Induced Nitric Oxide Production Research Articles

THE KALLIKREIN-KININ SYSTEM IS FALLING INTO PIECES: BRADYKININ FRAGMENTS ARE BIOLOGICAL ACTIVE PEPTIDES

Objective: Bradykinin [BK-(1–9)] is an endogenous peptide involved in many physiological and pathological processes, such as cardiovascular homeostasis and inflammation. The central dogma of the kallikrein-kinin system is that BK-(1–9) fragments are biologically inactive. In this work, we proposed to test whether these fragments were indeed inactive. Design and method: Nitric oxide (NO) was quantified in human, mouse and rat cells loaded with DAF-FM after stimulation with BK-(1–9), BK-(1–7), BK-(1–5) and BK-(1–3). We used adult male rat aortic ring preparation to test vascular reactivity mediated by BK-(1–9) fragments. Changes in blood pressure and heart rate was measured in conscious adult male rats by intraarterial catheter method. Results: BK-(1–9) induced NO production in all cell types tested by B2 receptor activation. BK-(1–7), BK-(1–5) and BK-(1–3) also induced NO production in all tested cell types but this response was independent of the activation of B1 receptor and/or B2 receptor. BK-(1–7), BK-(1–5) or BK-(1–3) induced only vasorelaxant effect and in a concentration-dependent fashion. Vasorelaxant effects for BK-(1–7), BK-(1–5) or BK-(1–3) were independent of the kinin receptors. BK-(1–9) induced in conscious adult male Wistar rats a dose-dependent hypotension while BK-(1–7), BK-(1–5) and BK-(1–3) induced a dose-independent hypotension. Importantly, these observations diverged from the BK-(1–9) results, highlighting that indeed the BK-(1–9) fragments do not seem to act via the classical kinin receptors. Conclusions: In conclusion, BK-(1–7), BK-(1–5) and BK-(1–3) are biologically active components of the kallikrein-kinin system. Importantly, observed pathophysiological outcomes of these peptides are independent of B1R and/or B2R activation.

Effect of skimmed milk on intestinal tract: Prevention of increased reactive oxygen species and nitric oxide formation

The capacity of skimmed milk to neutralise increased reactive oxygen species (ROS) and to attenuate nitric oxide (NO) production, as well as to present cytoprotective effect at the intestinal level was assessed after in vitro gastro-intestinal digestion. The impact on ROS modulation was evaluated at a non-cytotoxic concentration of hydrogen peroxide (H2O2) in a co-culture of Caco-2 and HT-29 intestinal cells. In parallel, a cytotoxic concentration of H2O2 was used to study the effect of digested milk against induced cell apoptosis. Concerning induced NO production, it was evaluated using the model lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Results showed that digested milk prevented the increase of basal ROS level in the intestinal epithelium and attenuated NO production by LPS-stimulated macrophage cells. In the H2O2-induced cytotoxicity assay, digested milk had no protection against apoptosis, confirmed by the failure in attenuating activated caspase-3/7.

Inhibition of inducible nitric oxide production by Caryota urens and its active constituents umbelliferone and rutin

Inducible Nitric Oxide Synthase (iNOS) is a key mediator in a variety of diseases, and thus it is interesting to discover new iNOS inhibitors. This study aimed to evaluate if the hydroalcoholic leaf extract of Caryota urens (CULHA) consisting of rutin and umbelliferone prevents inflammation in response to external stimuli. To demonstrate the therapeutic potential of CULHA against inflammatory diseases, nitric oxide production (NO) mediated by iNOS was evaluated. In lipopolysaccharide induced RAW 264.7 cells, CULHA, rutin and umbelliferone exhibited remarkable inhibition of NO at 61%, 30% and 41% respectively without affecting the cell viability at the highest concentration tested. In conclusion, results obtained in this work substantiate the traditional uses of this plant and brings a brief understanding of their anti inflammatory potential.

Immunomodulatory Effects of Recombinant Mycobacterium smegmatis Expressing Antigen-85B Epitopes in Infected J774A.1 Murine Macrophages.

