Nitric Oxide Synthase Production Research Articles

Inhibition of oxidative metabolism by nitric oxide restricts EMCV replication selectively in pancreatic beta-cells

Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1β, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for β-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in β-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a β-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.

Nardostachin from Nardostachys jatamansi exerts anti‑neuroinflammatory effects through TLR4/MyD88‑related suppression of the NF‑κB and JNK MAPK signaling pathways in lipopolysaccharide‑induced BV2 and primary microglial cells.

Through searching for anti‑neuroinflammatory metabolites from Nardostachys jatamansi extracts, nardostachin was revealed to exert anti‑neuroinflammatory effects against lipopolysaccharide (LPS)‑induced overproduction of nitric oxide and prostaglandin E2 in BV2 and rat primary microglial cells. Furthermore, nardostachin inhibited the production of inducible nitric oxide synthase and cyclooxygenase‑2 as well as pro‑inflammatory cytokines, including interleukin (IL)‑1β, IL‑6, IL‑12 and tumor necrosis factor‑α in LPS‑stimulated BV2 and rat primary microglial cells. In a mechanistic study, nardostachin exhibited inhibitory activity on the nuclear factor (NF)‑κB signaling pathway in LPS‑stimulated BV2 and rat primary microglial cells by repressing IκB‑α phosphorylation and blocking NF‑κB translocation. Furthermore, nardostachin exhibited inhibitory effects on LPS‑induced phosphorylation of c‑Jun N‑terminal kinase (JNK) mitogen‑activated protein kinase (MAPK). Additionally, nardostachin repressed protein expression of Toll‑like receptor4 (TLR4) and myeloid differentiation factor88 (MyD88) in LPS‑induced BV2 and rat primary microglial cells. These results suggested that nardostachin exerts anti‑neuroinflammatory effects on LPS‑induced BV2 and rat primary microglial cells by suppressing the TLR4‑MyD88‑NF‑κB and JNK MAPK pathways.

δ-Opioid receptor activation ameliorates lipopolysaccharide-induced inflammation and apoptosis by inhibiting the MAPK/caspase-3 pathway in BV2 microglial cells.

Delta-opioid receptor (DOR) is widely distributed in the central nervous system, and its activation protects against ischaemic/hypoxic brain injury. However, the role of DOR in microglia in ischaemic stroke has not yet been fully investigated. We found that DOR was expressed in both human and mouse cerebral microglia, besides, it was upregulated in activated BV2 microglial cells by immunofluorescence staining and Western blot. DOR activation by the specific agonist TAN-67 significantly enhanced BV2 microglial cell viability and reduced apoptosis, as evidenced by decreased cleaved caspase-3 levels and TdT-mediated aUTP-X nick end labelling (TUNEL) staining after LPS stimulation. Furthermore, activation of DOR significantly inhibited inducible nitric oxide synthase (iNOS) production and dose-dependently inhibited the mRNA and protein expression levels of other pro-inflammatory cytokines, including IL-1β and IL-6, whereas it increased the expression of the anti-inflammatory cytokine IL-10 in LPS-stimulated BV2 microglial cells; these effects were correlated with diminished phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. Moreover, these effects could be reversed by the DOR antagonist naltrindole. DOR activation can activate microglia to switch to the beneficial phenotype and inhibit LPS-induced inflammation and apoptosis via the mitogen-activated protein kinase (MAPK)/caspase-3 pathway in BV2 microglial cells. This study provides new insight into neuroprotection against and treatment of ischaemic stroke.

Morphological changes of the liver under conditions of hyperhomocysteinemia in the background of hypo- and hyperthyroidism

Thyroxine and Triiodothyronine are very important for normal growth, development and organ function. These hormones regulate the basal rate of metabolism of all cells, including hepatocytes, and thus modulate liver function. There is a close connection between hyperhomocysteinemia (HHCy) and the induction of oxidative processes, disruption of nitric oxide production of NO synthase, damage to the endoplasmic reticulum and activation of inflammatory processes in the liver. Disorders of homocysteine metabolism (HC) in thyroid dysfunction are also known. Therefore, it can be assumed that the violation of the structure and functions of the liver will be an important manifestation of the negative impact of HHCy on organs and tissues in hyper- and hypothyroidism. The aim of the study was to establish the reorganization of the structural components of the liver in the conditions of modelized HHCy, hyper- and hypothyroidism and their joint effects. Thiolactone HHCy was modelized by administering to animals an exogenous HC in the form of Thiolactone at a dose of 100 mg/kg body weight once a day for 28 days. Hyperthyroidism was modelized by daily administration of L-thyroxine at a dose of 200 μg/kg for the 21 days, hypothyroidism - daily administration of Thiamazole at a dose of 10 mg/kg for the 21 days. Individual groups of animals were administered L-thyroxine and Thiamazole in parallel with HC. It was found that in the conditions of simulated HHCy, hypo- and hyperthyroidism in the liver of experimental animals there is an incompleteness of hepatocyte beams, changes in hepatocytes of destructive, dystrophic and necrotic nature with signs of steatosis, vascular disorders. Conclusions: both HHCy and hypo- or hyperthyroidism lead to a violation of the structural organization of liver tissue. With the development of thyroid dysfunction on the background of HHCy, the disturbances of the histological structure of hepatocytes significantly increased.

