Excessive Nitric Oxide Production Research Articles

Hepatocellular Protein Arginine Methyltransferase 1 Suppresses Alcohol-Induced Hepatocellular Carcinoma Formation by Inhibition of Inducible Nitric Oxide Synthase.

Alcohol is a well‐established risk factor for hepatocellular carcinoma (HCC), but the mechanisms by which alcohol promotes liver cancer are not well understood. Studies suggest that ethanol may enhance tumor progression by increasing hepatocyte proliferation and through alcohol‐induced liver inflammation. Protein arginine methyltransferase 1 (PRMT1) is the main enzyme responsible for cellular arginine methylation. Asymmetric dimethyl arginine, produced by PRMT1, is a potent inhibitor of nitric oxide synthases. PRMT1 is implicated in the development of several types of tumors and cardiovascular disease. Our previous work has shown that PRMT1 in the liver regulates hepatocyte proliferation and oxidative stress and protects from alcohol‐induced liver injury. However, its role in HCC development remains controversial. In this study, we found that hepatocyte‐specific PRMT1‐knockout mice develop an increased number of tumors in an N‐nitrosodiethylamine (DEN) alcohol model of liver tumorigenesis in mice. This effect was specific to the alcohol‐related component because wild‐type and knockout mice developed similar tumor numbers in the DEN model without the addition of alcohol. We found that in the presence of alcohol, the increase in tumor number was associated with increased proliferation in liver and tumor, increased WNT/β‐catenin signaling, and increased inflammation. We hypothesized that increased inflammation was due to increased oxidative and nitrosative stress in knockout mice. By blocking excess nitric oxide production using an inducible nitric oxide synthase inhibitor, we reduced hepatocyte death and inflammation in the liver and prevented the increase in WNT/β‐catenin signaling, proliferation, and tumor number in livers of knockout mice. Conclusion: PRMT1 is an important protection factor from alcohol‐induced liver injury, inflammation, and HCC development.

Role of iNOS in osteoarthritis: Pathological and therapeutic aspects.

Accumulating evidence suggests that inflammation has a key role in the pathogenesis of osteoarthritis (OA). Nitric oxide (NO) has beenestablished as one of the major inflammatory mediators in OA and drives many pathological changes during the development and progression of OA. Excessive production of NO in chondrocytes promotes cartilage destruction and cellular injury. The synthesis of NO in chondrocytes is catalyzed by inducible NO synthase (iNOS), which is thereby an attractive therapeutic target for the treatment of OA. A number of direct and indirect iNOS inhibitors, bioactive compounds, and plant-derived small molecules have been shown to exhibit chondroprotective effects by suppressing the expression of iNOS. Many of these iNOS inhibitors hold promise for the development of new, disease-modifying therapies for OA; however, attempts to demonstrate their success in clinical trials are not yet successful. Many plant extracts and plant-derived small molecules have also shown promise in animal models of OA, though further studies are needed in human clinical trials to confirm their therapeutic potential. In this review, we discuss the role of iNOS in OA pathology and the effects of various iNOS inhibitors in OA.

Leocarpinolide B attenuates LPS-induced inflammation on RAW264.7 macrophages by mediating NF-κB and Nrf2 pathways

Macrophages-mediated inflammation is involved in the regulation of rheumatoid arthritis (RA). Sigesbeckiae Herba (SH) has been traditionally used for rheumatism. However, the bioactive ingredients of SH are still unclear. Recently, we isolated a compound (Leocarpinolide B, LB) from SH and identified its potent anti-inflammatory and antioxidant effects on RAW264.7 macrophages for the first time. LB effectively inhibited excessive production of nitric oxide (NO), prostaglandin E2 (PGE2), cytokines (IL-6, TNF-α and MCP-1), and the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthases (iNOS) in lipopolysaccharide (LPS)-induced RAW264.7 cells. LB blocked the degradation of inhibitor of kappa B (IκBα) and translocation of nuclear factor kappa B (NF-κB) p65. Additionally, LB reduced the intracellular reactive oxygen species, and increased the expression of heme oxygenase-1 (HO-1) and the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in the presence or absence of LPS. The results suggested that LB might be one of the bioactive components of SH, and be potential for the treatment of RA and valuable to be further investigated.

