Inhibition of inducible nitric oxide synthase reduces lipopolysaccharide-induced renal injury in the rat

1. Gram-negative bacterial lipopolysaccharide (LPS) release and subsequent septic shock is a major cause of death in intensive care units. Lipopolysaccharide has been reported to increase the production of nitric oxide (NO) and the formation of oxygen-derived free radicals (OFR) in different organs. The aim of the present study was to evaluate the role of an inducible form of NO synthase (iNOS) and OFR production in LPS-induced renal impairment. 2. Measurement of vitamin E as the most important fat-soluble anti-oxidant was used as a marker of tissue oxidative stress. Lipopolysaccharide (10 mg/kg), L-iminoethyl lysine (L-Nil; 3 mg/kg, i.p.; a specific inhibitor of iNOS activity) and dimethyl thiourea (DMTU; 500 mg/kg i.p.; a well-known OFR scavenger) were used. Four groups of eight rats were studied. One group received LPS, whereas a second group received LPS + L-Nil. A third group received LPS + DMTU and the fourth group, receiving saline, acted as a control group. To evaluate renal function, plasma creatinine and blood urea nitrogen (BUN) were measured. High-pressure liquid chromatography and ultraviolet detection were used to measure plasma and tissue vitamin E levels. Light microscopy was used to examine histopathological changes in the four groups. 3. Lipopolysaccharide markedly decreased the vitamin E content of renal plasma and tissue (P < 0.05). Administration of L-Nil attenuated renal dysfunction and preserved vitamin E levels. However, DMTU failed to prevent renal injury, as indicated by plasma BUN levels and renal histology, despite the fact that it maintained renal vitamin E levels and increased plasma vitamin E levels. Thus, the overproduction of NO by iNOS may have a role in this model of LPS-induced renal impairment.     

The lung is the major site that produces nitric oxide to induce acute pulmonary oedema in endotoxin shock

1. The present study was undertaken to determine the locus of nitric oxide (NO) production that is toxic to the lung and produces acute pulmonary oedema in endotoxin shock, to examine and compare the effects of changes in lung perfusate on endotoxin-induced pulmonary oedema (EPE) and to evaluate the involvement of constitutive and inducible NO synthase (cNOS and iNOS, respectively). 2. Experiments were designed to induce septic shock in anaesthetized rats with the administration of Escherichia coli lipopolysaccharide (LPS). Exhaled NO, lung weight (LW)/bodyweight (BW) ratio, LW gain (LWG) and lung histology were measured and observed to determine the degree of EPE 4 h following LPS. The EPE was compared between groups in which LPS had been injected either into the systemic circulation or into the isolated perfused lung. The lung perfusate was altered from whole blood to physiological saline solution (PSS) with 6% albumin to test whether different lung perfusions affected EPE. Pretreatment with various NOS inhibitors was undertaken 10 min before LPS to investigate the contribution of cNOS and iNOS to the observed effects. 3. Endotoxin caused profound systemic hypotension, but little change in pulmonary arterial pressure. The extent of EPE was not different between that induced by systemic injection and that following administration to isolated lungs preparations. Replacement of whole blood with PSS greatly attenuated (P < 0.05) EPE. In blood-perfused lungs, pretreatment with NOS inhibitors, such as Nomega-nitro-L-arginine methyl ester, aminoguanidine and dexamethasone, significantly prevented EPE (P < 0.05). 4. The major site of NO production through the whole blood is in the lung. The NO production mediated by the iNOS system is toxic to the endothelium in the pulmonary microvasculature. Inhalation of NO for patients with sepsis may be used with clinical caution. Therapeutic consideration of lung extracorporeal perfusion with PSS and pharmacological pretreatment with iNOS inhibitors may be warranted.     