Tuberculosis (TB) causes more than 1.5 million deaths each year, remaining a significant global health problem. Mycobacterium smegmatis (M. smegmatis) and Mycobacterium tuberculosis (M. tuberculosis) share features, which support the use of the former use in new generation TB vaccine development. In a previous study, the specific humoral and cellular immunogenicity of a recombinant M. smegmatis strain expressing epitopes from M. tuberculosis Ag85B protein (rMs064), was demonstrated in mice. In the current study, the immunomodulatory capacity of rMs064 was determined in a J774A.1 murine macrophage cell line. To determine the immunomodulatory effect of rMs064 in J774A.1 macrophages, the expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) was evaluated. The expression of activation surface markers (MHC-II, CD40, CD80 and CD86) and the production of cytokines (IL-1β, TNF-α, IL-12p70 and IL-6) was also determined in rMs064 infected J774A.1 macrophages. Our findings showed the ability of rMs064 to induce substantial increases in macrophage activation markers expression; MHC class II and CD40, compared with M. smegmatis transformed with the empty vector (rMs012) and uninfected cells. rMs064 induced significant increases in IL-12p70 compared to uninfected cells. The expression of iNOS and CD86, and the production of IL-1β, and TNF-α were increased in rMs064 and rMs012, compared to uninfected cells. rMs064 demonstrated its immunomodulatory ability by stimulating the innate immune response, which supports its further evaluation as a TB vaccine candidate.

Troxerutin attenuates inflammatory response in lipopolysaccharide-induced sepsis in mice

Troxerutin (Tx), known as vitamin P4 is a derivative of natural bioflavonoid rutin. Tx possesses different biological activities such as antioxidant, anticancer, and anti-inflammatory. The current study was conducted to determine potential therapeutic effect of Tx in lipopolysaccharides (LPS)-induced sepsis in mice. In LPS-induced sepsis, the mice were treated intraperitoneally (ip) with Tx twice daily. Therapeutic effect was assessed by measuring serum level of cytokines, alanine aminotransferase (ALT) and lactate dehydrogenase (LDH). Level of nitric oxide (NO), superoxide dismutase (SOD), catalase (CAT), Myeloperoxidase (MPO) and Malondialdehyde (MDA) was measured. Expression of CD40 receptor on leucocytes was measured using flowcytometry. Splenocyte proliferation was evaluated using MTT assay. The effect of Tx on survival rate during administration of lethal dose of LPS was investigated. The results showed that Tx inhibited LPS induced NO production. Inflammatory pathways were suppressed by reduction of inflammatory cytokines production. Further, elevated CD40 expression of leucocytes and proliferation of splenocytes markedly reduced in Tx treated group. Antioxidant defense system was enhanced by increased activity of SOD and CAT and decreased level of MDA. MPO, ALT and LDH activity. Additionally, treatment with Tx significantly increased the mean survival time of mice compared with the LPS treated group. Histologically, Tx treatment decreased inflammatory cells infiltration and histopathologicl changes in the liver. Our findings showed that reduced inflammatory parameters, improved antioxidant activity, reduced histological lesions and increased survival rate. These findings suggest that Tx is an effective anti-inflammatory agent for the treatment of LPS-induced sepsis.

WDR5a functions in cadmium-inhibited root meristem growth by regulating nitric oxide accumulation in Arabidopsis.

Cadmium stress induces WDR5a expression to promote NO accumulation to repress root meristem growth via suppressing auxin transport and synthesis in Arabidopsis. Nitric oxide (NO) synthase (NOS)-like activity plays a vital role in toxic cadmium (Cd)-induced NO production and inhibition of root meristem growth, while factor(s) regulating NOS-like activity and root meristem growth in plant response to Cd has not been identified yet. Here, we report that WD40 repeat 5a (WDR5a) functions in Cd-induced NOS-like activity, NO accumulation and root meristem growth suppression. We found that wdr5a-1 mutant root has increased root meristem growth with lower NOS-like activity and NO accumulation than wild type upon Cd exposure, and exogenous NO donors sodium nitroprusside or nitrosoglutathione can restore its reduced Cd sensitivity. In addition, Cd activates WDR5a expression in roots, and overexpressing WDR5a results in increased NO accumulation and suppressed root meristem growth similar to Cd-stressed wild-type roots, while scavenging NO or inhibiting NOS-like activity significantly reverts these effects of Cd. Furthermore, WDR5a acts in Cd-repressed auxin accumulation through reducing the levels of auxin efflux carriers PIN1/3/7 and biosynthetic enzyme TAA1, and reduced sensitivity of wdr5a-1 root meristem to Cd can be partially reverted by inhibiting TAA1 activity pharmaceutically or mutating TAA1 genetically. This study identified WDR5a as a key factor modulating NO accumulation and root meristem growth in plant response to Cd.