Inhibitory Effects of Gynura procumbens Ethanolic Extract on Nitric Oxide Production and Inducible Nitric Oxide Synthase (iNOS) Protein Expression in Macrophages

Nitric oxide (NO) overproduction by inducible nitric oxide synthase (iNOS) may be associated with acute and chronic inflammations. Macrophages as important cells in the innate immune system are able to be stimulated and can lead to iNOS activation and excessive NO production. Gynura procumbens is a medicinal plant traditionally used in treating various ailments including inflammation but the mechanism of anti-inflammatory activity of this plant is still elusive. This study was carried out to investigate the anti-inflammatory therapeutic effects of Gynura procumbens ethanolic extract on NO production and iNOS protein expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). Cell viability of RAW 264.7 macrophages treated with Gynura procumbens ethanolic extract was determined by MTT assay. NO production was determined by Griess assay following Gynura procumbens ethanolic extract treatment alone or in combination with LPS stimulation. Protein expression of iNOS was determined by western blot. RAW 264.7 macrophages viability of more than 90% was observed after 24 h treatment with Gynura procumbens ethanolic extract concentration range of 3.9 μg/mL to 500 μg/mL. Significant inhibition of NO production level has been identified in LPS-stimulated RAW 264.7 cells pre-treated with 250 μg/mL Gynura procumbens ethanolic extract (p<0.05) while all selected concentrations of Gynura procumbens ethanolic extract showed no significant alteration of NO production in the absence of LPS stimulation. Pre-treatment of 250 μg/mL Gynura procumbens ethanolic extract also demonstrated significant suppression of iNOS protein expression in LPS-stimulated RAW 264.7 cells (p<0.05). In conclusion, this study demonstrates that Gynura procumbens ethanolic extract exhibits anti-inflammatory potential through inhibition of NO production and iNOS protein expression in LPS-stimulated macrophages, suggesting that this plant could be further researched for its beneficial use in inflammatory disorders.

Potential Utility of Protein Targets of Cysteine-S-Nitrosylation in Identifying Clinical Disease Status in Human Chagas Disease.

Trypanosoma cruzi (Tc) infection causes Chagas disease (ChD) presented by dilated cardiomyopathy and heart failure. During infection, oxidative and nitrosative stresses are elicited by the immune cells for control the pathogen; however, excess nitric oxide and superoxide production can result in cysteine S-nitrosylation (SNO) of host proteins that affects cellular homeostasis and may contribute to disease development. To identify the proteins with changes in SNO modification levels as a hallmark of ChD, we obtained peripheral blood mononuclear cells (PBMC) from seronegative, normal healthy (NH, n = 30) subjects, and from seropositive clinically asymptomatic (ChD CA, n = 25) or clinically symptomatic (ChD CS, n = 28) ChD patients. All samples were treated (Asc+) or not-treated (Asc−) with ascorbate (reduces nitrosylated thiols), labeled with the thiol-labeling BODIPY FL-maleimide dye, resolved by two-dimensional electrophoresis (total 166 gels), and the protein spots that yielded significant differences in abundance or SNO level at p-value of ≤ 0.05t−test/Welch/BH were identified by MALDI-TOF/TOF MS or OrbiTrap LC-MS/MS. Targeted analysis of a new cohort of PBMC samples (n = 10–14/group) was conducted to verify the differential abundance/SNO levels of two of the proteins in ChD (vs. NH) subjects. The multivariate adaptive regression splines (MARS) modeling, comparing differences in relative SNO level (Asc−/Asc+ ratio) of the protein spots between any two groups yielded SNO biomarkers that exhibited ≥90% prediction success in classifying ChD CA (582-KRT1 and 884-TPM3) and ChD CS (426-PNP, 582-KRT1, 486-ALB, 662-ACTB) patients from NH controls. Ingenuity Pathway Analysis (IPA) of the SNO proteome dataset normalized to changes in protein abundance suggested the proteins belonging to the signaling networks of cell death and the recruitment and migration of immune cells were most affected in ChD CA and ChD CS (vs. NH) subjects. We propose that SNO modification of the select panel of proteins identified in this study have the potential to identify ChD severity in seropositive individuals exposed to Tc infection.

Optimization of a microchip electrophoresis method with electrochemical detection for the determination of nitrite in macrophage cells as an indicator of nitric oxide production.