Pharmacological profile of FR260330, a novel orally active inducible nitric oxide synthase inhibitor

In this study, we examined effects of a newly synthesized chemical compound, FR260330, (2E)-3-(4-chlorophenyl)-N-[(1S)-2-oxo-2-{[2-oxo-2-(4-{[6-(trifluoromethyl)-4-pyrimidinyl]oxy}-1-piperidinyl)ethyl]amino}-1-(2-pyridinylmethyl)ethyl]acrylamide on nitric oxide (NO) production in rat splenocytes and human colon cancer cell line, DLD-1 cells. FR260330 inhibited NOx production dose dependently in both cells. In lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) treated murine macrophage cell line, RAW264.7, Western blot analysis with gel filtration chromatography revealed FR260330 might prevent dimerization of inducible nitric oxide synthase (iNOS), but had no effect on the expression of iNOS protein. Furthermore, oral administration of FR260330 reduced NOx production dose dependently in plasma from rats exposed to LPS (IC50=1.6 mg/kg). Meanwhile, higher dose (100 mg/kg) of oral administration of FR260330 did not change mean arterial blood pressure in rats. These results suggest that FR260330 might be a useful therapeutical approach to various inflammatory diseases, in which superoxide or peroxynitrite formed from iNOS-derived NO are involved.     

Self-administration of heroin produces alterations in the expression of inducible nitric oxide synthase

Nitric oxide plays a critical role in the immune response, and our studies have shown that heroin induces a reduction in the expression of iNOS, the enzyme responsible for nitric oxide production. The present study evaluated the effect of heroin self-administration on iNOS expression using a three-group design. Group one (self-administration) was trained to press a lever for i.v. administration of heroin. Group two (yoked heroin) received a simultaneous equivalent infusion of heroin determined by the responses of a 'partner' animal in the first group. A third group (yoked saline) also was yoked to the first group, but received i.v. injections of saline. Immediately following the last session, all rats received an injection of lipopolysaccharide (LPS) to induce iNOS expression. About 6 h after the injection of LPS, iNOS mRNA and protein expression were determined in spleen, lung, and liver. Additionally, the accumulation of plasma nitrite/nitrate, the more stable end products of nitric oxide degradation were measured. Although there was not a consistent difference between the self-administering and yoked-heroin animals, the results show that rats will self-administer a sufficient amount of heroin to induce a pronounced, widespread reduction in the expression of iNOS.     

Manganese potentiates in vitro production of proinflammatory cytokines and nitric oxide by microglia through a nuclear factor kappa B-dependent mechanism.

Recent evidence suggests that the mechanism of manganese (Mn) neurotoxicity involves activation of microglia and/or astrocytes; as a consequence, neurons adjacent to the activated microglia may be injured. Mn modulation of proinflammatory cytokine expression by microglia has not been investigated. Therefore, the objectives of this research were to (1) assess whether Mn induces proinflammatory cytokine expression and/or modulates lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines and (2) investigate possible mechanisms for such an induction. N9 microglia were exposed in vitro to increasing concentrations (50-1000 microM) of Mn in the presence or absence of LPS (10, 100, or 500 ng/ml). After various incubation times (up to 48 h), media levels of several cytokines and nitric oxide (NO) were determined, as was the expression of the inducible form of NO synthase (iNOS). Lactate dehydrogenase (LDH) release into the medium and the cellular uptake of Neutral Red were used as general measures for cytotoxicity. In the absence of LPS, Mn moderately increased interleukin-6 and tumor necrosis factor alpha (TNF-a) production only at higher Mn concentrations, which were cytotoxic. At all LPS doses, however, proinflammatory cytokine production was dose-dependently increased by Mn. Similarly, LPS-induced NO production and iNOS expression were substantially enhanced by Mn. Pharmacological manipulations indicated that nuclear factor kappa B (NFkappaB) activation is critical for the observed enhancement of cytokine and NO production. Within the context of inflammation, increased production of proinflammatory cytokines and NO by Mn could be an important part of the mechanism by which Mn exerts its neurotoxicity.     