Electroconvulsive stimulation attenuates chronic neuroinflammation.

Electroconvulsive therapy is highly effective in resistant depression by unknown mechanisms. Microglial toxicity was suggested to mediate depression and plays key roles in neuroinflammatory and degenerative diseases, where there is critical shortage in therapies. We examined the effects of electroconvulsive seizures (ECS) on chronic neuroinflammation and microglial neurotoxicity. Electric brain stimulation inducing full tonic-clonic seizures during chronic relapsing–progressive experimental autoimmune encephalomyelitis (EAE) reduced spinal immune cell infiltration, reduced myelin and axonal loss, and prevented clinical deterioration. Using the transfer EAE model, we examined the effect of ECS on systemic immune response in donor mice versus ECS effect on CNS innate immune activity in recipient mice. ECS did not affect encephalitogenicity of systemic T cells, but it targeted the CNS directly to inhibit T cell–induced neuroinflammation. In vivo and ex vivo assays indicated that ECS suppressed microglial neurotoxicity by reducing inducible NOS expression, nitric oxide, and reactive oxygen species (ROS) production, and by reducing CNS oxidative stress. Microglia from ECS-treated EAE mice expressed less T cell stimulatory and chemoattractant factors. Our findings indicate that electroconvulsive therapy targets the CNS innate immune system to reduce neuroinflammation by attenuating microglial neurotoxicity. These findings signify a potentially novel therapeutic approach for chronic neuroinflammatory, neuropsychiatric, and neurodegenerative diseases.

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.

Moringa oleifera leaf extract enhances endothelial nitric oxide production leading to relaxation of resistance artery and lowering of arterial blood pressure

A mass of evidence has identified a promoting of nitric oxide (NO) production in endothelial cells using natural products as a potential strategy to prevent and treat hypertension. This study investigated whether the aqueous extract of Moringa oleifera leaves (MOE) could lower mean arterial pressure (MAP) and relax mesenteric arterial beds in rats via stimulating endothelium-derived NO production. Intravenous administration of MOE (1–30 mg/kg) caused a dose-dependent reduction in MAP in anesthetized rats. In rats pretreated with the NO-synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg, i.v.), the effect of MOE on MAP was significantly reduced. MOE (0.001–3 mg) induced relaxation in methoxamine (10 μM) pre-contracted mesenteric arterial beds, which was abolished by endothelium denudation. This endothelium-dependent vasorelaxation was reduced by L-NAME (100 μM) or the NO-sensitive guanylyl cyclase inhibitor, 1H- [1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (10 μM). In primary human pulmonary artery endothelial cells, MOE (3–30 μg/mL) induced NO production, which was inhibited by L-NAME (100 μM) pretreatment. These findings show that MOE stimulates the endothelium-derived NO release for driving its vasorelaxation to lower arterial blood pressure. These suggest the development of MOE as a natural antihypertensive supplement.

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.

The contributions of hydroxylamine and nitrite to NO and N2O production in alkaline and acidic vegetable soils

The contribution of hydroxylamine (NH2OH) and nitrite (NO2−) to nitric oxide (NO) and nitrous oxide (N2O) production remains unclear in vegetable production soils. Soils collected from six typical greenhouse vegetable fields were incubated for 48 h following amendment with 1 mM NaNO2, 10 μM NH2OH, or 1 mM NaNO2 + 10 μM NH2OH. The importance of abiotic processes on the NO and N2O formation from the NH2OH and NO2− were studied by irradiating the soil samples with γ-irradiation. NO2− amendment significantly stimulated NO production, while the NH2OH-dependent NO production was minimal. NH2OH stimulated more abiotic N2O production in alkaline soils than in acidic soils (p < 0.05), while NO2− stimulated more biotic N2O production in acidic soils than in alkaline soils (p < 0.05). The NH2OH- and NO2−-dependent sources produced biotic or abiotic N2O with site preference (SP) values of 27.4–36.5‰, which is similar to those from ammonia-oxidizing archaea (AOA) or ammonia-oxidizing bacteria (AOB) sources (25.1–34.2‰), indicating that abiotic N2O production were closely linked with biotic NH3 oxidation. The variability of NO2−+NH2OH-induced N2O production can be explained by the soil organic carbon and iron concentrations, whereas NO2−-induced NO production can be explained by the soil pH. NO2− addition dominated NO production in all soils. Furthermore, NO2− addition increased biotic N2O production in acidic soils, while NH2OH addition increased abiotic N2O production in alkaline soils. The presence of NO2− could significantly stimulate the abiotic conversion of NH2OH to N2O in soils with low soil organic carbon and high iron concentrations. Thus, assessing the abundance of NH2OH and NO2− could provide crucial information for understanding NO and N2O production procedures in vegetable soils. • The chemical decomposition of NO2− dominated NO production in all soils. • The NH2OH stimulated abiotic N2O production in alkaline soils. • The NO2− stimulated biotic N2O production in acidic soils. • The SP for abiotic NO2−-/NH2OH-related N2O were in the same range as AOB/AOA sources.

