苦参碱对四氯化碳所致小鼠慢性肝纤维化肝组织一氧化氮合酶表达的干预及意义

目的:研究苦参碱对四氯化碳(CCl4)所致的小鼠慢性肝纤维化肝组织诱生型一氧化氮合酶(iNOS)表达的干预及意义.方法:将健康昆明小鼠50只,随机分为4组:CCl4组(A组,n=15)、CCl4 苦参碱治疗组(B组,n=15)、生理盐水对照组(C组,n=10)、苦参碱对照组(D组,n=10).各组分别于造模4wk、8 wk后取材,常规HE染色法观察比较不同组别小鼠肝组织病理变化,免疫组化方法检测各组肝组织内iNOS的表达.结果:A组小鼠4wk肝组织出现脂肪变,随CCL4给药时间延长,肝组织损伤加重,第8周出现空泡变及点状坏死,多见于门管区,小叶结构紊乱.B组各时间点肝组织结果A组有明显改善、肝细胞坏死和脂肪变性程度显著减轻.C组、D组肝小叶结构完整,肝细胞结构清晰.免疫组化结果显示,A组、B组各时间点受损肝细胞均有iNOS表达,A组与B组iNOS表达分值相比差异有显著性(2.20±0.12 vs 1.20±0.02,P＜0.01;1.2±0.06 vs 0.60±0.02,P＜0.01).C组、D组肝细胞iNOS表达阴性.结论:iNOS参与CC14诱导的小鼠肝慢性纤维化损伤.苦参碱可通过干预受损肝细胞iNOS表达,有效降低CCl4所致肝脏损伤.     

Altered response to inflammatory cytokines in hepatic energy metabolism in inducible nitric oxide synthase knockout mice

BACKGROUND/AIMS: Production of nitric oxide (NO) in the liver is believed to be a critical factor for carbohydrate and energy metabolism in endotoxin shock. The present study focuses on the involvement of NO produced by inducible nitric oxide synthase (iNOS) in glycogen synthesis and energy metabolism stimulated by insulin.METHODS: Primary hepatocytes prepared from wild-type and iNOS knockout (iNOS(-/-)) mice were employed.RESULTS: Incubation of wild-type hepatocytes with a combination of cytokines (interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma) and lipopolysaccharide (cytokines/LPS) inhibited insulin-stimulated glycogen synthesis and adenosine triphosphate (ATP) increase, and decreased the ketone body ratio (KBR) at 8-12 h, concomitant with expression of iNOS protein and NO production. While the glycogen synthesis was suppressed by cytokines/LPS, reduction of the ATP increase and a decrease in KBR by cytokines/LPS were not observed in iNOS(-/-) hepatocytes. Further, N(G)-monomethyl-L-arginine, a NOS inhibitor, reversed the inhibition of ATP increase and decrease in KBR by cytokines/LPS, but not the inhibition of glycogen synthesis. Conversely, addition of S-nitroso-N-acetylpenicillamine, a NO donor, inhibited the insulin-stimulated ATP increase synthesis in iNOS(-/-) hepatocytes, but not the insulin-stimulated glycogen synthesis.CONCLUSIONS: These results demonstrate that NO mediates the suppression of insulin-stimulated energy metabolism, but not glycogen synthesis, in cytokines/LPS-treated hepatocytes.     

Overexpression of hepatic inducible nitric oxide synthase in biliary atresia

Aims: Biliary atresia (BA) is a rare and serious liver disease in infants characterized by progressive inflammatory cholangiopathy. The aims of this study were to investigate hepatic expression of inducible nitric oxide synthase (iNOS) in BA and to associate the iNOS expression with their early therapeutic outcome. Methods: Hepatic iNOS expression was determined using immunohistochemistry from liver biopsies of 24 BA patients, and 16 non‐BA patients whose liver tissues were needed in the treatment process. Six months after surgery, the BA patients were categorized into two groups;good and poor outcome. The iNOS expression of hepatocyte areas was evaluated based on its intensity using ImageJ software. Unpaired t‐tests were used for the comparisons of iNOS expression between groups. Results: Hepatic iNOS expression of BA patients was significantly stronger than that of non‐BA patients (P < 0.0001). The largest area of hepatic iNOS expression was the area of hepatocytes. Subgroup analysis of BA patients at 6 months post‐op revealed that there was no difference in iNOS expression between the patients with good outcome and those with poor outcome (P = 0.732). Conclusions: Overexpression of hepatic iNOS in BA patients was demonstrated. Within liver tissues, hepatocytes were the major source of hepatic iNOS production. However, the expression was not associated with the early therapeutic outcome. These results suggest that iNOS plays a role in the liver pathology of BA but its expression cannot be used as a predictor for therapeutic outcome.     

