Excessive Nitric Oxide Production Research Articles

Anti-inflammatory effects of flavonoids and phenylethanoid glycosides from Hosta plantaginea flowers in LPS-stimulated RAW 264.7 macrophages through inhibition of the NF-\u03baB signaling pathway

BackgroundThe flower of Hosta plantaginea (Lam.) Aschers has traditionally been used in China as an important Mongolian medicine for the treatment of inflammatory diseases with limited scientific evidence. In previous studies, 16 flavonoids and 3 phenylethanoid glycosides (1–19) were isolated from the ethanolic extract of H. plantaginea flowers. Nevertheless, the anti-inflammatory effects of these constituents remain unclear. In the present study, the anti-inflammatory effects of these 19 constituents and their underlying mechanisms were assessed in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.MethodsThe viability of RAW 264.7 macrophages was detected by Cell Counting Kit-8 (CCK-8) assay. Meanwhile, nitric oxide (NO) production was measured by Griess assay, while the secretion of tumor necrosis factor α (TNF-α), prostaglandin E2 (PGE2), interleukin 1β (IL-1β) and IL-6 in LPS-induced macrophages was determined by enzyme-linked immunosorbent assay (ELISA). Furthermore, the protein expression of nuclear factor kappa B (NF-κB) p65 and phosphorylated NF-κB p65 was evaluated by Western blot analysis.ResultsAll constituents effectively suppressed excessive NO production at a concentration of 40 μM with no toxicity to LPS-induced RAW 264.7 macrophages. Among them, five flavonoids (1, 4–6 and 15) and one phenylethanoid glycoside (17) remarkably prevented the overproduction of NO with median inhibitory concentration (IC50) values in the range of 12.20–19.91 μM. Moreover, compounds 1, 4–6, 15 and 17 potently inhibited the secretion of TNF-α, PGE2, IL-1β and IL-6, and had a prominent inhibitory effect on the down-regulation of the phosphorylated protein level of NF-κB p65.ConclusionTaken together, compounds 1, 4–6, 15 and 17 may be useful in managing inflammatory diseases by blocking the NF-κB signaling pathway and suppressing the overproduction of inflammatory mediators.

Anti-Inflammatory Effects of Heliangin from Jerusalem Artichoke (Helianthus tuberosus) Leaves Might Prevent Atherosclerosis.

Atherosclerosis is considered the major cause of cardiovascular and cerebrovascular diseases, which are the leading causes of death worldwide. Excessive nitric oxide production and inflammation result in dysfunctional vascular endothelial cells, which are critically involved in the initiation and progression of atherosclerosis. The present study aimed to identify a bioactive compound from Jerusalem artichoke leaves with anti-inflammatory activity that might prevent atherosclerosis. We isolated bioactive heliangin that inhibited NO production in LPS-induced macrophage-like RAW 264.7 cells. Heliangin suppressed ICAM-1, VCAM-1, E-selectin, and MCP-1 expression, as well as NF-κB and IκBα phosphorylation, in vascular endothelial cells stimulated with TNF-α. These results suggested that heliangin suppresses inflammation by inhibiting excessive NO production in macrophages and the expression of the factors leading to the development of atherosclerosis via the NF-κB signaling pathway in vascular endothelial cells. Therefore, heliangin in Jerusalem artichoke leaves could function in the prevention of atherosclerosis that is associated with heart attacks and strokes.

ROLE OF ΚB AND AP-1 TRANSCRIPTION FACTORS IN CHANGES IN NITRIC OXIDE PRODUCTION AND UTILIZATION IN THE HEART OF RATS UNDER CHRONIC FLUORIDE INTOXICATION