Nitric oxide (NO) is involved in many biological functions, including blood pressure regulation, the immune response, and neurotransmission. However, excess production of NO can lead to the generation of reactive nitrogen species and nitrosative stress and has been linked to several neurodegenerative diseases and cardiovascular disorders. Because NO is short-lived and generally difficult to detect, its primary stable degradation product, nitrite, is frequently monitored in its place. In this paper, an improved method using microchip electrophoresis with electrochemical detection (ME-EC) was developed for the separation and detection of nitrite in cell lysates. A separation of nitrite from several electroactive cell constituents and interferences was optimized, and the effect of sample and buffer conductivity on peak efficiency was explored. It was found that the addition of 10 mM NaCl to the run buffer caused stacking of the nitrite peak and improved limits of detection. A platinum black working electrode was also evaluated for the detection of nitrite and other electroactive cellular species after electrophoretic separation. The use of a modified platinum working electrode resulted in 2.5-, 1.7-, and 7.2-fold signal enhancement for nitrite, ascorbic acid, and hydrogen peroxide, respectively, and increased the sensitivity of the method for nitrite twofold. The optimized ME-EC method was used to compare nitrite production by native and lipopolysaccharide-stimulated RAW 264.7 macrophage cells.

Effect of Ethanol Extracts from DefattedPerilla frutescenson LPS-induced Inflammation in Mouse BV2 Microglial Cells

To evaluate the antioxidant and anti-neuroinflammatory effects of defatted Perilla frutescens extract (DPE) in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Cell viabilities were estimated by MTT assay. LPS-stimulated BV-2 microglia were used to study the expression and production of inflammatory mediators such as nitric oxide (NO), inducible NO synthase (iNOS), Cyclooxygenase-2 (COX-2), and prostaglandin E₂ (PGE₂). Pretreatment with DPE prior to LPS treatment significantly inhibited excessive production of NO (10, 25, 50, 75, and 100 μg/mL) in a dose-dependent manner, and was associated with down regulation of expression of iNOS and COX-2. DPE also suppressed the LPSinduced increase in PGE₂ level (10, 25, 50, 75, and 100 μg/mL) in BV-2 cells. Therefore, DPE can be considered as a useful therapeutic and preventive approach for the treatment of several neurodegenerative diseases.

Deep-red fluorogenic probe for rapid detection of nitric oxide in Parkinson’s disease models

Nitric oxide (NO), as one of important gaseous signaling molecule in human body, has been associated with a variety of physiologic processes. However, excessive production of NO has been profoundly implicated in the pathogenesis of neurodegenerative disorders in particular, Parkinson's disease (PD). Therefore, accurate and facile detection of NO is of great significance for investigating its functions in PD, and the subsequent diagnosis and/or treatment. Herein, we developed a deep-red fluorogenic probe (BT-NH), with high sensitivity and good selectivity to detect NO, which was successfully used to visualize exogenous/endogenous NO level in living cells, and further applied to in vitro and in vivo PD models. Therefore, BT-NH could be a potential biological fluorogenic tool to explore the roles of NO in PD.

Promising neuroprotective effects of β-caryophyllene against LPS-induced oligodendrocyte toxicity: A mechanistic study

Myelin loss subsequent to oligodendrocyte death has been reported in a variety of myelin-associated disorders such as multiple sclerosis (MS). Lipopolysaccharide (LPS) has been shown to elicit cellular responses in the central nervous system (CNS) and trigger immune infiltrates and glial cells to release a variety of inflammatory cytokines and mediators. LPS-induced oligodendrocytes toxicity may be chosen as an efficient model to evaluate the role of oligodendrocytes in neuroprotective activities of compounds. β-Caryophyllene (BCP) is a selective type 2 cannabinoid (CB2) receptor agonist. However, the mechanisms underlying the anti-inflammatory effects of BCP are not completely understood. On this basis, we aimed to investigate the protective effects of a wide range of BCP concentrations against LPS-induced toxicity in a proliferative oligodendrocyte cell line (OLN-93) and evaluate the possible correlation between BCP concentration and selective modulation of CB2, Nrf2, sphingomyelinase (SMase) and peroxisome proliferator-activated receptors (PPAR)-γ signaling pathways. We found that LPS significantly increases the levels of reactive oxygen species (ROS), nitric oxide (NO) metabolite and tumor necrosis factor (TNF)-α production while decreases the level of GSH. BCP could prevent LPS-induced cytotoxicity and excessive production of NO, ROS, and TNF-α. Also, we demonstrated that BCP’s protective effects against LPS-induced oligodendrocytes toxicity were mediated via the CB2 receptor through different pathways including Nrf2/HO-1/anti-oxidant axis, and PPAR-γ, at low (0.2 and 1 µM), and high (10–50 µM) concentrations, respectively. Additionally, we observed that the addition of SMase inhibitors imipramine (IMP) and fluoxetine (FLX) synergistically increased the protective effects of BCP. Finally, BCP at low concentrations exerted promising protective effects that could be considered for the treatment of neurodegenerative disorders such as MS. However, more studies using other models of neurodegenerative diseases should be undertaken to assess different parameters such as the activity or expression of SMase.