Temporal expression of hepatic inducible nitric oxide synthase in liver cirrhosis

AIM: Nitric oxide (NO) has been implicated in the pathogenesis of liver cirrhosis. We have found inducible nitric oxide synthase (iNOS) can be induced in hepatocytes of cirrhotic liver. This study further investigated the temporal expression and activity of hepatic iNOS in cirrhosis development. METHODS: Cirrhosis was induced in rats by chronic bile duet ligatjon (BDL). At different time points after the operation, samples were collected to examine NO concentration, liver function, and morphological changes. Hepatocytes were isolated for determination of iNOS mRNA, protein and enzymatic activity. RESULTS: Histological examination showed early cirrhosis 1-2 wk after BDL, with advanced cirrhosis at 3-4 wk. Bilirubin increased dramatically 3 d after BDL, but decreased by 47% on d 14. Three weeks after BDL, it elevated again. Systemic NO concentration did not increase significantly until 4 wk after BDL, when ascites developed. Hepatocyte iNOS mRNA expression was identified 3 d after BDL, and enhanced with time to 3 wk, but reduced thereafter. iNOS protein showed a similar pattern to mRNA expression. iNOS activity decreased from d 3 to d 7, but increased again thereafter till d 21. CONCLUSION: Hepatic iNOS can be induced in the early stage, which increases with time as cirrhosis develops. lts enzymatic activity is significantly correlated with protein expression and histological alterations of the liver, but not with systemic NO levels, nor with absolute values of liver function markers.     

Decreased neointimal thickening after arterial wall injury in inducible nitric oxide synthase knockout mice

Mechanical injury in vivo results in the expression of the inducible form of nitric oxide synthase (iNOS) in vascular smooth muscle cells. However, the role of iNOS in modulating neointima formation after arterial wall injury is not clear. To determine whether the induction of iNOS gene expression promotes or attenuates the neointimal response to injury, we used a murine model of perivascular injury induced by placing a periadventitial collar around the carotid arteries in both wild-type and iNOS knockout mice (iNOS-KO mice). Periadventitial injury induced iNOS expression in the wild-type but not the iNOS-KO mice. Neointimal area and the intima/media ratio were significantly less in the iNOS-KO mice compared with the wild-type mice at 21 days. Injury-induced proliferation of medial cells and vascular cell adhesion molecule-1 expression were also attenuated in iNOS-KO mice compared with wild-type mice. The induction of iNOS and the activation of the nuclear factor-kappaB-mediated pathway were also demonstrated in an in vitro injury model. We conclude that mechanical injury in vivo and in vitro induces iNOS expression and that lack of iNOS expression attenuates neointima formation after perivascular arterial injury. Taken together, these findings suggest that iNOS expression after vascular injury may promote neointima formation.     

Lack of inducible nitric oxide synthase does not prevent aging-associated insulin resistance

Inducible nitric oxide synthase (iNOS) is involved in obesity-induced insulin resistance. Since aging is accompanied by increased iNOS expression, the effect of iNOS gene deletion on aging-associated insulin resistance was investigated in 7-month-old (adult) and 22-month-old (old) iNOS knockout and wild-type mice using the hyperinsulinemic–euglycemic clamp. While body weight and fat mass were increased, muscle mass was reduced with aging in wild-type mice. However, body composition was not changed with aging in iNOS knockout mice due to increased locomotor activity. NO metabolites in plasma, and protein levels of iNOS and nitrotyrosine in skeletal muscle increased with aging in wild-type mice. Deletion of iNOS gene attenuated NO metabolites and nitrotyrosine with aging in iNOS knockout mice. Glucose uptake in whole body and skeletal muscle was reduced with aging in both wild-type and iNOS knockout mice and there was no difference between two groups. Plasma level of tumor necrosis factor-α and gene expression of proinflammatory cytokines in peripheral tissues were increased with aging in both groups, and that was more heightened in iNOS knockout mice. These results suggest that lack of iNOS does not prevent aging-associated insulin resistance in mice and heightened production of proinflammatory cytokines may be involved.     