Fluorides, being hazardous contaminants of soil and drinking water, can get in excessive amounts into human and animal bodies. This is especially true for regions where the fluoride content in soils is very high, for example, Poltava, Dnipropetrovsk, and Kirovohrad regions in Ukraine. Excessive fluoride intake can change the rate of nitric oxide production. The impact of fluorides on changes in nitric oxide production and metabolism in the heart and the role of redox-sensitive transcription factors in these changes are poorly understood. The aim of this study was to determine the effect of activation of κB transcription factors and activator protein 1 on the activity of inducible NO-synthase, constitutive isoforms of NO-synthase, nitrite and nitrate reductase, arginase, concentration of nitrites, peroxynitrite and nitrosothiols in the heart of rats during chronic fluoride intoxication. Materials and methods. The study was performed on 24 adult male Wistar rats weighing 220-260 grams. Animals were randomly divided into 4 groups of 6 animals in each (control, chronic fluoride intoxication group, κB blockade group and activator protein 1 blockade group). The experiment lasted 30 days. We determined the activity of inducible NO-synthase, constitutive isoforms of NO-synthase, the concentration of peroxynitrite alkali and alkaline earth metals, the concentration of nitrites and nitrosothiols, the activity of nitrite reductase, nitrate reductase and arginase. Results. Chronic fluoride intoxication increases the activity of inducible NO-synthase by 1.74 times, does not affect the activity of constitutive isoforms and reduces the activity of arginase by 35.68% compared with the control group of animals. The concentration of nitrites in the heart of rats increases 1.73 times, peroxynitrite 1.43 times, and the concentration of nitrosothiols doubled. The use of κB transcription factor blockers and activator protein 1 reduces nitric oxide production from NO synthases and reduces the concentrations of all nitric oxide metabolites in the heart of rats under conditions of chronic fluoride intoxication. Conclusions. Activation of κB transcription factors and activator protein 1 during chronic excessive intake of fluoride leads to hyperproduction of nitric oxide in the heart of rats due to increased activity of inducible NO-synthase and nitrite reductases. Excess production of nitric oxide under chronic fluoride intoxication leads to the accumulation of nitrites, peroxynitrite and nitrosothiols in the heart of rats.

VISTA Stimulation of VSIG4-Positive Macrophages Strongly Suppresses T Cell Proliferation via Excessive Nitric Oxide Production in Sepsis.

Organ damage and immune deficiency are important problems in sepsis caused by an excessive immune response. There is controversy about the cause of immune suppression. In this study, we investigated the roles of macrophages that exhibit excessive activity on T cell immunity. Peritoneal macrophages from mice with cecal ligation and puncture (CLP)-induced sepsis migrated to different organs. In particular, V-set immunoglobulin (Ig)-domain-containing 4 (VSIG4) positive macrophages appeared in the spleen 48 h after CLP induction. When cocultured with splenic T cells, VSIG4(+) cells inhibited the proliferation of activated T cells through the release of nitric oxide (NO) compared to VSIG4(-) cells. Stimulation of VSIG4(+) cells with V-domain Ig suppressor of T cell activation (VISTA) antibody increased the expression of several cytokine genes and the release of NO, but not phagocytosis, compared to those of hamster IgG-stimulated VSIG4(+) cells. When cocultured with splenic T cells, VISTA-stimulated VSIG4(+) cells induced excessive T cell suppression via more NO secretion compared to hamster IgG-stimulated VSIG4(+) cells. Taken together, the current study demonstrates that VSIG4(+) peritoneal macrophages play important roles in inducing immunosuppression and that VISTA acts as a costimulatory receptor in these cells. These data suggest that blocking the migration of VSIG4(+) cells might alleviate excessive immune activity and that blocking VISTA on VSIG4(+) macrophages might play a crucial role in the development of new therapies to prevent T cell suppression in sepsis.

Gut Microbiota in NSAID Enteropathy: New Insights From Inside.