Caffeine modulates brain purinergic signaling in Nile tilapia (Oreochromis niloticus) under hypoxia conditions: improvement of immune and inflammatory responses.

Purinergic signaling is linked to neurodegenerative and proinflammatory damage during pathological conditions such as hypoxia, but involvement of this pathway in brain damage in fish exposed to environmental hypoxia remains unknown, and we propose dietary supplementation with caffeine in order to improve the immune response. Therefore, the aim of the study was to evaluate whether the enzymatic purinergic signaling pathway is associated with inflammatory brain damage in Nile tilapia (Oreochromis niloticus) exposed to environmental hypoxia and whether dietary supplementation with caffeine (5% and 8%) can prevent these changes in purinergic signaling. Animals were randomly divided into six groups (A-F, n = 6 per group, in triplicate), as follows: groups A-C were submitted to normoxia, while groups D-F were submitted to hypoxia. Groups A and D received the basal diet, while groups B and D and groups C and F received a diet containing 5% and 8% caffeine, respectively, and fed with their respective diets for 21days. After 21days, aeration was disconnected (groups D-F) and the dissolved oxygen levels were maintained as follows: group A (6.55 ± 0.23mg/L), group B (6.51 ± 0.24mg/L), group C (6.58 ± 0.22mg/L), group D (1.23 ± 0.11mg/L), group E (1.20 ± 0.15mg/L), and group F (1.18 ± 0.13mg/L). Cerebral triphosphate diphosphohydrolase (NTPDase) using adenosine triphosphate (ATP) as a substrate and 5'-nucleotidase activities decreased in fish exposed to 72h of hypoxia compared with the normoxia group, while adenosine deaminase (ADA) activity and levels of nitric oxide (NOx) metabolites were higher. Dietary supplementation with 5% and 8% caffeine prevented all alterations elicited by hypoxia, with the exception of ADA activity in the case of 5% caffeine. Based on this evidence, our findings reveal that nucleotide/nucleoside hydrolysis is modified in the brains of fish exposed to 72h of hypoxia, contributing to inflammatory damage, which apparently is mediated by excessive ATP content in the extracellular medium and by excessive NOx production. Also, the use of a diet containing 5% and 8% caffeine prevented these alterations (except 5% of dietary caffeine on ADA activity) and can be considered an interesting approach to preventing the impairment of immune and inflammatory responses elicited by hypoxia, principally the inclusion of 8% caffeine.

216 - Inducible nitric oxide synthase-derived nitric oxide promotes mitochondrial dysfunction, altered nutrient metabolism, and apoptosis in Ctns null kidney proximal tubular epithelial cells

Nitric oxide (NO) has been shown to play an important role in renal physiology and pathophysiology partly through its influence on various solute transport systems in the kidney proximal tubule. Coincidentally, the same transport systems are affected in the lysosomal storage disorder, cystinosis, which manifest clinically as renal Fanconi syndrome. Cystinosis is caused by one or more mutations in the CTNS gene, which codes for a lysosomal transmembrane protein, cystinosin. Several in vitro and in vivo studies have implicated oxidative stress in the pathogenesis of cystinosis. However, the role of NO in the kidney dysfunction associated with cystinosis is largely unknown. In the present study, the effects of NO on Na+,K+-ATPase activity and expression, mitochondrial integrity and function, nutrient metabolism, and apoptosis were investigated in Ctns null proximal tubular epithelial cells (PTECs). Ctns null PTECs exhibited an increase in inducible nitric oxide synthase (iNOS) expression which resulted in the augmentation of intracellular NO levels and extracellular nitrite/nitrate production. This was accompanied by a reduction in Na+,K+-ATPase expression and activity. In addition, Ctns null PTECs displayed depolarized mitochondria, reduced ATP production, altered nutrient metabolism, and elevated apoptosis. Treatment of Ctns null PTECs with an iNOS-selective inhibitor, 1400W, abolished these effects which culminated in the mitigation of apoptosis. These findings indicate that excessive NO production by iNOS may constitute an upstream event that leads to the molecular and biochemical alterations observed in Ctns null PTECs. This may explain, at least in part, the generalized kidney proximal tubular dysfunction associated with cystinosis. Further studies are needed to realize the potential benefits anti-nitrosative therapies in improving renal function and/or attenuating renal injury in cystinosis.