Influence of leptin in the development of hepatic fibrosis produced in mice by Schistosoma mansoni infection and by chronic carbon tetrachloride administration

BACKGROUND/AIMS: Leptin, a product of the obese (ob) gene is present in activated stellate cells. This study investigated whether leptin is essential for the development of hepatic fibrosis caused by various agents. METHODS: Control and ob/ob mice were infected with Schistosoma mansoni or were administered chronic carbon tetrachloride to cause hepatic fibrosis. RESULTS: Fibrosis developed in both ob/ob and control mice. However, the amount of histologically detectable fibrosis and the increase in liver hydroxyproline content was significantly greater in both models of fibrosis for treated controls than for treated ob/ob mice. Fibrosis was associated with higher secretion of TGFbeta1 from spleen cells of treated control than treated ob/ob mice. Chronic leptin administration in ob/ob mice infected with Schistosoma mansoni resulted in an increase in the amount of fibrosis caused by Schistosoma mansoni, eliminating any significant differences in the amount of fibrosis between infected ob/ob mice and control mice. It also eliminated any significant difference in TGFbeta1 secretion between the infected ob/ob and infected control mice. CONCLUSIONS: This study shows that leptin deficiency decreases but does not eliminate hepatic fibrosis produced by Schistosoma mansoni and carbon tetrachloride administration. The effect of leptin in potentiating fibrogenesis is most likely mediated by TGFbeta1.     

Activation of inducible nitric oxide synthase/nitric oxide by curli fibers leads to a fall in blood pressure during systemic Escherichia coli infection in mice

Septic shock, a major cause of death, is characterized by a pathophysiologic increased production of nitric oxide (NO), which leads to vasodilation and myocardial toxicity. Septic Escherichia coli frequently express proteinaceous curli fibers. In this study, curliated E. coli induced high levels of NO by directly inducing type 2 nitric oxide synthase (NOS2) both in vitro and in vivo. More severe hypotension and higher plasma nitrite/nitrate levels were seen in wild type mice systemically infected with curliated E. coli than in animals infected with E. coli mutants that lacked curli proteins. Blood pressure remained stable in NOS2-deficient mice with curliated bacteria. Increased heart rates, transient hypothermia, and loss of gross activity were seen in all mice, regardless of curli expression. Study results suggest that expression of curli fibers by E. coli activates the NO/NOS2 arm of the innate immune system, which leads to a significant fall in blood pressure.     

Development of lyme arthritis in mice deficient in inducible nitric oxide synthase

Nitric oxide (NO) is a powerful antimicrobial agent and an important regulatory molecule of the innate immune response. To determine if NO has a role in experimental Lyme disease, arthritis-resistant DBA/2J and arthritis-susceptible C3H/HeJ mice were bred to be genetically deficient for inducible NO synthase (iNOS). Following footpad injection of Borrelia burgdorferi, arthritis was similar between iNOS-deficient and control animals regardless of their genetic background. Histologic examination and arthritis severity scores of ankles revealed no differences in arthritis development between iNOS-deficient and control animals. Despite being deficient in a key antimicrobial agent, iNOS-deficient mice had tissue levels of B. burgdorferi similar to those in control mice. Thus, NO does not have a critical role in susceptibility to Lyme arthritis through tissue damage via an overexuberant inflammatory response, nor is it required in resistance through the clearance of spirochetes from tissues.     

Inducible nitric oxide synthase gene promoter polymorphism is associated with increased gastric mRNA expression of inducible nitric oxide synthase and increased risk of gastric carcinoma

BACKGROUND AND AIMS: Stimulation of inducible nitric oxide synthase gene expression by Helicobacter pylori, with subsequent overproduction of nitric oxide, has been implicated in gastric carcinogenesis. We investigated whether inducible nitric oxide synthase promoter gene polymorphisms are associated with (a) inducible nitric oxide synthase mRNA expression in the gastric mucosa, and (b) the risk of gastric carcinoma. MATERIALS AND METHODS: The relationship between gastric inducible nitric oxide synthase mRNA expression and inducible nitric oxide synthase promoter polymorphisms (CCTTT repeat polymorphism and -2445 C-->G SNP) was examined in 74 H. pylori-infected patients with gastric cancer, peptic ulcer, or functional dyspepsia. In a case-control study the prevalence of the polymorphisms was examined in H. pylori-infected gastric carcinomas (n=77) and noncancerous controls (n=154). RESULTS: Inducible nitric oxide synthase mRNA levels were significantly higher in long CCTTT repeat (either allele>11) carriers than in short ones (P=0.015). Multivariate regression analysis showed that inducible nitric oxide synthase mRNA expression was significantly linked to long CCTTT repeat and gastric cancer (P=0.026), but not to -2445 C-->G SNP and other parameters. The case-control study showed that long CCTTT repeat carriers had an increased risk of gastric cancer with an odds ratio of 2.0 (P=0.021). -2445 C-->G SNP was not associated with the risk. CONCLUSIONS: Helicobacter pylori induces higher inducible nitric oxide synthase mRNA expression in carriers of long CCTTT repeats of inducible nitric oxide synthase promoter, and this polymorphism is associated with an increased risk of gastric carcinoma.     