As a class of the commonly used drugs in clinical practice, non-steroidal anti-inflammatory drugs (NSAIDs) can cause a series of adverse events including gastrointestinal injuries. Besides upper gastrointestinal injuries, NSAID enteropathy also attracts attention with the introduction of capsule endoscopy and double balloon enteroscopy. However, the pathogenesis of NSAID enteropathy remains to be entirely clarified. Growing evidence from basic and clinical studies presents that gut microbiota is a critical factor in NSAID enteropathy progress. We have reviewed the recent data about the interplay between gut microbiota dysbiosis and NSAID enteropathy. The chronic medication of NSAIDs could change the composition of the intestinal bacteria and aggravate bile acids cytotoxicity. Meanwhile, NSAIDs impair the intestinal barrier by inhibiting cyclooxygenase and destroying mitochondria. Subsequently, intestinal bacteria translocate into the mucosa, and then lipopolysaccharide released from gut microbiota combines to Toll-like receptor 4 and induce excessive production of nitric oxide and pro-inflammatory cytokines. Intestinal injuries present in the condition of intestinal inflammation and oxidative stress. In this paper, we also have reviewed the possible strategies of regulating gut microbiota for the management of NSAID enteropathy, including antibiotics, probiotics, prebiotics, mucosal protective agents, and fecal microbiota transplant, and we emphasized the adverse effects of proton pump inhibitors on NSAID enteropathy. Therefore, this review will provide new insights into a better understanding of gut microbiota in NSAID enteropathy.

Dichlorophene activates aryl hydrocarbon receptor (AhR) and indoleamine 2, 3-dioxygenase 1 (IDO1) to mediate splenotoxicity in rat

Dichlorophene (DCP) is a halogenated phenolic compound, widely used as fungicide, bactericide and antiprotozoan and also exhibit therapeutic application in several pathological conditions. Taking account of broad use of DCP, its possible effect on spleen (an important immune organ) was investigated in this study. Male albino rats were treated with graded doses of DCP (10%, 20% and 30% of LD50) and spleen and blood were obtained at 24, 48 and 72 hours post treatment. Oxidative stress parameters, proinflammatory cytokines and protein expression of aryl hydrocarbon receptor (AhR), indoleamine-2, 3-Dioxygenase 1 (IDO1) and nuclear factor erythroid 2–related factor 2 (Nrf2) were measured along with histopathological evaluation of spleen. In the present study, DCP perturbs redox status of splenocytes of rats as evidenced by excess ROS generation, lipid peroxidation and nitric oxide production simultaneously with reduction of antioxidant level [glutathione (GSH)] and inhibition of antioxidative enzymes [superoxide dismutase (SOD) and catalase (CAT)]. Two important proinflammatory cytokines, IL-6 and TNF-α were found to be elevated upon DCP treatment. Moreover, DCP also caused activation of AhR and IDO1 with simultaneous down regulation of Nrf2. All these effects of DCP were found to be dose and duration dependent. DCP also affects the spleen micro-architecture in the present study and these alterations were more prominent in high dose group at 72 hours post treatment. Taken together, all these results suggested that DCP induces oxidative stress and also increases proinflammatory cytokine levels to mount its toxic effect on spleen.

Nitric Oxide as a Target for Phytochemicals in Anti-Neuroinflammatory Prevention Therapy.

Nitric oxide (NO) is a neurotransmitter that mediates the activation and inhibition of inflammatory cascades. Even though physiological NO is required for defense against various pathogens, excessive NO can trigger inflammatory signaling and cell death through reactive nitrogen species-induced oxidative stress. Excessive NO production by activated microglial cells is specifically associated with neuroinflammatory and neurodegenerative conditions, such as Alzheimer’s and Parkinson’s disease, amyotrophic lateral sclerosis, ischemia, hypoxia, multiple sclerosis, and other afflictions of the central nervous system (CNS). Therefore, controlling excessive NO production is a desirable therapeutic strategy for managing various neuroinflammatory disorders. Recently, phytochemicals have attracted considerable attention because of their potential to counteract excessive NO production in CNS disorders. Moreover, phytochemicals and nutraceuticals are typically safe and effective. In this review, we discuss the mechanisms of NO production and its involvement in various neurological disorders, and we revisit a number of recently identified phytochemicals which may act as NO inhibitors. This review may help identify novel potent anti-inflammatory agents that can downregulate NO, specifically during neuroinflammation and neurodegeneration.

