by loss of reductase activities. These events can be prevented when the enzyme is preincubated with melatonin prior to HOCl addition. Melatonin supplementation to patients experiencing low NO levels due to inflammatory diseases may be helpful to restore physiological NO functions.

Cysteine-Rich Whey Protein Isolate (CR-WPI) Ameliorates Erectile Dysfunction by Diminishing Oxidative Stress via DDAH/ADMA/NOS Pathway.

Nitric oxide synthase- (NOS-) dependent endothelial dysfunction induced by oxidative stress (OS) is assumed to play a pivotal role in the pathogenesis and progression of diabetes mellitus-related erectile dysfunction (DMED). Cysteine-rich whey protein isolate (CR-WPI) is a widely used protein supplement and has been confirmed to reduce reactive oxygen species (ROS) by increasing cellular antioxidant glutathione (GSH). However, it is currently unknown whether CR-WPI elicits therapeutic effects in DMED. Here, we provide diabetic rats with CR-WPI to determine its effect on DMED and the underlying mechanisms. The results suggest that CR-WPI supplementation increased GSH biosynthesis and reduced ROS content and simultaneously upregulated the dimethylarginine dimethylaminohydrolase (DDAH)/asymmetrical dimethylarginine (ADMA)/nitric oxide synthase (NOS) metabolic pathway. Evaluation of intracavernous pressure (ICP) also showed an improvement of penile erectile function in CR-WPI-treated rats. The results of the vitro cell culture showed that glutathione pretreatment protected corpus cavernosum smooth muscle cells (CCSMC) from H2O2-induced apoptosis by decreasing Caspase 9 and Caspase 3 expressions. These results augur well for the potential therapeutic application of dietary CR-WPI supplementation for treating diabetic erectile dysfunction.

Shufeiya Recipe Improves Monocrotaline-Induced Pulmonary Hypertension in Rats by Regulating SIRT3/FOXO3a and Its Downstream Signaling Pathways.

Pulmonary hypertension (PH) is a chronic and progressive disease caused by obstructions and functional changes of small pulmonary arteries. Current treatment options of PH are costly with patients needing long-term taking medicine. The traditional Chinese medicine (TCM) compound "Shufeiya Recipe" was used to intervene in monocrotaline- (MCT-) induced pulmonary hypertension in rats. The rats were randomly divided into the control group, model group, positive drug (Sildenafil) group, and Shufeiya Recipe low-, moderate-, and high-dose groups. The improvement effect of the Shufeiya Recipe on the mean pulmonary artery pressure (mPAP) was assessed in PH rats, and pathological staining was used to observe the pathological changes of lung tissue. The impact of the Shufeiya Recipe on oxidative stress damage in rats with pulmonary hypertension and the regulation of SIRT3/FOXO3a and its downstream signaling pathways were determined. The results showed that Shufeiya Recipe could significantly downregulate mPAP and improve lung histopathological changes; downregulate serum levels of reactive oxygen species (ROS); upregulate the concentrations of COX-1 and COX-2 and the activity of Mn-SOD; inhibit oxidative response damage; promote the protein expression of SIRT3, FOXO3a, p-PI3K, p-AKT, and p-eNOS; increase the level of expression of NO, sGC, cGMP, and PKG; and downregulate the level of protein expression of Ras, p-MEK1/2, p-ERK1/2 and c-fos. These results indicate that Shufeiya Recipe can improve MCT-induced pulmonary hypertension in rats by regulating SIRT3/FOXO3a and its downstream PI3K/AKT/eNOS and Ras/ERK signaling pathways.

Anti-Oxidation and Anti-Inflammatory Potency Evaluation of Ferulic Acid Derivatives Obtained through Virtual Screening.