Nitrotyrosine formation, iNOS and the Na /K -ATPase activities in sepsis: The possible effects of CAPE

Sepsis is a response to infection characterized by the formation of highly reactive oxygen and nitrogen substances. The rat kidney was chosen for this purpose because many important inflammatory mediators, including inducible nitric oxide synthase (iNOS) and nitrotyrosine (nTyr) production, are expressed by kidney cells following either lipopolysaccharide (LPS) or bacterial challenge. The present study was aimed at investigating the relationship between nTyr formation with iNOS and Na+,K+-ATPase activities. We were also aimed at investigating the possible role of caffeic acid phenethyl ester (CAPE) on endogenous nTyr production, Na+,K+-ATPase and iNOS activities in the kidney. In this study, kidney Na+,K+-ATPase activity were maximally inhibited 6h after LPS injection and LPS treatment significantly increased iNOS activity of kidney. The regression analysis negative correlation between Na+,K+-ATPase activity and nTyr levels of LPS treated animals. Na+,K+-ATPase activity were also negatively correlated with iNOS activity in LPS-treated rats. These data suggest that nitric oxide (NO.) and peroxynitrite (ONOO-) contribute to the development of oxidant injury. Furthermore, the source of NO. may be iNOS. iNOS are expressed by the kindey, and their activity may increase following LPS administration. In addition, NO. and ONOO- formation inhibited Na+,K+-ATPase activity. This results also have strongly suggested that bacterial LPS disturbs activity of membran Na+,K+-ATPase that may be an important component leading to the pathological consequences such as renal dysfunction in which the production of reactive nitrogen substance (RNS) are increased as in the case of LPS challenge. CAPE treatment was decreased nTyr production and iNOS activites, was increased Na+,K+-ATPase activity. These data suggest that CAPE treatment contribute to the decrease of oxidant injury.

Brassinin, a brassica-derived phytochemical, regulates monocyte-to-macrophage differentiation and inflammatory responses in human monocytes and murine macrophages.

The effects and molecular mechanisms of brassinin (BR), an indole phytoalexin from cruciferous vegetables, on monocyte-to-macrophage differentiation and inflammatory responses were investigated in this study. Inflammatory responses from RAW264.7 cells and THP-1 were stimulated by lipopolysaccharide (1µg/ml), and monocyte-to-macrophage differentiation of THP-1 was induced by phorbol myristate acetate (50ng/ml). The production of inflammatory mediators was determined by ELISA, Western blot or real-time PCR. Reactive oxygen species were examined by DCFH-DA assay. Brassinin at 50µm suppressed lipopolysaccharide-induced production of nitric oxide synthase, cyclooxygenase-2, prostaglandin E2 and reactive oxygen species by 90%, 69%, 52% and 41%, respectively, in RAW264.7 cells. In THP-1 cells, BR inhibited phorbol myristate acetate-induced monocyte-to-macrophage differentiation by suppressing cluster of differentiation molecule β and CD36. In addition, BR suppressed translocation of nuclear factor 'kappa-light-chain-enhancer' of activated B cells (NF-κB) into the nucleus. However, BR activated the nuclear factor erythroid-derived 2-like 2 (Nrf2) and its target molecules hemoxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1), with an increase in nuclear translocation of Nrf2. Brassinin suppressed monocyte-to-macrophage differentiation and inflammatory responses by differentially regulating Nrf2 and NF-κB signallings.