Heuristics in Role of Human Glutathione S-transferase Mu 1 as Nitric Oxide Carrier and its Engineered Variants for Enhanced Activity.

Human glutathione S-transferases (hGSTs) are phase-II detoxification enzymes that catalyze the conjugation of electrophilic compounds and glutathione. Anomalous excess production of NO in the cellular environment under diseased or stressed condition results in lethal effects to the cell. Studies have reported that the evolution of tyrosine-based GSTs as a defense mechanism by the cell to mitigate Nitric Oxide (NO) toxicity. The dual role of hGSTP1 as NO carrier and scavenger is a prelude for the research forthwith. A plausible role of hGSTM1 as NO carrier is considered. Being a prominent cellular messenger and secondary metabolite, excess production of NO is lethal to the cell. Moreover, hGSTM1 polymorphisms lead to diminished catalytic activity that promotes a diseased state. Hence, it is compelling to generate hGSTM1 mutants that have more catalytic efficacy compared to Wild Type (WT). hGSTM1 mutants with enhanced efficiency were generated using in silico and in vitro Site-Directed Mutagenesis (SDM). WT and mutant proteins were overexpressed and purified using affinity chromatography. The catalytic activity and binding efficiency of WT and mutant proteins towards CDNB (1-chloro-2, 4-dinitrobenzene) & NO were determined. NO assay reveals the probable interaction of WT hGSTM1 with NO. In silico, SDM studies provided E129K and Q109K mutants with superior NO binding efficiency as compared to WT. The catalytic activity (GST and NO assays) of the mutants corroborate the in silico results. WT hGSTM1 is recognized as a positive NO carrier. The novel mutant enzymes E129K and Q109K are inferred to possess superior NO carrying capacity.

PHYTOCHEMICAL COMPOSITION AND ANTIINFLAMMATORY ACTIVITY OF NELUMBO NUCIFERA GAERTN

Objective: Inflammation is a process of injuries caused by physical, chemical, and biological factors. Nitric oxide (NO) plays an important role in theregulation of various pathological and pathophysiological processes. Overproduction of NO induces tissue damage associated with acute and chronicinflammations. This study was conducted to determine the phytochemical composition and the NO inhibitory properties of Nelumbo nucifera extractsin lipopolysaccharide(LPS)-stimulated macrophage cell line.Methods: The dried leaf, stalk, and flower materials of roseum plenum and album plenum (AP) were extracted with 95% ethanol solvents. Thephytochemical compounds of the extraction were analysed by gas chromatography-mass spectrometry. The cytotoxic assay of extracts againstmacrophage cells was conducted using resazurin. The NO was determined using LPS-induced RAW264.7 cells to measure inhibitory activity of extracton the production of NO.Results: The extracts from Lotus, which exhibited the non-cytotoxic to the RAW264.7 cells. The AP-stalk extracts were capable to reduce the NO levelin LPS-activated RAW264.7 cells. GC-MS analysis of AP-stalk extraction revealed pharmacologically active compounds.Conclusion: The results conduct that the AP-stalk extract effectively inhibited the NO production and may be useful in preventing inflammatorydiseases mediated by excessive production of NO. Bio-active phytoconstituents from AP stalk extract could potentially be used for anti-inflammation.These data also suggest that AP-stalk extract may serve as a good indicator of the pharmacological activities of medicinal plants.

Scarred Lung. An Update on Radiation-Induced Pulmonary Fibrosis.

Radiation-induced pulmonary fibrosis is a common severe long-time complication of radiation therapy for tumors of the thorax. Current therapeutic options used in the clinic include only supportive managements strategies, such as anti-inflammatory treatment using steroids, their efficacy, however, is far from being satisfactory. Recent studies have demonstrated that the development of lung fibrosis is a dynamic and complex process, involving the release of reactive oxygen species, activation of Toll-like receptors, recruitment of inflammatory cells, excessive production of nitric oxide and production of collagen by activated myofibroblasts. In this review we summarized the current state of knowledge on the pathophysiological processes leading to the development of lung fibrosis and we also discussed the possible treatment options.