Protein Arginine Methyltransferase 1 (PRMT1) Selective Inhibitor, TC-E 5003, Has Anti-Inflammatory Properties in TLR4 Signaling.

Protein arginine methyltransferase 1 (PRMT1) is the most predominant PRMT and is type I, meaning it generates monomethylarginine and asymmetric dimethylarginine. PRMT1 has functions in oxidative stress, inflammation and cancers, and modulates diverse diseases; consequently, numerous trials to develop PRMT1 inhibitors have been attempted. One selective PRMT1 inhibitor is N,N′-(Sulfonyldi-4,1-phenylene)bis(2-chloroacetamide), also named TC-E 5003 (TC-E). In this study, we investigated whether TC-E regulated inflammatory responses. Nitric oxide (NO) production was evaluated by the Griess assay and the inflammatory gene expression was determined by conducting RT-PCR. Western blot analyzing was carried out for inflammatory signaling exploration. TC-E dramatically reduced lipopolysaccharide (LPS)-induced NO production and the expression of inflammatory genes (inducible NO synthase (iNOS), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α and interleukin (IL)-6) as determined using RT-PCR. TC-E downregulated the nuclear translocation of the nuclear factor (NF)-κB subunits p65 and p50 and the activator protein (AP)-1 transcriptional factor c-Jun. Additionally, TC-E directly regulated c-Jun gene expression following LPS treatment. In NF-κB signaling, the activation of IκBα and Src was attenuated by TC-E. Taken together, these data show that TC-E modulates the lipopolysaccharide (LPS)-induced AP-1 and NF-κB signaling pathways and could possibly be further developed as an anti-inflammatory compound.

Induction of peroxisome proliferator activated receptor \u03b3 (PPAR\u03b3) mediated gene expression and inhibition of induced nitric oxide production by Maerua subcordata (Gilg) DeWolf

BackgroundThe health benefits of botanicals is linked to their phytochemicals that often exert pleiotropic effects via targeting multiple molecular signaling pathways such as the peroxisome proliferator-activated receptors (PPARs) and the nuclear factor kappaB (NFκB). The PPARs are transcription factors that control metabolic homeostasis and inflammation while the NF-κB is a master regulator of inflammatory genes such as the inducible nitric-oxide synthase that result in nitric oxide (NO) overproduction.MethodsExtracts of Maerua subcordata (MS) and selected candidate constituents thereof, identified by liquid chromatography coupled to mass spectroscopy, were tested for their ability to induce PPARγ mediated gene expression in U2OS-PPARγ cells using luciferase reporter gene assay and also for their ability to inhibit lipopolysaccharide (LPS) induced NO production in RAW264.7 macrophages. While measuring the effect of test samples on PPARγ mediated gene expression, a counter assay that used U2OS-Cytotox cells was performed to monitor cytotoxicity or any non-specific changes in luciferase activity.ResultsThe results revealed that the fruit, root, and seed extracts were non-cytotoxic up to a concentration of 30 g dry weight per litre (gDW/L) and induced PPARγ mediated gene expression but the leaf extract showed some cytotoxicity and exhibited minimal induction. Instead, all extracts showed concentration (1–15 gDW/L) dependent inhibition of LPS induced NO production. The root extract showed weaker inhibition. Among the candidate constituents, agmatine, stachydrine, trigonelline, indole-3-carboxyaldehyde, plus ethyl-, isobutyl-, isopropyl, and methyl-isothiocyanates showed similar inhibition, and most showed increased inhibition with increasing concentration (1–100 μM) although to a lesser potency than the positive control, aminoguanidine.ConclusionThe present study demonstrated for the first time the induction of PPARγ mediated gene expression by MS fruit, root, and seed extracts and the inhibition of LPS induced NO production by MS fruit, leaf, root, and seed extracts and some candidate constituents thereof.