N-acetylcysteine abrogates acute lung injury induced by endotoxin

1. Acute lung injury (ALI) or acute respiratory distress syndrome is a serious clinical problem with high mortality. N-Acetylcysteine (NAC) is an anti-oxidant and a free radical scavenger. It has been reported recently that NAC ameliorates organ damage induced by endotoxin (lipopolysaccharide; LPS) in conscious rats. The present study was designed to evaluate the effects of NAC on LPS-induced ALI and other changes in anaesthetized rats. 2. Sprague-Dawley rats were anaesthetized with pentobarbital (40 mg/kg, i.p.). Endotracheal intubation was performed to provide artificial ventilation. Arterial pressure and heart rate were monitored. The extent of ALI was evaluated with the lung weight (LW)/bodyweight ratio, LW gain, exhaled nitric oxide (NO) and protein concentration in bronchoalveolar lavage (PCBAL). Haematocrit, white blood cells, plasma nitrate/nitrite, methyl guanidine (MG), tumour necrosis factor (TNF)-alpha and interleukin (IL)-1b were measured. Pathological changes in the lung were examined and evaluated. 3. Endotoxaemia was produced by injection of 10 mg/kg, i.v., LPS (Escherichia coli). Animals were randomly divided into three groups. In the vehicle group, rats received an i.v. drip of physiological saline solution (PSS) at a rate of 0.3 mL/h. The LPS group received an i.v. drip of PSS for 1 h, followed by LPS (10 mg/kg by slow blous injection, i.v., over 1-2 min). Rats in the LPS + NAC group received NAC by i.v. drip at a rate of 150 mg/kg per h (0.3 mL/h) for 60 min starting 10 min before LPS administration (10 mg/kg by slow blous injection, i.v., over 1-2 min). Each group was observed for a period of 6 h. 4. N-Acetylcysteine treatment improved the LPS-induced hypotension and leukocytopenia. It also reduced the extent of ALI, as evidenced by reductions in LW changes, exhaled NO, PCBAL and lung pathology. In addition, NAC diminished the LPS-induced increases in nitrate/nitrite, MG, TNF-a and IL-1b. 5. In another series of experiments, LPS increased the mortality rate compared with the vehicle group (i.v. drip of PSS at a rate of 0.3 mL/h) during a 6 h observation period. N-Acetylcysteine, given 10 min prior to LPS, significantly increased the survival rate. 6. The results of the present study suggest that NAC exerts a protective effect on the LPS-induced ALI. The mechanisms of action may be mediated through the reduction of the production of NO, free radicals and pro-inflammatory cytokines.     

S-nitroso human serum albumin given after LPS challenge reduces acute lung injury and prolongs survival in a rat model of endotoxemia

Endotoxemia leads to the induction of inducible nitric oxide synthase (NOS-2) and increased expression of numerous inflammatory mediators contributing to endotoxin-induced acute lung injury. We tested the hypothesis that supplementation of nitric oxide (NO) by the novel NO donor S-nitroso human serum albumin (S-NO-HSA) given after lipopolysaccharide (LPS) challenge may reduce NOS-2 expression, lung inflammation and acute lung injury. Rats were divided into four groups: sham-operated (no treatment), LPS, LPS+HSA (human serum albumin), and LPS+S-NO-HSA. LPS was administered intravenously (20 mg kg(-1)) resulting in acute lung injury and a high mortality rate within 6 h (>90%). LPS-induced lung injury was characterized by an increased lung edema (lung wet/dry weight ratio), pulmonary neutrophil infiltration (myeloperoxidase activity, MPO) as well as a robust inflammatory response [increased expression of intercellular adhesion molecule-1 (ICAM-1), NOS-2, and cyclooxygenase-2 (COX-2)]. Infusion of S-NO-HSA or HSA was started 2 h after LPS and continued for 4 h (total dose of 72 mg kg(-1)) at a rate of 300 mug kg(-1) min(-1). S-NO-HSA but not HSA prolonged survival of endotoxemic rats, reduced the hypotensive response to LPS, minimized LPS-induced lung edema and injury, normalized MPO activity as well as diminished lung expression of pro-inflammatory molecules such as ICAM-1, NOS-2, and COX-2. Continuous supplementation of NO by S-NO-HSA after LPS challenge prevents induction of NOS-2, provides significant protection of endotoxin-induced acute lung injury, and prevents early mortality in endotoxic shock in rats. Our results suggest a potential therapeutic role for S-NO-HSA in endotoxemia.     