INTENSITY OF NITRIC OXIDE SYNTHESIS IN THE BLOOD OF RATS UNDER THE INFLUENCE OF SODIUM FLUORIDE

The influence of fluoride ion as a chemical agent affects the metabolism, the change of which is manifested in functional and structural changes. The final stable metabolites of NO include nitrite and nitrate anions (NO2, NO3), the concentration of which serves as an indirect method for estimating the intensity of NO synthesis. The concentration of NO is a major factor in its numerical biological effects (regulatory, protective). At low NO concentrations, direct effects aimed at maintaining homeostasis, such as the vascular, respiratory, and nervous systems, predominate; at high concentrations of NO, indirect effects prevail, due primarily to the formation of peroxynitrite. It is known that when the rate of free radical reactions in the body increases, NO can react with a superoxide anion radical to form peroxynitrite, a highly reactive compound with destructive properties against proteins and lipids. Oral administration of FN to rats in doses of 1/10 and 1/100 DL50 leads to an increase in the content of nitrite and nitrate anions in the blood plasma during the first 30 days, which indirectly indicates excess production of nitric oxide, which in the initial stages of intoxication may play a compensatory role, but subsequently cause pathological reactions associated with the activation of oxidative stress. The decrease in nitrite and nitrate anions at the end of long-term action of FN indirectly indicates a decrease in nitric oxide generation, which may be associated, in particular, with an increase in peroxynitrite concentration due to the use of nitric oxide in reaction with superoxide anion radical and antioxidant deficiency.

Suppressive effects of sulfated polysaccharide ascophyllan isolated from Ascophyllum nodosum on the production of NO and ROS in LPS-stimulated RAW264.7 cells.

In this study, we found that a sulfated polysaccharide isolated from the brown alga Ascophyllum nodosum, ascophyllan, showed suppressive effects on stimulated RAW264.7 cells. Ascophyllan significantly inhibited expression of inducible nitric oxide synthase mRNA and excessive production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in a dose-dependent manner without affecting the viability of RAW264.7 cells. Ascophyllan also reduced the elevated level of intracellular reactive oxygen species (ROS) in LPS-stimulated RAW264.7 cells. Furthermore, preincubation with ascophyllan resulted in concentration-dependent decrease in ROS production in phorbol 12-myristate-13-acetate-stimulated RAW264.7 cells. Our results suggest that ascophyllan can exhibit anti-inflammatory effects on stimulated macrophages mainly through the attenuation of NO and ROS productions.

Potential roles of neuronal nitric oxide synthase and the PTEN-induced kinase 1 (PINK1)/Parkin pathway for mitochondrial protein degradation in disuse-induced soleus muscle atrophy in adult rats.

Excessive nitric oxide (NO) production and mitochondrial dysfunction can activate protein degradation in disuse-induced skeletal muscle atrophy. However, the increase in NO production in atrophied muscles remains controversial. In addition, although several studies have investigated the PTEN-induced kinase 1 (PINK1)/Parkin pathway, a mitophagy pathway, in atrophied muscle, the involvement of this pathway in soleus muscle atrophy is unclear. In this study, we investigated the involvement of neuronal nitric oxide synthase (nNOS) and the PINK1/Parkin pathway in soleus muscle atrophy induced by 14 days of hindlimb unloading (HU) in adult rats. HU lowered the weight of the soleus muscles. nNOS expression showed an increase in atrophied soleus muscles. Although HU increased malondialdehyde as oxidative modification of the protein, it decreased 6-nitrotryptophan, a marker of protein nitration. Additionally, the nitrosocysteine content and S-nitrosylated Parkin were not altered, suggesting the absence of excessive nitrosative stress after HU. The expression of PINK1 and Parkin was also unchanged, whereas the expression of heat shock protein 70 (HSP70), which is required for Parkin activity, was reduced in atrophied soleus muscles. Moreover, we observed accumulation and reduced ubiquitination of high molecular weight mitofusin 2, which is a target of Parkin, in atrophied soleus muscles. These results indicate that excessive NO is not produced in atrophied soleus muscles despite nNOS accumulation, suggesting that excessive NO dose not mediate in soleus muscle atrophy at least after 14 days of HU. Furthermore, the PINK1/Parkin pathway may not play a role in mitophagy at this time point. In contrast, the activity of Parkin may be downregulated because of reduced HSP70 expression, which may contribute to attenuated degradation of target proteins in the atrophied soleus muscles after 14 days of HU. The present study provides new insights into the roles of nNOS and a protein degradation pathway in soleus muscle atrophy.