Neuroprotective Effect of Alpha-Linolenic Acid against Aβ-Mediated Inflammatory Responses in C6 Glial Cell.

Therapeutic approaches for neurodegeneration, such as Alzheimer's disease (AD), have been widely studied. One of the critical hallmarks of AD is accumulation of amyloid beta (Aβ). Aβ induces neurotoxicity and releases inflammatory mediators or cytokines through activation of glial cell, and these pathological features are observed in AD patient's brain. The purpose of this study is to investigate the protective effect of alpha-linolenic acid (ALA) on Aβ25-35-induced neurotoxicity in C6 glial cells. Exposure of C6 glial cells to 50 μM Aβ25-35 caused cell death, overproduction of nitric oxide (NO), and pro-inflammatory cytokines release [interleukin (IL)-6 and tumor necrosis factor-α], while treatment of ALA increased cell viability and markedly attenuated Aβ25-35-induced excessive production of NO and those inflammatory cytokines. Inhibitory effect of ALA on generation of NO and cytokines was mediated by down-regulation of inducible nitric oxide synthase and cyclooxygenase-2 protein and mRNA expressions. In addition, ALA treatment inhibited reactive oxygen species generation induced by Aβ25-35 through the enhancement of the nuclear factor-erythroid 2-related factor-2 (Nrf-2) protein levels and subsequent induction of heme-oxygenase-1 (HO-1) expression in C6 glial cells dose- and time-dependently. Furthermore, the levels of neprilysin and insulin-degrading enzyme protein expressions, which contribute to degradation of Aβ, were also increased by treatment of ALA compared to Aβ25-35-treated control group. In conclusion, effects of ALA on Aβ degradation were shown to be mediated through inhibition of inflammatory responses and activation of antioxidative system, Nrf-2/HO-1 signaling pathway, in C6 glial cells. Our findings suggest that ALA might have the potential for therapeutics of AD.

Effects of dietary arginine on antioxidant status and immunity involved in AMPK-NO signaling pathway in juvenile blunt snout bream

The present study assessed the effects of dietary arginine on antioxidant status and immunity involved in AMPK-NO signaling pathway in juvenile blunt snout bream. Fish were fed six practical diets with graded arginine levels ranging from 0.87% to 2.70% for 8 weeks. The results showed that compared with the control group (0.87% dietary arginine level), significantly higher mRNA levels of adenosine monophosphate activated protein kinase (AMPK) and nitric oxide synthetase (NOS), activities of total nitric oxide synthetase (T-NOS) and nitric oxide synthetase (iNOS), and plasma nitric oxide (NO) contents were observed in fish fed with 1.62%–2.70% dietary arginine levels. Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%–2.70% dietary arginine levels in enzyme-linked immune sorbent assay. At dietary arginine levels of 1.22%–2.70%, the mRNA levels of iNOS were significantly improved. Dietary arginine also significantly influenced plasma interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α) contents. Furthermore, dietary arginine significantly affected the activity and mRNA level of glutathione peroxidase (GPx), the mRNA levels of pro-inflammatory factor including IL-8 and TNF-α and plasma malondialdehyde (MDA) content. However, total superoxide dismutase (T-SOD) activity, plasma complement component 3 (C3) content, plasma immunoglobulin M (IgM) content, plasma interleukin 1β (IL-1β) content and the mRNA levels of copperzinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD) and IL-1β were not significantly affected by dietary arginine. After Aeromonas hydrophila challenge, the death rate was significantly lowered in fish fed with 1.62%–1.96% dietary arginine levels. Furthermore, the mRNA levels of AMPK, NOS and iNOS, plasma NO content and the activities of T-NOS and iNOS showed an upward trend with increasing dietary arginine levels. Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%–2.70% dietary arginine levels in enzyme-linked immune sorbent assay. At dietary arginine levels of 1.96%–2.31%, T-SOD activities were significantly improved. Significantly higher GPx activities were observed in fish fed with 1.22%–2.70% dietary arginine levels. At dietary arginine levels of 1.22%–2.31%, the plasma TNF-α and IL-8 contents were significantly decreased. Significantly lower plasma IL-1β contents were observed in fish fed 1.62%–1.96% dietary arginine levels. Dietary arginine significantly influenced the mRNA levels of antioxidant and pro-inflammatory genes including Cu/Zn-SOD, Mn-SOD, GPx, IL-8, TNF-α and IL-1β. Significantly higher plasma C3 contents and significantly lower plasma MDA contents were observed in fish fed with 1.62%–1.96% arginine levels. Furthermore, plasma IgM contents were significantly improved at dietary arginine levels of 1.62%–2.31%. However, high dietary arginine group (2.70%) significantly improved the mRNA levels of pro-inflammatory genes including IL-8, TNF-α and IL-1β and plasma MDA, IL-8, TNF-α and IL-1β contents as compared with optimal dietary arginine levels (1.62% and 1.96%). The present results indicate that optimal arginine level (1.62% and 1.96%) could improve antioxidant capacity, immune response and weaken tissues inflammatory involved in arginine-AMPK-NO signaling pathway, while high arginine level resulted in excessive NO production, leading to increase oxidative stress damage and inflammatory response in juvenile blunt snout bream.