Biomedical applications of copper-impregnated bioactive glass

Bioactive glass (BG) is generally reckoned as an active constituent that both bonds with skeleton and connects with cartilage. This active material often releases the active ions in a controlled fashion via degradation, which is of particular interest in clinical medicine. However, monodispersed bioactive glass without modification still exists limitations, e.g., aggregation or lower activities both in vitro and in vivo . Owing to its unique morphology, distinctive cell uptake process, excellent degradation and biomineralization ability, as well as certain high biosafety, functionalized bioactive glass plays a key role in diverse biomedical applications such as bioimaging, gene/drug delivery and wound healing, facilitating the self-repair and regeneration of tissues. The interesting physicochemical properties of bioactive glass can be adjusted by appropriately selecting active ions, especially metal ions, as impregnated components to alter original bridging oxygen (BO) network to non-bridging oxygen (NBO) structure. In terms of the difference in matrix, bioactive glass is generally devided into three types, i.e., silicate, borate, and phosphate-based architectures. As a universal element in human physiological fluids, copper ions can also be impregnated in the structure of bioactive glass, in which these species not only affect the signal pathway and metabolic activity, but also serve as essential cofactors for many enzymes. After the ion exchange with the surrounding fluidic environment, copper ions can be precisely released at the desired site, minimizing the adverse effects. On the one hand, the impregnated copper ions inhibit the activity of osteoclast and stimulate the generation and aggregation of collagens, which significantly impact on the formation and healing of skeleton and cartilage. On the other hand, these copper species not only upregulate the expresssion of fibroblast growth factor (FGF)-2 and hypoxia inducible factor (HIF)-1α, but also enhance the production of endothelial nitric oxide synthase, subsequently promoting the proliferation and migration of human endothelial cells. In addition, the doped copper species are capable of bonding with mercapto, imidazole, carboxyl and amine, among others, to make changes in the membrane structure, including permeability augmentation and membrane transport dysfunction, indicating efficient antibacterial ability. Besides, hyperthermia process appears due to the photothermal effect after the irridation of near-infrared. In another case, copper ions participate in Fenton-like reaction under the acidic microenvironment of tumors, converting glutathione to oxidized glutathione, catalyzing the generation of reactive oxygen species (ROS), thus leading to the damage of mitochondria, ultimately inducing the apoptosis of cancer cells. Currently, it is required to achieve clinical translation from laboratory outcomes. Despite of a series of advantegeous features, the biomedical applications of copper-impregnated bioactive glass are still facing great challenges. To further facilitate the superiority of this prospective field, the developmental proposals are systemically discussed in this review. Further, critical discussions on effectively solving the aggregation caused by high-temperature sintering process and the denaturation induced by organic solvent utilization are emphasized. Moreover, the encapsulation mechanisms of certain molecules and compensate for the dificient exertion in cancer therapeutics are elaborated, providing an innovative and uninvasive platform in the field of prosperous biomedicine.

Hydroxytyrosol ameliorates oxidative challenge and inflammatory response associated with lipopolysaccharide-mediated sepsis in mice.

Hydroxytyrosol (HT) is among the main bioactive ingredients isolated from olive tree with a variety of biological and pharmacological activities. In the current study, the antioxidative and anti-inflammatory activities of HT were distinguished in the splenic tissue following lipopolysaccharide (LPS)-mediated septic response. Thirty-five Swiss mice were divided into five groups (n = 7): control, HT (40 mg/kg), LPS (10 mg/kg), HT 20 mg+LPS and HT 40 mg+LPS. HT was administered for 10 days, while a single LPS dose was applied. The obtained findings demonstrate that HT administration enhanced the survival rate and decreased lactate dehydrogenase level in LPS-challenged mice. Treatment with HT inhibited the incidence of oxidative damage in splenic tissue through decreasing lipoperoxidation and increasing antioxidant molecules, namely glutathione, superoxide dismutase and catalase. HT also decreased total leukocytes count, C-reactive protein, monocyte chemoattractant protein-1, and myeloperoxidase levels. Additionally, HT suppressed the production levels of tumor necrosis factor-α, interleukin-1β, and interleukin-6. Moreover, mRNA expression of inducible nitric oxide synthase and nitric oxide production were increased after HT administration. Furthermore, HT supplementation resulted in a downregulation of p38 mitogen-activated protein kinase, inhibited the activation of the nuclear factor kappa-B from the nucleus to the cytoplasm, and attenuated infiltration of activated immune cells and tissue injury following LPS injection. Collectively, these findings demonstrate the antioxidative and anti-inflammatory properties of HT against LPS-mediated inflammation and sepsis. Therefore, HT could be applied as an alternative anti-inflammatory agent to minimize or prevent the development of systemic inflammatory response associated with septic shock.