β-hydroxybutyrate (β-HB) exerts anti-inflammatory and antioxidant effects in lipopolysaccharide (LPS)-stimulated macrophages in Liza haematocheila

Poly-β-hydroxybutyrate (PHB) can be hydrolyzed to β-hydroxybutyrate (β-HB) in the intestinal tract of animals, and dietary PHB supplementation could enhance the immunity and disease resistance of aquatic animals. Antioxidant system is responsive to PHB stimuli via MAPK/PI3K-Akt/TNF/NF-κB/TCR/TLR signaling pathways. However, the precise immunopotentiation mechanism needs further study. In this study, macrophages from spleen in Liza haematocheila was used to study the effect of β-HB on cell viability and antioxidant function to illustrate the immunopotentiation mechanism of PHB. The results showed that β-HB (100 μg/mL) promoted the viability of macrophages and balanced the production of reactive oxygen species, but inhibited the excessive production of intracellular nitric oxide. In order to further explore the immunopotentiation mechanism of β-HB, LPS (100 μg/mL) was used to induce the inflammation and investigated the inhibitory effect of β-HB on inflammation. The results showed that LPS could induce inflammation successfully, and β-HB exerted anti-inflammatory and antioxidant effects in LPS-stimulated macrophages. Compared with LPS stimuli alone, the expression of anti-inflammatory genes NF-κBIA, MAP3K8 and TLR5 in β-HB pretreatment group was up-regulated, and the expression of pro-inflammatory genes TNFSF6, TNF-α, PI3K, NF-κB and TLR1 down-regulated. It suggested that β-HB inhibited the inflammatory response by up-regulation of anti-inflammatory genes such as NF-κBIA, thereby enhancing the immunity of the body.

Berry Phenolic and Volatile Extracts Inhibit Pro-Inflammatory Cytokine Secretion in LPS-Stimulated RAW264.7 Cells through Suppression of NF-κB Signaling Pathway.

Berries are a rich source of phytochemicals, especially phenolics well known for protective activity against many chronic diseases. Berries also contain a complex mixture of volatile compounds that are responsible for the unique aromas of berries. However, there is very limited information on the composition and potential health benefits of berry volatiles. In this study, we isolated phenolic and volatile fractions from six common berries and characterized them by HPLC/HPLC-MS and GC/GC-MS, respectively. Berry phenolic and volatile fractions were evaluated for an anti-inflammatory effect using lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells by measuring levels of pro-inflammatory cytokines and the nuclear factor-kappa B (NF-κB) signaling pathway. Results showed that LPS-induced excessive production of nitric oxide (NO), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which were inhibited by berry phenolic and volatile extracts. Moreover, berry phenolic and volatile extracts reduced the nuclear translocation of NF-κB by blocking the phosphorylation of p65 and degradation of IκBα. These findings showed that berry volatiles from six berries had comparable anti-inflammatory effects to berry phenolics through the suppression of pro-inflammatory mediators and cytokines expression via NF-κB down-regulation, despite being present in the fruit at a lower concentration.

HDAC6 Mediates Macrophage iNOS Expression and Excessive Nitric Oxide Production in the Blood During Endotoxemia.