Chitosan Nanoparticles: Shedding Light on Immunotoxicity and Hemocompatibility.

Nanoparticles (NPs) assumed an important role in the area of drug delivery. Despite the number of studies including NPs are growing over the last years, their side effects on the immune system are often ignored or omitted. One of the most studied polymers in the nano based drug delivery system field is chitosan (Chit). In the scientific literature, although the physicochemical properties [molecular weight (MW) or deacetylation degree (DDA)] of the chitosan, endotoxin contamination and appropriate testing controls are rarely reported, they can strongly influence immunotoxicity results. The present work aimed to study the immunotoxicity of NPs produced with different DDA and MW Chit polymers and to benchmark it against the polymer itself. Chit NPs were prepared based on the ionic gelation of Chit with sodium tripolyphosphate (TPP). This method allowed the production of two different NPs: Chit 80% NPs (80% DDA) and Chit 93% NPs (93% DDA). In general, we found greater reduction in cell viability induced by Chit NPs than the respective Chit polymers when tested in vitro using human peripheral blood monocytes (PBMCs) or RAW 264.7 cell line. In addition, Chit 80% NPs were more cytotoxic for PBMCs, increased reactive oxygen species (ROS) production (above 156 μg/mL) in the RAW 264.7 cell line and interfered with the intrinsic pathway of coagulation (at 1 mg/mL) when compared to Chit 93% NPs. On the other hand, only Chit 93% NPs induced platelet aggregation (at 2 mg/mL). Although Chit NPs and Chit polymers did not stimulate the nitric oxide (NO) production in RAW 264.7 cells, they induced a decrease in lipopolysaccharide (LPS)-induced NO production at all tested concentrations. None of Chit NPs and polymers caused hemolysis, nor induced PBMCs to secrete TNF-α and IL-6 cytokines. From the obtained results we concluded that the DDA of the Chit polymer and the size of Chit NPs influence the in vitro immunotoxicity results. As the NPs are more cytotoxic than the corresponding polymers, one should be careful in the extrapolation of trends from the polymer to the NPs, and in the comparisons among delivery systems prepared with different DDA chitosans.

In Vitro Antiosteoporosis Activity and Hepatotoxicity Evaluation in Zebrafish Larvae of Bark Extracts of Prunus jamasakura Medicinal Plant.

Osteoporosis is one of the main health problems in the world today characterized by low bone mass and deterioration in bone microarchitecture. In recent years, the use of natural products approach to treat it has been in the increase. In this study, in vitro antiosteoporosis activity and hepatotoxicity of P. jamasakura bark extracts were evaluated. Methods. Mouse bone marrow macrophage (BMM) cells were incubated with tartrate-resistant acid phosphate (TRAP) buffers and p-nitrophenyl phosphate and cultured with different P. jamasakura bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 μg/ml in the presence of the receptor activator of nuclear factor kappa-Β ligand (RANKL) for 6 days. The osteoclast TRAP activity and cell viability were measured. Nitric oxide (NO) assay was conducted using murine macrophage-like RAW 264.7 cells treated with P. jamasakura ethanolic and methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, 50, 100, and 200 μg/ml. For hepatotoxicity assessment, zebrafish larvae were exposed to P. jamasakura bark extracts, 0.05% dimethyl sulfoxide as a negative control, and 5 μM tamoxifen as a positive control. The surviving larvae were anesthetized and assessed for hepatocyte apoptosis. Results. TRAP activity was significantly inhibited (p < 0.001) at all concentrations of P. jamasakura extracts compared to the control treatment. At 50 μg/ml, both ethanolic and methanolic extracts of P. jamasakura exhibited significant (p < 0.01) BMM cell viability compared to the control treatment. P. jamasakura ethanolic and methanolic extracts had significant inhibitory (p < 0.01) effects on lipopolysaccharide (LPS)-induced NO production at 200 μg/ml and exhibited significant (p < 0.01) and (p < 0.05) stimulative effects, respectively, on RAW 264.7 cell viability. No overt hepatotoxicity was observed in the liver of zebrafish larvae in any of the treatments. Conclusion. The TRAP activity of P. jamasakura bark gives a foundation for further studies to enhance future development of antiosteoporosis drug.