Various factors such as ultraviolet rays can cause a continuous threat to our skin, resulting in inflammation or oxidation problems. Ferulic acid (FA), with certain antioxidant and anti-inflammatory properties, is widely used in many cosmetics, even used to treat various diseases in the clinic. In this study, the FA structural skeleton was used to search for FA derivatives. Then, molecular docking, the rule of five, and Veber rules were performed to virtually screen compounds that can bind to proteins with a good drug likeness. DPPH and ABTS were used to evaluate their antioxidant potency and an MTT assay was employed to investigate the toxicities of the compounds, while Griess Reaction System and ELISA were used to judge the concentration variations of NO and different inflammatory factors (TNF-α, IL-1ß, and IL-6). Western blotting featured nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression levels. The trend of the intracellular changes of reactive oxygen species (ROS) was detected by the DCFH-DA method and fluorescence staining. As a result, we found that the ferulic acid derivative S-52372 not only had certain scavenging effects on free radicals in biochemical experiments, but also prevented inflammation and oxidative stress in LPS-stimulated RAW264.7 cells in the cellular environment; intracellular ROS and inflammatory mediators, including iNOS, COX-2, TNF-α, and IL-6, were also suppressed. In a computer prediction, S-52372 owned better water solubility and lower toxicity than FA. This compound deserves further research to find an ideal FA derivative.

Potential of Ageratum conyzoides in Inhibiting Nitric Oxide Synthase.

<b>Background and Objective:</b> Inflammation occurs <i>via</i> several mechanisms, one of which includes the production of Nitric Oxide (NO) catalyzed by inducible nitric oxide synthase (iNOS), which is inhibited selectively by isothioureas. <i>Ageratum conyzoides</i> L. has shown activity in reducing pain and inflammation, although the molecular mechanism had not been undertaken. The objectives of this work were (1) to study the mechanism of anti-inflammatory activity of <i>A. conyzoides</i> through inhibition of iNOS, (2) to correlate the iNOS inhibitory activity of the plant with the total flavonoid content of the plants and (3) to identify the flavonol synthase (FLS), an enzyme that catalyzes the production of quercetin. <b>Materials and Methods:</b> The inhibitory activity against iNOS was assayed by <i>in vitro</i> method. The total flavonoids (calculated as quercetin) of <i>A. conyzoides</i> were determined by fluorometry. The protein extraction of the leaves was carried out by employing Laing and Christeller's (2004) method, followed with SDS-PAGE. <b>Results:</b> The inhibitory activity (IC₅₀) of ethanol extract and ethyl acetate fraction of <i>A. conyzoides</i> against iNOS was 92.05 and 4.78 µg mL¹, respectively. Pearson correlation analysis resulted in 0.548 (ethanol extract) and 0.696 (ethyl acetate fraction). The total flavonoids (calculated as quercetin) contained in the ethanol extract and ethyl acetate fraction of <i>A. conyzoides</i> were 0.71 and 7.65%, respectively. The FLS in <i>A. conyzoides</i> leaves was identified at 31 kDa. <b>Conclusion:</b> <i>A. </i>c<i>onyzoides</i> L. is potential in inhibiting iNOS due to quercetin contained in the leaves. This report will add a scientific insight of <i>A. conyzoides</i> for biological sciences.

Inhibition of nNOS in the paraventricular nucleus of hypothalamus decreases exercise-induced hyperthermia.

The paraventricular nucleus of the hypothalamus (PVN) is an important site for autonomic control, which integrates thermoregulation centers and sympathetic outflow to thermoeffector organs. PVN neurons express the neuronal isoform of nitric oxide synthase (nNOS) whose expression is locally upregulated by physical exercise. Thus, the aim of the present study was to evaluate the role of nNOS in the PVN in the exercise-induced hyperthermia. Seven days after surgery, male Wistar rats received bilateral intra-PVN microinjections of the selective nNOS inhibitor Nw-Propyl-L-Arginine (NPLA) or vehicle (saline) and were submitted to an acute progressive exercise session on a treadmill until fatigue. Abdominal and tail skin temperature (Tabd and Ttail, respectively) were measured, and the threshold (Hthr; °C) and sensitivity (Hsen) for heat dissipation calculated. Performance variables were also collected. During the progressive exercise protocol, all animals displayed an increase in the Tabd. However, compared to vehicle group, the microinjection of NPLA in the PVN attenuated the exercise-induced hyperthermia. There was no difference in Ttail or Hthr between NPLA and control rats. In contrast, Hsen was increased in the NPLA group compared to vehicle. In addition, heat storage was lower in NPLA-treated animals. Despite the temperature differences, inhibition of nNOS in the PVN did not affect running performance on the treadmill. These results suggest that nitrergic signaling within the PVN, under nNOS activation, drives the increase of body temperature, being necessary for the proper thermal regulatory mechanisms during progressive exercise-induced hyperthermia.