Excessive nitric oxide (NO) production and NO-mediated nitrative stress contribute to vascular dysfunction, inflammation, and tissue injury in septic shock. New therapeutic targets are urgently needed to provide better control of NO level during septic shock. In the present study, we investigated the role of HDAC6 in the regulation of NO production and nitrative stress in a mouse model of endotoxin-induced septic shock. HDAC6 deficient mice and a specific HDAC6 inhibitor were utilized in our studies. Our data clearly indicate that HDAC6 is an important mediator of NO production in macrophages. HDAC6 mediates NO production through the regulation of iNOS expression in macrophages. HDAC6 up-regulates iNOS expression in macrophages by modulating STAT1 activation and IRF-1 expression. HDAC6 inhibition potently blocked endotoxin-induced STAT1 activation and iNOS expression in macrophages. Furthermore, HDAC6 contributes to excessive NO production and nitrotyrosine level in the blood and promotes iNOS expression in the lung tissues during septic shock. Our data reveal a novel HDAC6/STAT1/iNOS pathway that mediates excessive NO production and nitrative stress in septic shock.

Vasopressor-Sparing Action of Methylene Blue in Severe Sepsis and Shock: A Narrative Review.

Shock is a serious acute circulatory failure leading to inadequate oxygen delivery to the cells. Its treatment is mainly based on circulating fluid optimization, and vasopressors to provide an adequate mean arterial pressure and microcirculatory flow. Norepinephrine is the drug of choice, but high dosages may be responsible for several side effects, including increased myocardial oxygen consumption, dysrhythmias, and peripheral and organ ischemia. Moreover, some patients are “non-responders” to first-line norepinephrine treatment. Hence, other drugs have been proposed to reach and maintain the hemodynamic target. In general, they are described as catecholamine-sparing agents. Among others, the most used are vasopressin, corticosteroids, and angiotensin II. Methylene blue (MB) represents a further option, even though its use is still a topic of controversy. This review article tries to summarize what is known and unknown about the actions of MB in patients in shock. It reduces excessive production of nitric oxide via blockade of guanylate cyclase in shock states. At present, it appears the MB provides positive results in septic shock, if administered early. Further randomized controlled trials are warranted regarding its use to provide more precise indications to physicians involved in the treatment of such patients.

Low-dose hydralazine improves endotoxin-induced coagulopathy and multiple organ dysfunction via its anti-inflammatory and anti-oxidative/nitrosative properties

Coagulopathy is the major cause of organ injury as well as a strong predictor of mortality in septic patients. Systemic inflammatory response and redox imbalance are regarded as the major causes of sepsis-induced coagulopathy. There is growing evidence that a vasodilator hydralazine has beneficial effects on heart failure, hypertension, and ischemia/reperfusion injury via its antioxidant and anti-inflammatory properties. However, the effects of hydralazine on sepsis have not been examined. Therefore, we evaluated the effects of low-dose hydralazine on coagulopathy and multiple organ dysfunction in septic rats induced by endotoxin. Sepsis-induced coagulopathy was established by intravenous injection of rats with lipopolysaccharide (LPS). The changes of blood pressure, heart rate, blood glucose, hemostatic variables, prothrombin time, organ function indices, interleukin-6 (IL-6) concentration, and nitric oxide (NO) level were assessed during the experimental period. In addition, the aortas, lungs, livers, and kidneys were dissected to analyze superoxide levels and protein expressions. LPS induced (i) coagulopathy, multiple organ dysfunction, and circulatory failure successfully, and (ii) excessive superoxide, NO, and IL-6 production, accompanied by the overexpression of iNOS and Wnt5a in animals. Treatment of LPS-induced septic rats with low-dose hydralazine not only improved coagulopathy but also ameliorated multiple organ dysfunction. These could be due to attenuation of the overproduction of superoxide, NO, and IL-6, which were attributed to reduction of the overexpression of iNOS and Wnt5a. Thus, these findings indicate that low-dose hydralazine could be a potential therapy for sepsis-induced coagulopathy and multiple organ dysfunction via its anti-inflammatory and anti-oxidative/nitrosative properties.