Nitrite may serve as a combination partner and a biomarker for the anti-cancer activity of RRx-001.

RRx-001 is an anti-cancer immunotherapeutic that increases the sensitivity of drug resistant tumors via multiple mechanisms which involve binding to hemoglobin and enhancing nitrite reductase activity of deoxyhemoglobin. In the present study, the effect of clinically used doses of RRx-001 on erythrocyte deformability was examined. A dose dependent effect of RRx-001 (1-1000 micro molar) on erythrocyte deformability was measured by ektacytometer under hypoxia (n =8). Low dose RRx-001 (20 micro molar) in the presence of ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one), L-NAME (L-NG-Nitroarginine methyl ester) or nitrite were examined both in normoxia and hypoxia. Intracellular nitric oxide (NO) levels were measured fluorometrically with DAF-FM-DA. Higher doses of RRx-001 (100, 1000 micro molar) significantly decreased erythrocyte deformability under hypoxia (p <0.01; p <0.05, respectively). RRx-001 (20 micro molar), alone or in combination with ODQ or L-NAME, did not change deformability. However, RRx-001 and nitrite caused an increase in deformability (p <0.01) under hypoxia. RRx-001 induced NO production was more pronounced in the presence of nitrite (p <0.05). Co-administration of RRx-001 and nitrite under hypoxic conditions results in a significant increase in erythrocyte deformability that is related to increased NO production. We suggest that measurement of serum nitrite level in RRx-001 treated cancer patients should be routinely undertaken and supplemented if levels are low for maximal activity.

Methane-induced lateral root formation requires the participation of nitric oxide signaling

Although methane (CH4)-induced lateral root (LR) formation has been discovered, the identification of downstream signaling compounds has yet to be fully elucidated. Here, we report a unique mechanism for the involvement of nitric oxide (NO) in the above CH4-mediated pathway in tomato (Solanum lycopersicum L.) and Arabidopsis thaliana. NO was produced rapidly in the root tissues of tomato seedlings when CH4 was administrated exogenously. The scavenging of NO with its scavengers prevented lateral root primordia formation and thereafter lateral rooting triggered by CH4. Gene expression analysis revealed that similar to the responses of sodium nitroprusside (SNP; a NO-releasing compound), CH4-induced SlCYCA2;1, SlCYCA3;1, and SlCDKA1 transcripts, and -downregulated SlKRP2 mRNA, were differentially abolished when endogenous NO was removed by its scavengers. Changes in the lateral root-related miRNA genes (SlmiR160 and SlmiR390a) and their target genes (SlARF16 and SlARF4), exhibited similar tendencies. Similar to those results in tomato, the addition of CH4 and SNP could obviously induce NO production and LR formation in Arabidopsis seedlings, which were correlated with the transcriptional profiles of representative LR-related genes. Combine with these findings in tomato and Arabidopsis thaliana, our results showed that NO might act, at least partially, as the downstream signaling molecule for CH4 control of lateral rooting.

Inducible Nitric Oxide Synthase is required for parasite restriction and inflammatory modulation during Neospora caninum infection

Neospora caninum infection is an important cause of neuromuscular disease in dogs and abortion in cattle, leading to significant economic losses in beef and dairy industries. The protective immunity against apicomplexan parasites, specifically Toxoplasma gondii and N. caninum, is typically achieved by inducing an IL-12-driven Th1 immune response. IL-12 stimulates IFN-γ production, which activates Inducible Nitric Oxide Synthase (iNOS) and promotes consequent Nitric Oxide (NO) synthesis, classically described as one of the main effector mechanisms for parasite elimination. Here, we aimed to evaluate the role played by iNOS during N. caninum infection. Our results show that N. caninum infection in C57BL/6 wild type (WT) mice induce NO production in vivo and in vitro. In agreement, iNOS deficient mice, as well as WT mice treated with iNOS inhibitor aminoguanidine, succumbed during acute infection with a dose lethal to 50 % of the WT mice, and presented significant increase in parasite load when submitted to sub-lethal infection protocols. Interestingly, the lack of control of parasite proliferation observed in iNOS−/− mice was associated with notable CNS inflammation and increased production of the main systemic proinflammatory cytokines (IL-12, IFN-γ, IL-6, TNF and IL-17A). Taken together, our findings show that iNOS plays an important role in restricting N. caninum replication, while also modulates the inflammatory process induced by the infection.