Prenylated Polyphenols from Broussonetia kazinoki as Inhibitors of Nitric Oxide Production.

Excessive nitric oxide (NO) production by macrophages has been involved in inflammatory diseases. Seven polyphenols (1–7) were isolated from Broussonetia kazinoki (B. kazinoki) and investigated as potential inhibitors of NO overproduction in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Among them, four prenylated polyphenols (2–4 and 6) with a catechol moiety efficiently suppressed the LPS-induced high level of NO with IC50 values of less than 6 µM. The compounds 2–4 and 6 also attenuated protein and mRNA levels of inducible nitric oxide synthase (iNOS). Moreover, they suppressed the nuclear factor κB (NF-κB) activity by inhibiting the degradation of inhibitory-κB-α (I-κB-α) and the translocation of NF-κB into the nucleus in LPS-activated macrophages. Taken together, these findings suggest that polyphenols from B. kazinoki might be beneficial for treatment of inflammatory diseases.

Resveratrol protects against sodium nitroprusside induced nucleus pulposus cell apoptosis by scavenging ROS

Oxidative stress induced disc cell apoptosis plays an important role in intervertebral disc (IVD) degeneration. The present study aims to investigate effects of resveratrol (RV), a natural polyphenol compound, on sodium nitroprusside (SNP) induced nucleus pulposus (NP) cell apoptosis and related mechanism. Rat NP cells were pretreated with RV, N-acetyl cysteine (NAC) and carboxy-PTIO (PTIO) before SNP treatment. Cell Counting Kit-8 assay was carried out for cell viability evaluation. Annexin V/propidium iodide (PI), Hoechst 33258 and Actin-Tracker Green and Tubulin-Tracker Red staining were conducted to detect NP cell apoptosis and apoptotic structural changes. Mitochondrial membrane potential (ΔΨm) was analyzed with tetramethylrhodamine methyl ester staining. DCFH-DA and DAF-FM DA staining was used to determine intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels. An ex vivo experiment was also carried out followed by TUNEL assay of sections of discs. SNP induced NP cell apoptosis, excessive production of intracellular ROS and NO, reduction of ΔΨm as well as disruption of cytoskeletal and morphological structure. Meanwhile, organ culture results showed that SNP induced NP cell apoptosis ex vivo. RV and NAC siginificantly inhibited SNP induced NP cell apoptosis, production of intracellular ROS, deline of ΔΨm as well as disruption of cytoskeletal and morphological structure, while RV did not suppress NO production. RV and NAC could also suppress SNP induced NP cell apoptosis ex vivo. However, PTIO did not prevent SNP induced NP cell apoptosis, though it scavenged NO significantly. In conclusion, RV protects against SNP induced NP cell apoptosis by scavenging ROS but not NO, suggesting a promising prospect of RV in IVD degeneration retardation.