Role of inducible nitric oxide synthase-derived nitric oxide in lipopolysaccharide plus interferon-gamma-induced pulmonary inflammation

Exposure of mice to lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) increases nitric oxide (NO) production, which is proposed to play a role in the resulting pulmonary damage and inflammation. To determine the role of inducible nitric oxide synthase (iNOS)-induced NO in this lung reaction, the responses of inducible nitric oxide synthase knockout (iNOS KO) versus C57BL/6J wild-type (WT) mice to aspirated LPS + IFN-gamma were compared. Male mice (8-10 weeks) were exposed to LPS (1.2 mg/kg) + IFN-gamma (5000 U/mouse) or saline. At 24 or 72 h postexposure, lungs were lavaged with saline and the acellular fluid from the first bronchoalveolar lavage (BAL) was analyzed for total antioxidant capacity (TAC), lactate dehydrogenase (LDH) activity, albumin, tumor necrosis factor-alpha (TNF-alpha), and macrophage inflammatory protein-2 (MIP-2). The cellular fraction of the total BAL was used to determine alveolar macrophage (AM) and polymorphonuclear leukocyte (PMN) counts, and AM zymosan-stimulated chemiluminescence (AM-CL). Pulmonary responses 24 h postexposure to LPS + IFN-gamma were characterized by significantly decreased TAC, increased BAL AMs and PMNs, LDH, albumin, TNF-alpha, and MIP-2, and enhanced AM-CL to the same extent in both WT and iNOS KO mice. Responses 72 h postexposure were similar; however, significant differences were found between WT and iNOS KO mice. iNOS KO mice demonstrated a greater decline in total antioxidant capacity, greater BAL PMNs, LDH, albumin, TNF-alpha, and MIP-2, and an enhanced AM-CL compared to the WT. These data suggest that the role of iNOS-derived NO in the pulmonary response to LPS + IFN-gamma is anti-inflammatory, and this becomes evident over time.     

Endotoxin-induced acute lung injury and organ dysfunction are attenuated by pentobarbital anaesthesia

1. Acute lung injury (ALI) as a result of sepsis is a major cause of mortality. Certain anaesthetic agents have been reported to suppress pro-inflammatory cytokines and inducible nitric oxide (NO) synthase (iNOS) activities. We investigated the effects of pentobarbital on ALI and organ functions after the administration of endotoxin. 2. Intravenous (i.v.) pentobarbital (20 or 40 mg/kg) was administered 5 min after lipopolysaccharide (LPS; 10 or 30 mg/kg via i.v. infusion). To avoid hypoxia and/or hypercapnia following anaesthesia, we installed a special chamber connected to a rodent ventilator to provide ventilation with 95% oxygen content and 5% nitrogen. The animal was kept at eucapnic conditions (arterial PCO2 at an average of 38 +/- 2 mmHg). 3. We monitored the arterial pressure (AP) and heart rate (HR). Acute lung injury was evaluated by lung weight changes, protein concentration in bronchoalveolar lavage, and Evans blue leakage. Plasma nitrate/nitrite, methyl guanidine and biochemical factors were determined. Pathological and immunofluorescent examinations were performed to observe the lung changes and to determine the activities of pro-inflammatory cytokines, nitrotyrosine and iNOS. 4. Lipopolysaccharide caused dose-dependent systemic hypotension with an increase in the extent of ALI. The lung pathology included oedema and inflammatory cell infiltration. Accompanying the ALI, LPS elevated plasma nitrate/nitrite, methyl guanidine, blood urea nitrogen, lactic dehydrogenase, creatinine phosphokinase, glutamic transaminase and amylase. The lung tissue content of tumour necrosis factor-alpha, interleukin-lbeta, iNOS and nitrotyrosine was increased following LPS administration. These changes were abrogated by pentobarbital anaesthesia. 5. Our results indicated that pentobarbital anaesthesia significantly augmented the LPS-induced systemic hypotension. However, it attenuated the LPS-induced ALI and organ dysfunctions. This agent also improved the survival rate following LPS at high and low doses. This mechanism may be related to the inhibitory effects on the increases in the production or activity of NO, free radicals, pro-inflammatory cytokines, nitrotyrosine and iNOS.     