Red-Light (670 nm) Therapy Reduces Mechanical Sensitivity and Neuronal Cell Death, and Alters Glial Responses after Spinal Cord Injury in Rats.

Individuals with spinal cord injury (SCI) often develop debilitating neuropathic pain, which may be driven by neuronal damage and neuroinflammation. We have previously demonstrated that treatment using 670 nm (red) light irradiation alters microglia/macrophage responses and alleviates mechanical hypersensitivity at 7 days post-injury (dpi). Here, we investigated the effect of red light on the development of mechanical hypersensitivity, neuronal markers, and glial response in the subacute stage (days 1-7) following SCI. Wistar rats were subjected to a mild hemi-contusion SCI at vertebra T10 or to sham surgery followed by daily red-light treatment (30 min/day; 670 nm LED; 35 mW/cm2) or sham treatment. Mechanical sensitivity of the rat dorsum was assessed from 1 dpi and repeated every second day. Spinal cords were collected at 1, 3, 5, and 7 dpi for analysis of myelination, neurofilament protein NF200 expression, neuronal cell death, reactive astrocytes (glial fibrillary acidic protein [GFAP]+ cells), interleukin 1 β (IL-1β) expression, and inducible nitric oxide synthase (iNOS) production in IBA1+ microglia/macrophages. Red-light treatment significantly reduced the cumulative mechanical sensitivity and the hypersensitivity incidence following SCI. This effect was accompanied by significantly reduced neuronal cell death, reduced astrocyte activation, and reduced iNOS expression in IBA1+ cells at the level of the injury. However, myelin and NF200 immunoreactivity and IL-1β expression in GFAP+ and IBA1+ cells were not altered by red-light treatment. Thus, red-light therapy may represent a useful non-pharmacological approach for treating pain during the subacute period after SCI by decreasing neuronal loss and modulating the inflammatory glial response.

Salsola collina ethyl acetate extract alleviates diabetic gastroparesis possibly through oxidative stress inhibition

Objective: To investigate the therapeutic significance of ethyl acetate extract of Salsola collina (EES) on diabetic gastroparesis (DGP) and its underlying mechanisms. Methods: The composition of EES was analyzed by HPLC and LC/MS. A DGP model was established by streptozotocin injection and irregularly feeding a high-sugar, high-fat (HSHF) diet. Serum nitric oxide (NO), total cholesterol (TC), and triglyceride (TG) and the gastric superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), and glutathione peroxidase (GSH-Px) concentrations were measured by colorimetry and ELISA. The expression of neuronal nitric oxide synthase (nNOS) and protein gene product 9.5 (PGP9.5) in the gastric tissue were examined by Western blot and immunohistochemistry. Results: EES promoted gastric emptying delayed by DGP, which was mainly composed of 10 organic acids. Furthermore, EES increased serum NO, decreased glucose, TC and TG, increased gastric SOD, CAT, and GSH-Px, while decreased MDA, increased nNOS and PGP9.5 expression in the gastric tissue, and showed a concentration dependence. Conclusion: EES promoted gastric emptying in the DGP rats, which might be related to its inhibition of oxidative stress and the associated increase in the gastric neuron population, as well as its hypoglycemic and lipid-lowering activities. These findings suggest that Salsola may have potential benefits in the treatment of DGP.

Erythropoietin prevents LPS-induced preterm birth and increases offspring survival.

ProblemPreterm delivery is the leading cause of neonatal mortality and contributes to delayed physical and cognitive development in children. At present, there is no efficient therapy to prevent preterm labor. A large body of evidence suggests that infections might play a significant and potentially preventable cause of premature birth. This work assessed the effects of erythropoietin (EPO) in a murine model of inflammation‐associated preterm delivery, which mimics central features of preterm infections in humans.Method of studyBALB/c mice were injected i.p. with 20 000 IU/kg EPO or normal saline twice on gestational day (GD) 15, with a 3 hours time interval between injections. An hour after the first EPO or normal saline injection, all mice received two injections of 50 μg/kg LPS, also given 3 hours apart.ResultsEPO significantly prevented preterm labor and increased offspring survival in an LPS induced preterm delivery model. EPO prevented LPS‐induced leukocyte infiltration into the placenta. Moreover, EPO inhibited the expression of pro‐inflammatory cytokines, interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), and tumour necrosis factor‐α (TNF‐α) in maternal serum and amniotic fluid. EPO also prevented LPS‐induced increase in placental prostaglandin (PG)E2 and uterine inducible nitric oxide synthase (iNOS) production, while decreasing nuclear factor kappa‐B (NF‐κβ) activity in the myometrium. EPO also increased the gene expression of placental programmed cell death ligand 1 (PD‐L1) in LPS‐treated mice.ConclusionsOur results suggest that EPO could be a potential novel therapeutic strategy to tackle infection‐related preterm labor.