Green Soybean Extract Ameliorates Dextran Sodium Sulfate-Induced Colitis

Green-mature soybean ( Glycine max) extract (GSE) inhibits the progression of immunoglobulin E-mediated inflammation more than the ordinary yellow-pigmented soybean extract (YSE). The inhibition of nitric oxide (NO) production by GSE in lipopolysaccharide-stimulated RAW264.7 cells has been found to be lower than that by YSE. However, this inhibitory ability became markedly pronounced by visible light irradiation. We investigated the anti-inflammatory effects of GSE and visible light-irradiated GSE (L-GSE) on dextran sodium sulfate (DSS)-induced colitis, immunoglobulin E-independent inflammation, and assessed whether light irradiation affects their anti-inflammatory properties. The severity of DSS-induced colitis was moderated by the ingestion of any of the soybean extracts, and L-GSE was the most effective in moderating the progression in colitis. GSE and L-GSE, except for YSE, inhibited the mRNA expressions of pro-inflammatory cytokines and chemokine in mice colons. All soybean extracts suppressed the mRNA expression of inducible nitric oxide synthase (iNOS), whereas GSE and L-GSE also inhibited the expression of the iNOS protein in the early phase of colitis. These results suggest that GSE is likely to suppress NO production, unlike in vitro results. Unfortunately, a significant difference in the anti-inflammation ability of GSE and L-GSE was not observed in the DSS-induced colitis mice. However, GSE and L-GSE are likely to become new candidate agents for the inhibition of inflammation and excessive NO production.

Crocin Attenuates Oxidative Stress and Myocardial Infarction Injury in Rats.

Oxidative stress and excessive nitric oxide (NO) production play considerable roles in infarction-induced injury impairing cardiac functions. Crocin is the active constituent of Crocus sativus (saffron) with antioxidant properties and has protective effects against disturbances induced by ischemia in many organs. The present study aimed to investigate the protective effects and the underlying mechanisms of crocin on myocardial infarction (MI)-induced injury in rats in vivo. MI rats were intraperitoneally injected with crocin at different doses for seven successive days after coronary ligation. Infarct size, hemodynamic studies, and capillary density were evaluated. Levels of oxidative stress, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and their corresponding phosphorylation expressions were then measured. Crocin decreased infarct size, left ventricular (LV) end-diastolic pressure, and LV minimum dP/dt while increased LV maximum dP/dt and percentage of LV fractional shortening dose dependently. Capillary density was markedly increased after crocin treatment. Crocin enhanced superoxide dismutase activity and reduced malondialdehyde levels as well as inhibited excessive production of NO through downregulating iNOS as well as upregulating eNOS during MI-induced injury. This study reveals that crocin improves MI-induced impairments in cardiac function, which is associated with its antioxidant properties.

Hypoxic Signaling and Cholesterol Lipotoxicity in Fatty Liver Disease Progression.

Cholesterol is the only lipid whose absorption in the gastrointestinal tract is limited by gate-keeping transporters and efflux mechanisms, preventing its rapid absorption and accumulation in the liver and blood vessels. In this review, I explored the current data regarding cholesterol accumulation in liver cells and key mechanisms in cholesterol-induced fatty liver disease associated with the activation of deleterious hypoxic and nitric oxide signal transduction pathways. Although nonalcoholic fatty liver disease (NAFLD) affects both obese and nonobese individuals, the mechanism of NAFLD progression in lean individuals with healthy metabolism is puzzling. Lean NAFLD individuals exhibit normal metabolic responses, implying that liver damage is not associated with impaired metabolism per se and that direct lipotoxic effects are crucial for disease progression. Several redox and oxidant signaling pathways involving cholesterol are at play in fatty liver disease development. These include impairment of the mitochondrial and lysosomal function by cholesterol loading of the inner-cell membranes; formation of cholesterol crystals and hepatocyte degradation; and crown-like structures surrounding degrading hepatocytes, activating Kupffer cells, and evoking inflammation. The current review focuses on the induction of liver inflammation, fibrosis, and steatosis by free cholesterol via the hypoxia-inducible factor 1α (HIF-1α), a main oxygen-sensing transcription factor involved in all stages of NAFLD. Cholesterol loading in hepatocytes can result in chronic HIF-1α activity because of the decreased oxygen availability and excessive production of nitric oxide and mitochondrial reactive oxygen species.