Anti-inflammatory and anti-osteoarthritis effects of Cm-02 and Ck-02

Osteoarthritis (OA) is a common degenerative joint disease characterized by progressive deterioration of articular cartilage. There have been reports that small molecule inhibitors have anti-osteoarthritis effects; however, the effects of 3-(4-chloro-2-fluorophenyl)-6-(2,4-difluorophenyl)-2H-benzo[e] [1,3]oxazine-2,4(3H)-dione (Cm-02) and 6-(2,4-difluorophenyl)-3-(3,4-difluorophenyl)-2H-benzo[e] [1,3]oxazine-2,4(3H)-dione (Ck-02), small molecule inhibitors which share many structural similarities with quercetin (a potent anti-inflammatory flavonoid), remain unclear. In this study, TNF-α-stimulated porcine and human chondrocyte models were used to investigate the inhibitory effects of Cm-02 and Ck-02 on the molecular mechanisms underlying the anti-OA effects. TNF-α was used to stimulate porcine and human chondrocytes to mimic immunomodulatory potency in-vitro. Anti-osteoarthritic effects were characterized in terms of protein and mRNA levels associated with the pathogenesis of OA. We also examined (1) the inducible nitric oxide synthase (iNOS)-nitric oxide (NO) system in cultured chondrocytes, (2) matrix metalloproteinases (MMPs) in cultured chondrocytes, and (3) aggrecan degradation in cartilage explants. Finally, we tested the activation of nuclear factor-kappaB (NF-κB), interferon regulatory factor-1 (IRF-1), and activate the protein-1 (AP-1), and we tested the signal transduction and activation of transcription-3 (STAT-3). Our results indicate that, in chondrocytes, Cm-02 and Ck-02 inhibit TNF-α induced NO production, iNOS, MMP, the expression of disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), and the enzyme activity of MMP-13. Furthermore, both Cm-02 and Ck-02 were found to stimulate TNF-α, which has been shown to suppress the activation of several transcription factors, including NF-κB, STAT-3, and IRF-1 in porcine and human chondrocytes. Cm-02 and Ck-02 were also found to help prevent the release of proteoglycans from cartilage explants. Our findings demonstrate that both Cm-02 and Ck-02 have potent anti-inflammatory activities and the ability to protect cartilage in an OA cell model. These findings indicate that Cm-02 and Ck-02 have the potential to be further developed for the therapeutic treatment of OA.

Anti-inflammatory potential of South African medicinal plants used for the treatment of sexually transmitted infections

Inflammation associated with sexually transmitted infection (STIs) often results from delayed diagnosis or treatment, and may result in life-threatening complications. The root or stem bark of plants selected in this study are used by traditional healers in the management of STIs and to treat the associated inflammation. This study evaluated the anti-inflammatory activities of 12 South African medicinal plants used traditionally in the treatment of STIs and the associated inflammation. Acetone extracts were tested for anti-inflammatory activity against 15-lipoxygenase (15-LOX), xanthine oxidase (XO) and inducible nitric oxide production in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophage cells. The free radical and nitric oxide scavenging capacity was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and nitric oxide inhibition assays. The MTT assay was used to determine the cytotoxic effect of the extracts on RAW 264.7 macrophages and Vero cells. Preliminary phytochemical analysis was carried out using NMR spectroscopy, Gas Chromatography Mass Spectroscopy (GC–MS) analysis and the determination of total phenolic and flavonoid contents. The root bark extract of Lannea schweinfurthii had the highest inhibitory activity against 15-LOX (IC50 = 40 ± 3 μg/mL) while C. abbreviata best inhibited XO (IC50 = 46.8 ± 1.5 μg/mL). Extracts of Ficus abutilifolia, Faurea saligna and Zanthoxylum capense showed significant suppression of inducible nitric oxide production with IC50 values comparable to quercetin (IC50 < 30.0 μg/mL). The extracts of C. abbreviata and F. abutilifolia had significantly higher radical scavenging activities compared to ascorbic acid. Most of the extracts showed little or no cytotoxicity against the tested cell lines. Phytochemical analysis pointed to the presence of diverse classes of secondary metabolites as well as the presence of proanthocyanidins as the major constituents of the most active extracts in the anti-inflammatory assays. For most of the plants, this study provides the first report of 15-LOX inhibition and xanthine oxidase inhibition. The activity of most of the tested extracts supports the administration of these plants by traditional healers in the treatment of STIs and their associated inflammation.