Pharmacological Uses of the Plants Belonging to the Genus Commiphora.

Natural products have a unique place in the healthcare industry. The genus Commiphora emerged as a potential medicinal source with huge benefits as evidenced through its use in various traditional and modern systems of medicine. Therefore, we aimed to prepare a concise review on the pharmacological activities and the indigenous uses of various plant species belonging to the genus Commiphora along with the structural information of various active botanical ingredients present in these plants based on the published literatures and scientific reports. To collect the published literatures on Commiphora in various journals; to study and classify the available information on the pharmacological uses and chemical constituents; and to present the gathered information as a precise review to serve as a potential reference for future research. Pharmacological and phytochemical data on Commiphora plant species were collected from various journals, books, reference materials, websites including scientific databases, etc. for compilation. This review article describes the various pharmacological properties of plants of Commiphora species viz., anti-arthritic and anti-inflammatory, anti-atherogenic, antibacterial, anti-coagulant, antidicrocoeliasis, anti-epileptic, anti-fascioliasis, anti-fungal, anti-heterophyidiasis, anti-hypercholesterolemic, anti-hyperlipidemic, anti-hypothyroidism, anti-obesity, anti-osteoarthritic, antiosteoclastogenesis, anti-oxidant, anti-parasitic, anti-pyretic, anti-schistosomiasis, anti-septic, antithrombotic, anti-ulcer, cardioprotective, COX enzyme inhibitory, cytotoxic /anti-carcinogenic/anticancer, DNA cleavage, hypotensive, inhibits lipid peroxidation, inhibits NO and NO synthase production, insecticidal, local anesthetic, molluscicidal, smooth muscle relaxant, and tick repellent activities along with toxicity studies. Furthermore, the review also included various secondary metabolites isolated from various species of Commiphora genus along with their chemical structures serving as a ready resource for researchers. We conclude that the plant species belonging to the genus Commiphora possesses abundant pharmacological properties with a huge treasure of diverse secondary metabolites within themselves. This review indicates the necessity of further in-depth research, pre-clinical, and clinical studies with Commiphora genus, which may help to detect the unidentified potential of the Commiphora plant species.

Effect Of Epigallocatechin-3-Gallate (EGCG) On The Number Of Macrophage Cells In Inflammation Of Pulp With Mechanical Injury

Background: Pulpitis is an inflammatory pulp that can occur due to mechanical trauma that causes perforation of the pulp. Treatment of pulpitis Emergency frequently using Eugenol. High concentrations of Eugenol have a cytotoxic effect that causes local necrosis and inhibits the recovery process. An alternative consideration with Epigallocatechin 3-gallate (EGCG) which has good antioxidant properties and increases the complexity of inflammation by inhibiting the production of inducible nitric oxide synthase (iNOS) and nitric oxide so that levels migration of inflammatory cells to the area of injury will decrease and inflammation will occur shorter then initiates the proliferation so the recovery process and tissue repair will be more rapidly occurring. One of the cells that support tissue repair is macrophages. Purpose: this study aims to explain the effect of EGCG on the number of macrophage cells in pulp inflammation with mechanical injury. Methods: The study used 24 Wistar rats teeth divided into four groups, namely control (C), and 3 treatment groups T1, T2, and T3. Each group consisted of 6 rats prepared, then T1 was named EGCG hydrogel 60 ppm, T2 was given EGCG hydrogel 90 ppm, while T3 was given EGCG hydrogel 120 ppm. On the 3rd day, Wistar rats were decapitated to continue making HPA preparations. Results:This study showed a significant difference in each group (p< 0.05) using One-Way Anova analysis. Conclusion: EGCG hydrogel 90 ppm is effectivein increasing the number of macrophage cells.