塞来昔布对急性肺损伤大鼠肺组织NF-κB和iNOS的影响

【摘要】目的探讨脂多糖（LPS）所致大鼠急性肺损伤时环氧化酶-2（COX-2）抑制剂塞来昔布对肺组织的核因子-κB（NF-κB）p65和诱导型一氧化氮合酶（iNOS）表达的影响。方法将60只大鼠随机分为对照组、LPS组、治疗组和塞来昔布组,每组15只。LPS组尾静脉注射LPS（5mg/kg）复制急性肺损伤模型;治疗组注射LPS复制急性肺损伤模型后30min用塞来昔布灌胃（20mg/kg）;塞来昔布组不造模,于相同时间点用相同剂量塞来昔布灌胃;对照组不造模、不给药,给等量的生理盐水;各组均于3h后放血处死动物。采用蛋白质印迹（Western blot）和逆转录-聚合酶链反应（RT-PCR）方法分别检测肺组织COX-2、NF-κB p65、iNOS蛋白及NF-κB p65、iNOS mRNA的表达。结果LPS组与对照组相比COX-2、NF-κB p65和iNOS蛋白及NF-κB p65、iNOS mRNA表达均显著升高（P<0.01）;治疗组NF-κB p65、iNOS mRNA和蛋白表达较LPS组明显降低（P<0.01）。结论选择性COX-2抑制剂塞来昔布对急性肺损伤大鼠有保护作用。     

瑞芬太尼对脂多糖诱导的急性肺损伤大鼠肺脏Toll样受体4的影响

目的研究瑞芬太尼对LPS诱导的急性肺损伤大鼠肺组织的影响。方法24只Wistar大鼠随机分为3组(n=8):对照组(C组),肺损伤组(LPS组)和瑞芬太尼组(R组)。分别于气管内给药前、给药1h、3h和5h记录平均动脉压和心率,行动脉血气分析。注药、机械通气5h后测定TLR4mRNA和蛋白表达,观察肺组织病理学及支气管肺泡灌洗液(BALF)中肿瘤坏死因子(TNF-α)的表达。结果与C组比较,LPS组及R组TLR4mRNA表达和蛋白表达明显增加,BALF中TNF-α增加。与LPS组比较,R组TLR4mRNA表达增高,BALF中TNF-α增加。肺组织损伤程度从轻至重分别为C组、LPS组、R组。结论瑞芬太尼能加重LPS诱导的ALI大鼠肺组织的损伤,其作用机制与上调TLR4表达有关。     

The inhibition of inducible nitric oxide synthase and oxidative stress by agmatine attenuates vascular dysfunction in rat acute endotoxemic model

Vascular dysfunction leading to hypotension is a major complication in patients with septic shock. Inducible nitric oxide synthase (iNOS) together with oxidative stress play an important role in development of vascular dysfunction in sepsis. Searching for an endogenous, safe and yet effective remedy was the chief goal for this study. The current study investigated the effect of agmatine (AGM), an endogenous metabolite of l-arginine, on sepsis-induced vascular dysfunction induced by lipopolysaccharides (LPS) in rats. AGM pretreatment (10 mg/kg, i.v.) 1 h before LPS (5 mg/kg, i.v.) prevented the LPS-induced mortality and elevations in serum creatine kinase-MB isoenzyme (CK-MB) activity, lactate dehydrogenase (LDH) activity, C-reactive protein (CRP) level and total nitrite/nitrate (NOx) level after 24 h from LPS injection. The elevation in aortic lipid peroxidation illustrated by increased malondialdehyde (MDA) content and the decrease in aortic glutathione (GSH) and superoxide dismutase (SOD) were also ameliorated by AGM. Additionally, AGM prevented LPS-induced elevation in mRNA expression of iNOS, while endothelial NOS (eNOS) mRNA was not affected. Furthermore AGM prevented the impaired aortic contraction to KCl and phenylephrine (PE) and endothelium-dependent relaxation to acetylcholine (ACh) without affecting endothelium-independent relaxation to sodium nitroprusside (SNP). In conclusion: AGM may represent a potential endogenous therapeutic candidate for sepsis-induced vascular dysfunction through its inhibiting effect on iNOS expression and oxidative stress.     