The effect of capsicum oleoresin on nitric oxide production and nitric oxide synthase gene expression in macrophage cell line

Background: Nitric oxide (NO) is an inflammatory agent produced by macrophages. It also acts as a neurotransmitter. However, overproduction of NO results in acute or chronic inflammation. Capsicum is well known for its anti-oxidant, anti–inflammatory, and anticancer properties. Objective: The objective of this study was to evaluate the effect of capsicum oleoresin on NO production and NO synthase gene expression in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophage cell line. Materials and Methods: Macrophage RAW 264.7 cells were obtained from the National Centre for Cell Science, Pune with Passage no 16. RAW macrophages were treated with 12.5 μg/ml, 25 μg/ml, and 50 μg/ml of Capsicum extract with 1 μg/ml of LPS and incubated for 24 h. Results: When capsicum was added at three different doses of 12.5 μg/ml, 25 μg/ml, and 50 μg/ml, the inducible NO synthase (iNOS) levels was significantly suppressed, compared to that of LPS treatment only. The level of NO increased by LPS induction was significantly decreased in a dose-dependent manner when treated with different concentrations of capsicum extract and capsicum had a suppressing effect on iNOS gene expression in LPS – stimulated RAW 264.7 macrophage. Conclusion: This study concludes that capsicum oleoresin is good enough to suppress iNOS gene expression and NO production. Hence, it may be used in inflammatory conditions with excessive NO production. Abbreviations Used: NO: Nitric oxide, LPS: Lipopolysaccharide, iNOS: Inducible nitric oxide synthase, DMEM: Dulbecco's modified Eagle medium, MTT: 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide for, DMSO: Dimethyl sulfoxide, PBS: Phosphate buffer saline, FBS: Fetal bovine serum.

Effects of Ghrelin on iNOS-Derived NO Promoted LPS-Induced Pulmonary Alveolar Epithelial A549 Cells Apoptosis

Background/Aims: In the process of abnormal apoptosis of pulmonary alveolar type II epithelial A549 cells in acute respiratory distress syndrome (ARDS), inducible nitric oxide synthase (iNOS) activity in the lung, nitric oxide (NO) production, and the level of protein S-nitrosylation were increased. However, the role of excessive NO production in sepsis-induced ARDS is controversial. Additionally, ghrelin is a growth hormone that exerts an inhibitory role in cell apoptosis. We examined the effect of NO and S-nitrosylation on apoptosis of A549 cells induced by Lipopolysaccharide (LPS) and molecular mechanism underlying the anti-apoptotic effect of ghrelin in this process. Methods: Flow cytometry and qPCR were used to detect lentiviral infection efficiency and iNOS gene level, respectively. Extracellular and intracellular NO levels were observed by Griess assay kit and DAF-FM DA. Mitochondrial transmembrane potential, apoptosis rate and SNO levels were determined by flow cytometry, Biotin-Switch method and immunofluoresence staining. The expression of iNOS, apoptotic proteins and JNK were assessed by immunoblot analysis. Results: The results showed about two times increase in iNOS expression and intracellular NO levels response to LPS exposure at 24 hours (P< 0.05), while not in extracellular NO levels. NO donors, S-nitroso-N-acetylpenicillamine (SNAP) significantly raised (36.7%, P< 0.05; 38.4%, P< 0.05; 41.8%, P< 0.05) extracellular NO levels without influencing the intracellular NO levels. LPS increased the apoptosis rate (42.4%±2.6% vs 2.8%±1%, P< 0.05) of A549 accompanied by increased Bax levels and decreased Bcl-2 levels through activating JNK signaling, which was reversed when we diminished the iNOS expression in A549 cells using lentiviral vectors encoding iNOS shRNA in the presence of LPS (24.8%±3.8% vs 42.4%±2.6%, P< 0.05). However, the apoptosis rate was increased when SNAP was added (38.8%±1.3% vs 24.8%±3.8%, P< 0.05). Furthermore, we investigated whether ghrelin exert a protective role against LPS-induced apoptosis and the potential mechanism involved in. Ghrelin alone appeared to decrease iNOS expression (32.3%, P< 0.05; 42.3%, P< 0.05), which showed no signifiant difference between LPS+ghrelin group and LPS group. However, this study showed that ghrelin decreased the intracellular NO production (38.9%, P< 0.05), protein S-nitrosylation levels (33.5%, P< 0.05), Bax protein expression (70.2%, P< 0.05), whereas increasing Bcl-2 protein expression (14.1%, P< 0.05) and mitochondrial transmembrane potential (∆ΨM) (20.7%, P< 0.05) in the presence of LPS. Conclusion: The data suggested that NO derived from iNOS induced by LPS stimulation exerts an important role in promoting apoptosis of A549 cells, and ghrelin abolished intracellular NO production and protein S-nitrosylation levels, abrogating the apoptosis of A549 cells partly through inhibiting mitochondrial-dependent pathways.