Non-opioid Analgesics and the Endocannabinoid System

Non-steroidal anti-inflammatory drugs produce antinociceptive effects mainly through peripheral cyclooxygenase inhibition. In opposition to the classical non-steroidal anti-inflammatory drugs, paracetamol and dipyrone exert weak anti-inflammatory activity, their antinociceptive effects appearing to be mostly due to mechanisms other than peripheral cyclooxygenase inhibition. In this review, we classify classical non-steroidal anti-inflammatory drugs, paracetamol and dipyrone as “non-opioid analgesics” and discuss the mechanisms mediating participation of the endocannabinoid system in their antinociceptive effects. Non-opioid analgesics and their metabolites may activate cannabinoid receptors, as well as elevate endocannabinoid levels through different mechanisms: reduction of endocannabinoid degradation via fatty acid amide hydrolase and/or cyclooxygenase-2 inhibition, mobilization of arachidonic acid for the biosynthesis of endocannabinoids due to cyclooxygenase inhibition, inhibition of endocannabinoid cellular uptake directly or through the inhibition of nitric oxide synthase production, and induction of endocannabinoid release.

Fyn kinase mediates pro-inflammatory response in a mouse model of endotoxemia: Relevance to translational research

Systemic inflammation resulting from the release of pro-inflammatory cytokines and the chronic activation of the innate immune system remains a major cause of morbidity and mortality in the United States. After having demonstrated that Fyn, a Src family kinase, regulates microglial neuroinflammatory responses in cell culture and animal models of Parkinson's disease, we investigate here its role in modulating systemic inflammation using an endotoxic mouse model. Fyn knockout (KO) and their wild-type (WT) littermate mice were injected once intraperitoneally with either saline or 5 mg/kg lipopolysaccharide (LPS) and were killed 48 h later. LPS-induced mortality, endotoxic symptoms and hypothermia were significantly attenuated in Fyn KO, but not WT, mice. LPS reduced survival in Fyn WT mice to 49% compared to 84% in Fyn KO mice. Fyn KO mice were also protected from LPS-induced deficits in horizontal and vertical locomotor activities, total distance traveled and stereotypic movements. Surface body temperatures recorded at 24 h and 48 h post-LPS dropped significantly in Fyn WT, but not in KO, mice. Importantly, endotoxemia-associated changes to levels of the serum pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), splenocyte apoptosis and inducible nitric oxide synthase (iNOS) production in hepatocytes were also significantly attenuated in Fyn KO mice. Likewise, pharmacologically inhibiting Fyn with 10 mg/kg dasatinib (oral) significantly attenuated LPS-induced increases in plasma TNF-α and IL-6 protein levels and hepatic pro-IL-1β messenger ribonucleic acids (mRNAs). Collectively, these results indicate that genetic knockdown or pharmacological inhibition of Fyn dampens systemic inflammation, demonstrating for the first time that Fyn kinase plays a critical role in mediating the endotoxic inflammatory response.

Immunomodulatory potential of extracts, fractions and pure compounds from Phyllanthus amarus and Psidium guajava on striped catfish (Pangasianodon hypophthalmus) head kidney leukocytes

This study aimed to identify major phytochemical constituents, as well as compare the immunomodulatory effects of Psidium guajava L. and Phyllanthus amarus Schun and Thonn crude ethanol extracts and their fractions on striped catfish (Pangasianodon hypophthalmus) head kidney leukocytes (HKLs). Moreover, pure constituents were also investigated for their effects on those cells: hypophyllanthin, identified as a major constituent of P. amarus crude extracts and its hexane fraction; corosolic acid, ursolic acid, and oleanolic acid, identified in P. guajava crude extract, ethyl acetate and dichloromethane fractions; with other terpenic derivatives, as well as guajaverin and avicularin, identified with other flavonoids by LC-UV-MS in the crude P. guajava extract and its ethyl acetate fraction. Cell viability, respiratory burst assay (RBA), nitric oxide synthase (NOS) and lysozyme activity in HKLs were analyzed after 24 h stimulation with each extract (10, 20 and 40 μg/mL) or pure compound (7.5, 15 and 30 μM). Our results show that the hexane fraction of both plant extracts inhibited the viability of HKLs, while several other fractions enhanced the cell viability. All P. guajava fractions at all or some concentration considerably enhanced the RBA production in HKLs. Similarly, NOS production was also significantly increased by some or all concentrations of P. guajava dichloromethane and ethyl acetate fractions. However, the NOS production was dose-dependently inhibited in HKLs treated with Pa ethyl acetate and both plants aqueous fractions at 10 or 10 and 40 μg/mL respectively. The lysozyme activity in cells treated with P. guajava crude extracts and all its organic solvent fractions were stronger than those in P. amarus treatments. Pure compounds including corosolic acid, guajaverin, ursolic acid, hypophyllanthin inhibited the HKLs viability according to concentration and type of compound. All pure compounds except avicularin significantly stimulated, at certain or all concentrations, the RBA production and/or the lysozyme activity in HKLs. The NOS production was significantly reduced in HKLs treated with oleanolic acid (30 μM) and hypophyllanthin (7.5 μM) while its level was increased by hypophyllanthin at 30 μM. These results highlighted that the crude ethanol extracts of P. guajava and P. amarus, their fractions and some of their pure components at certain concentrations can potentially act as immunomodulators, and could be considered as valuable candidates in fishery sciences.