Protective effect of chitosan oligosaccharide against oxidative damage of peripheral blood mononuclear cells in dairy cows induced by diethylenetriamine/nitric oxide via NF-κB signalling pathway

Excessive production of nitric oxide (NO) during the lactation period of cows leads to oxidative stress and inflammatory reactions. Chitosan has enhanced effects on antioxidant and immune function, but the mechanism of this effect is unclear. The current study investigated the underlying mechanism of the protective effect chitosan oligosaccharides (COS) exert against oxidative damage of peripheral blood mononuclear cells (PBMCs) in dairy cows. PBMCs were allocated to receive one of treatments: CTR: control treatment without COS addition; COS40, COS80, COS160, and COS320 received 40, 80, 160, and 320 μg/mL of COS; PBMCs were then treated with 200 μmol/L diethylenetriamine/nitric oxide (DETA/NO) for 4 h. Compared to the CTR group, the cell viability of the COS pre-treated group increased with increasing COS concentration. The activities of antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxidase and the thioredoxin reductase content showed an upward trend in a dose-dependent manner and COS at a concentration of 160 μg/mL exhibited the strongest effect. COS decreased the content and gene expressions of interleukin-1β and tumour necrosis factor-α, the mRNA and protein expression and activity of the inducible nitric oxide synthase, and NO production compared with the values observed in the CTR group. The expression of genes and proteins related to the nuclear factor kappa-B (NF-κB) pathway also showed a downward trend. Based on these indicators, we concluded COS has a protective effect against oxidative damage caused by DETA/NO, and we speculated that COS may play a protective role by inhibiting the NF-κB pathway. Highlights In vitro experiments simulated changes in antioxidant and immune functions caused by increased NO in dairy cows’ PBMCs in vivo. We showed that chitosan has a protective effect against oxidative damage and a regulatory effect on immune function in vitro. The regulation of chitosan against oxidative stress and immune responses may be related to the NF-KB signalling pathway.

Anti-Inflammatory and Protein Tyrosine Phosphatase 1B Inhibitory Metabolites from the Antarctic Marine-Derived Fungal Strain Penicillium glabrum SF-7123.

A chemical investigation of the marine-derived fungal strain Penicillium glabrum (SF-7123) revealed a new citromycetin (polyketide) derivative (1) and four known secondary fungal metabolites, i.e, neuchromenin (2), asterric acid (3), myxotrichin C (4), and deoxyfunicone (5). The structures of these metabolites were identified primarily by extensive analysis of their spectroscopic data, including NMR and MS data. Results from the initial screening of anti-inflammatory effects showed that 2, 4, and 5 possessed inhibitory activity against the excessive production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated BV2 microglial cells, with IC50 values of 2.7 µM, 28.1 µM, and 10.6 µM, respectively. Compounds 2, 4, and 5 also inhibited the excessive production of NO, with IC50 values of 4.7 µM, 41.5 µM, and 40.1 µM, respectively, in LPS-stimulated RAW264.7 macrophage cells. In addition, these compounds inhibited LPS-induced overproduction of prostaglandin E2 in both cellular models. Further investigation of the most active compound (2) revealed that these anti-inflammatory effects were associated with a suppressive effect on the over-expression of inducible nitric oxide synthase and cyclooxygenase-2. Finally, we showed that the anti-inflammatory effects of compound 2 were mediated via the downregulation of inflammation-related pathways such as those dependent on nuclear factor kappa B and p38 mitogen-activated protein kinase in LPS-stimulated BV2 and RAW264.7 cells. In the evaluation of the inhibitory effects of the isolated compounds on protein tyrosine phosphate 1B (PTP1B) activity, compound 4 was identified as a noncompetitive inhibitor of PTP1B, with an IC50 value of 19.2 µM, and compound 5 was shown to inhibit the activity of PTP1B, with an IC50 value of 24.3 µM, by binding to the active site of the enzyme. Taken together, this study demonstrates the potential value of marine-derived fungal isolates as a bioresource for bioactive compounds.