Effect of Apremilast on LPS-induced immunomodulation and inflammation via activation of Nrf2/HO-1 pathways in rat lungs

Lipopolysaccharides (LPS), the lipid component of gram-negative bacterial cell wall, is recognized as the key factor in acute lung inflammation and is found to exhibit severe immunologic reactions. Phosphodiesterase-4 (PDE-4) inhibitor: “apremilast (AP)” is an immune suppressant and anti-inflammatory drug which introduced to treat psoriatic arthritis. The contemporary experiment designed to study the protective influences of AP against LPS induced lung injury in rodents. Twenty-four (24) male experimental Wistar rats selected, acclimatized, and administered with normal saline, LPS, or AP + LPS respectively from 1 to 4 groups. The lung tissues were evaluated for biochemical parameters (MPO), Enzyme Linked Immunosorbent Assay (ELISA), flowcytometry assay, gene expressions, proteins expression and histopathological examination. AP ameliorates the lung injuries by attenuating immunomodulation and inflammation. LPS exposure upregulated IL-6, TNF-α, and MPO while downregulating IL-4 which were restored in AP pretreated rats. The changes in immunomodulation markers by LPS were reduced by AP treatment. Furthermore, results from the qPCR analysis represented an upregulation in IL-1β, MPO, TNF-α, and p38 whereas downregulated in IL-10 and p53 gene expressions in disease control animals while AP pretreated rats exhibited significant reversal in these expressions. Western blot analysis suggested an upregulation of MCP-1, and NOS-2, whereas HO-1, and Nrf-2 expression were suppressed in LPS exposed animals, while pretreatment with AP showed down regulation in the expression MCP-1, NOS-2, and upregulation of HO-1, and Nrf-2 expression of the mentioned intracellular proteins. Histological studies further affirmed the toxic influences of LPS on the pulmonary tissues. It is concluded that, LPS exposure causes pulmonary toxicities via up regulation of oxidative stress, inflammatory cytokines and stimulation of IL-1β, MPO, TNF-α, p38, MCP-1, and NOS-2 while downregulation of IL-4, IL-10, p53, HO-1, and Nrf-2 at different expression level. Pretreatment with AP controlled the toxic influences of LPS by modulating these signaling pathways.     

The role of poly(ADP-ribose) synthetase inhibition in preventing endotoxemia-induced intestinal epithelial apoptosis.

In this lipopolysaccharide (LPS)-induced endotoxemia model, the effects of 3-aminobenzamide (3-AB), a poly(ADP-ribose) synthetase (PARS) inhibitor, on ileal apoptosis were evaluated by light microscopy and M30 cell death staining. Moreover, the relationship between Bcl-2, iNOS expression, and serum nitrate (NO(3)(-)) levels were investigated. Thirty-two male Wistar rats, weighing 180-220g were randomly divided into four groups. The group I (control; n=8) received saline and group II (sepsis; n=8) received 10 mg kg(-1) LPS intraperitoneally. 3-AB was given to the group IV (S+3-AB; n=8) 20 min before giving LPS and to the group III (C+3-AB; n=8) 20 min before giving saline. Six hours later, blood and ileum samples were taken. Endotoxemic group exhibited significant apoptosis in intestinal epithelial cells and the immunohistochemical examination with M30 was demonstrated that the 3-AB reduced the LPS-induced intestinal apoptosis. Serum NO(3)(-) level was increased in endotoxemic group, whereas the elevation of NO(3)(-) level was prevented in LPS+3-AB group (P<0.05). The increased iNOS expression observed in the LPS group was also prevented by 3-AB. Compared with the endotoxemic group, ileal epithelial columnar cells from LPS+3-AB group had a dense Bcl-2 staining which was almost identical with control. In conclusion, 3-AB decreases LPS-induced apoptosis in ileum by preventing LPS-induced depletion of Bcl-2 and blocking iNOS gene. Modification of Bcl-2 expression by PARS inhibitors should further be investigated as a new therapeutic alternatives in septic states.