Imperatorin suppresses IL-1β-induced iNOS expression via inhibiting ERK-MAPK/AP1 signaling in primary human OA chondrocytes

Joint inflammation is a key player in the pathogenesis of osteoarthritis (OA). Imperatorin, a plant-derived small molecule has been reported to have anti-inflammatory properties; however, its effect on chondrocytes is not known. Here, we investigated the effects of Imperatorin on interleukin-1β (IL-1β) induced expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in primary human OA chondrocytes and cartilage explants culture under pathological conditions and explored the associated signaling pathways. We pretreated chondrocytes or explants with Imperatorin (50 μM) followed by IL-1β (1 ng/ml), and the culture supernatant was used to determine the levels of nitrite production by Griess assay and chondrocytes were harvested to prepare cell lysate or RNA for gene expression analysis of iNOS by Western blot or qPCR and in explants by immunohistochemistry (IHC). Pretreatment of primary chondrocytes and cartilage explants with Imperatorin suppressed IL-1β induced expression of iNOS and NO production. Imperatorin blocked the IL-1β-induced phosphorylation of ERK-MAPK/AP1 signaling pathway to suppress iNOS expression. The role of ERK in the regulation of iNOS expression was verified by using ERK inhibitor. Interestingly, we also found that Imperatorin binds to iNOS protein and inhibits its activity in vitro. Our data demonstrated that Imperatorin possess strong anti-inflammatory activity and may be developed as a therapeutic agent for the management of OA.

Arctigenin exhibits hepatoprotective activity in Toxoplasma gondii-infected host through HMGB1/TLR4/NF-κB pathway

Toxoplasmosis is a parasitic zoonosis with the highest incidence in humans. Severe lesions due to acute toxoplasmosis have been recorded in the visceral organs including the liver, where hepatocytes and Kupffer cells are important innate immune cells. Arctigenin (AG) is a bioactive ingredient of Arctium lappa L. and increasing evidence suggests that AG exhibits anti-oxidant, anti-inflammatory and anti-Toxoplasma gondii (T. gondii) effects. However, the role of AG in acute liver damage induced by T. gondii infection remains unclear. In this study, we analyzed the effects of AG against T. gondii-induced liver damage by establishing an in vitro infection model using a murine liver cell line (NCTC-1469 cells) and an in vivo mouse model with acute T. gondii infection of virulent RH strain. In the current study, AG effectively attenuated hepatocytes apoptosis and inhibited the reproduction of T. gondii. The results of in vitro and in vivo studies showed that AG significantly reduced alanine aminotransferase/aspartate aminotransferase activities and lessened pathological damage of liver. Moreover, AG suppressed T. gondii-induced inducible nitric oxide synthase production. AG also attenuated liver inflammation by inhibiting T. gondii-induced activation of the high-mobility group box1/toll-like receptor 4/nuclear factor-kappa B (HMGB1/TLR4/NF-κB) signaling pathway. These findings demonstrated that AG exhibited prominent hepatoprotective activities in toxoplasmic liver injury with anti-inflammatory effects by inhibiting the HMGB1/TLR4/NF-κB signaling axis. Thus, this study provides the basis for the development of new drugs to treat toxoplasmic hepatitis.

Engineering blood outgrowth endothelial cells to optimize endothelial nitric oxide synthase and extracellular matrix production for coating of blood contacting surfaces