Arginine Supplementation Promotes Extracellular Matrix and Metabolic Changes in Keratoconus.

Keratoconus (KC) is a common corneal ectatic disease that affects 1:500-1:2000 people worldwide and is associated with a progressive thinning of the corneal stroma that may lead to severe astigmatism and visual deficits. Riboflavin-mediated collagen crosslinking currently remains the only approved treatment to halt progressive corneal thinning associated with KC by improving the biomechanical properties of the stroma. Treatments designed to increase collagen deposition by resident corneal stromal keratocytes remain elusive. In this study, we evaluated the effects of arginine supplementation on steady-state levels of arginine and arginine-related metabolites (e.g., ornithine, proline, hydroxyproline, spermidine, and putrescine) and collagen protein expression by primary human corneal fibroblasts isolated from KC and non-KC (healthy) corneas and cultured in an established 3D in vitro model. We identified lower cytoplasmic arginine and spermidine levels in KC-derived constructs compared to healthy controls, which corresponded with overall higher gene expression of arginase. Arginine supplementation led to a robust increase in cytoplasmic arginine, ornithine, and spermidine levels in controls only and a significant increase in collagen type I secretion in KC-derived constructs. Further studies evaluating safety and efficacy of arginine supplementation are required to elucidate the potential therapeutic applications of modulating collagen deposition in the context of KC.

A kaempferol derivative isolated from Lysimachia ramosa (Wall ex. Duby) induced alteration of acetyl cholinesterase and nitric oxide synthase in Raillietina echinobothrida.

Lysimachia ramosa has been used as a traditional medicine among the tribal population of Meghalaya, northeast India, for the control of helminthosis. The anthelmintic efficacy of L. ramosa has been documented earlier. In the present study, the active compound from L. ramosa has been isolated and identified using mass and NMR spectra. It's in vitro anthelmintic activity was evaluated against Raillietina echinobothrida, one of the most pathogenic cestode of domestic fowl. The isolated active compound was characterized to be a kaempferol derivative which showed potent anthelmintic activity against R. echinobothrida by changing surface ultrastructure and also inhibiting the activity of two neurotransmitter enzymes: acetyl cholinesterase (AChE) and nitric oxide synthase (NOS), both of which are known to perform dynamic roles in the intracellular communication mediated through neuromuscular system. Motility reduction, deformation in the surface architecture, extensive ultrastructural alterations and reduced histochemical stain intensity in both AChE and NOS was observed in the treated parasites. Biochemical result also revealed alteration in the enzyme activities in the treated parasites. Further, depletion in the nitric oxide (NO) production in the bioactive component exposed tissues of R. echinobothrida was also detected. The results provided evidence that the bioactive compound could be further explored to control helminthosis at a large scale.

Polyisoprenylated benzophenone derivatives from Garcinia cambogia and their anti-inflammatory activities.

Ten new polyisoprenylated benzophenone derivatives, 4,8-epi-uralione F (1), 4,8-epi-uralione G (2), uralione S (3), coccinone J (4), 6-epi-coccinone C (5), coccinone I (6), 36-hydroxy-guttiferone J (7), multiflorone I (8), garciniagifolone F (9) and 36-hydroxy-garciniagifolone F (10), were isolated from the fruits of Garcinia cambogia, along with seven known analogues. The structures of the new compounds were established based on the detailed analysis of 1D and 2D nuclear magnetic resonance (NMR) spectra and high resolution electrospray ionization mass spectrometra (HRESIMS), and their absolute configurations were determined from the electronic circular dichroism (ECD) spectra. All the isolates were tested for their inhibitory effects against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The results indicated that compound 1 displayed a potent NO inhibitory effect with an IC50 value of 41.60 ± 0.17 µM. Furthermore, compound 1 suppressed inducible NO synthase (iNOS) expression in a dose-dependent manner through inhibiting the activation of nuclear factor-κB (NF-κB).