Coverage of blood contacting surfaces by a functional endothelial layer is likely required to induce and maintain homeostasis. Blood outgrowth endothelial cells (BOECs), cultured from human peripheral blood monocytes, are readily available and functional autologous endothelial source that may represent a reasonable alternative to vascular derived cells. Endothelial nitric oxide synthase (eNOS) produces NO, an important factor that regulates homeostasis at the blood-contacting surface. We found that BOECs express markedly lower levels of eNOS protein (34% ± 13%, Western blot) and mRNA (29% ± 17%, qRT-PCR), as well as exhibiting reduced activity (49% ± 18%, Nitrite analysis) when compared to human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells. HUVECs grown on fibronectin, type I collagen, or laminin -coated surfaces exhibited significant reduction of eNOS mRNA and protein expression. However, no decrease in eNOS levels was observed in BOECs. Interestingly BOECs expressed significantly higher Collagen (Col) I compared to HUVECs, and blocking Col I synthesis significantly enhanced eNOS expression in BOECs. Inhibition of β1 integrin, focal adhesion kinase (FAK), or actin polymerization increased eNOS in both BOECs and HUVECs suggesting involvement of a signaling pathway culminating in stabilization of the cytoskeleton. Finally, we demonstrated that a Rho-associated protein kinases (ROCK) inhibitor, as a disruptor of actin stabilization, enhanced both eNOS expression and bioactivity. Taken together, our findings demonstrate that cell-ECM interactions are fundamental to the regulation of eNOS in BOECs and suggest that disruption of key intracellular pathways (such as ROCK) may be necessary to enhance functional activity of an endothelialized surface. Statement of SignificanceDevelopment of biocompatible blood-contacting biomaterial surfaces has not been possible to date, leading many investigators to believe that a complete autologous endothelial layer will be necessary. Blood outgrowth endothelial cells (BOECs), cultured from human peripheral blood monocytes, are readily available and functional autologous endothelial source. Endothelial nitric oxide synthase (eNOS) produces NO, an important factor that regulates homeostasis at the blood-contacting surface. In this study, we show that eNOS displays limited expression in cultured BOECs. We further demonstrate that a strong negative regulation of eNOS is mediated by collagen substrates and that treatment with ROCK inhibitor could enhance both eNOS expression and activity in BOECs and help to rapidly establish a functional autologous endothelial layer on cardiovascular biomaterials.

Structure and immunostimulating activity of a galactofuranose-rich polysaccharide from the bamboo parasite medicinal fungus Shiraia bambusicola

Ethnopharmacological relevanceShiraia bambusicola is a parasitic fungus on the twigs of bamboos. Its relatively large stroma has high medicinal value and can treat a variety of diseases such as rheumatoid arthritis, cold stomach pain, sciatica, injuries, chronic bronchitis, and infantile. It is widely distributed in many provinces in Southern China and also is also found in Japan. Aim of the studyMedicinal fungi were important resources for bioactive polysaccharides. To explore bioactive polysaccharides from Shiraia bambusicola, a heteropolysaccharide SB2-1 was purified and obtained from S. bambusicola and its immunostimulating activity was researched. Materials and methodsThe polysaccharide from S. bambusicola was extracted and purified using enzyme assisted extraction, ethanol precipitation, anion-exchange and size-exclusion chromatography. Molecular weight of polysaccharide was estimated by high performance gel permeation chromatography. Monosaccharide compositions were determined by high performance liquid chromatography after pre-column derivatization and UV detection. Structure information was elucidated by IR spectrum, GC-MS analysis after methylation and gradual acid hydrolysis of the polysaccharide. The RAW264.7 cells were used to study the immunostimulating activity in vitro. ResultsPhysicochemical and structural analyses showed that SB2-1 was a neutral heteropolysaccharide with molecular weight at 22.2 kDa and consisted of glucose, galactose and mannose at a ratio of 2.0:1.5:1.0. The structure of SB2-1 was a branched polysaccharides composed of a mannan core and side chains consisted of glucose and galactose. The mannan core was composed of (1→2)-Manp as the main chain. Glucose with (1→4)-D-Glcp, (1→2)-D-Glcp and (1→6)-D-Glcp at different degrees of polymerization were linked at C-6 and C-3 of the (1→2)-Manp as the side chains. The galactose with the linages of (1→6)-D-Galf, →2)-D-Galf(1→ and terminal D-Galf(1→ also existed in the side chain. The study on the immunostimulating activities of SB2-1 and its core structure P-2 were investigated on RAW264.7 macrophages. The results showed that SB2-1 could activate RAW264.7 macrophage and significantly improve its phagocytic ability by neutral red uptake experiment. Meanwhile, SB2-1 increased significantly higher inducible nitric oxide synthase (iNOS) production and the productions of IL-1, IL-6, IL-12 and TNF-α. The effect of SB2-1 was better than its core structure P-2 produced by gradual acid hydrolysis, which meant the side chains played an important role in the immunostimulating activities. ConclusionsThe investigation demonstrated that the galactofuranose-containing mannogalactoglucan was characteristic polysaccharides in S. bambusicola and could enhance the activation of macrophages.