BXSB小鼠肾组织诱生型一氧化氮合酶的异常表达及甲泼尼龙干预效应

目的探讨诱生型一氧化氮合酶(iNOS)是否参与介导BXSB小鼠狼疮肾炎(LN)及甲泼尼龙(MPS)是否可通过影响iNOS表达而缓解LN。方法用MPS对BXSB自发狼疮小鼠进行体内实验,利用反转录-聚合酶链反应(RT-PCR)和免疫组织化学技术研究了MPS对狼疮小鼠肾脏iNOS表达的影响,并观察了狼疮小鼠MPS治疗前后24h尿蛋白和NO3-/NO2-排泄量。结果BXSB狼疮小鼠肾组织检测到了iNOSmRNA的表达,而BALB/C小鼠未见表达;免疫组织化学示iNOS表达于BXSB小鼠肾小管上皮细胞、血管内皮细胞、肾间质及肾小球浸润的炎症细胞胞质内。MPS治疗组BXSB狼疮小鼠肾组织iNOSmRNA表达量显著低于未治疗组(P<0.01);治疗组24h尿蛋白及NO3-/NO2-排泄量显著低于未治疗组(P<0.01,P<0.05)。结论iNOS及其合成的一氧化氮(NO)可能参与介导了LN。MPS可能通过抑制肾组织iNOS的表达缓解LN。     

NF-kappaB stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha- and Fas-mediated apoptosis

BACKGROUND AND AIMS: Hepatocyte apoptosis is induced by tumor necrosis factor alpha (TNF-alpha) and Fas ligand. Although nuclear factor-kappaB (NF-kappaB) activation protects hepatocytes from TNF-alpha-mediated apoptosis, the NF-kappaB responsive genes that protect hepatocytes are unknown. Our aim was to study the role of NF-kappaB activation and inducible nitric oxide synthases (iNOSs) in TNF-alpha- and Fas-mediated apoptosis in hepatocytes. METHODS: Primary cultures of hepatocytes from wild-type and iNOS knockout mice were treated with TNF-alpha, the Fas agonistic antibody Jo2, a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillamine), an NO inhibitor (N(G)-methyl-L-arginine acetate), and/or adenovirus-expressing NF-kappaB inhibitors. RESULTS: The IkappaB superrepressor and a dominant-negative form of IkappaB kinase beta (IKKbeta) inhibited NF-kappaB binding activity by TNF-alpha or Jo2 and sensitized hepatocytes to TNF-alpha- and Jo2-mediated apoptosis. TNF-alpha and Jo2 induced iNOS messenger RNA and protein levels through the induction of NF-kappaB. S-nitroso-N-acetylpenicillamine inhibited Bid cleavage, the mitochondrial permeability transition, cytochrome c release, and caspase-8 and -3 activity, and reduced TNF-alpha- and Fas-mediated death in hepatocytes expressing IkappaB superrepressor. N(G)-methyl-L-arginine acetate partially sensitized hepatocytes to TNF-alpha- and Fas-mediated cell killing. TNF-alpha alone or Jo2 alone induced moderate cell death in hepatocytes from iNOS(-)/(-) mice. CONCLUSIONS: NO protects hepatocytes from TNF-alpha- and Fas-mediated apoptosis. Endogenous iNOS, which is activated by NF-kappaB via IKKbeta, provides partial protection from apoptosis.     

Monomeric inducible nitric oxide synthase localizes to peroxisomes in hepatocytes

Hepatocytes are capable of repeated inducible NO synthase (iNOS) expression, which occurs under inflammatory and stress conditions. This iNOS expression regulates a number of cellular functions as well as cell viability. To better understand the posttranslational mechanisms that regulate the fate of iNOS in these cells, we characterized the iNOS distributed within peroxisomes. The selective permeabilization of membranes (plasma vs. peroxisomal) confirmed that there are cytosolic and peroxisomal pools of iNOS in cytokine-stimulated hepatocytes and that the iNOS protein associates with peroxisome. Detergent solubilization of the membrane fraction released iNOS to the soluble fraction. iNOS localized to membrane fraction is predominantly monomeric, but dimerization is partially reconstituted rapidly upon incubation with tetrahydrobiopterin. The reconstituted iNOS exhibits a lower specific activity than iNOS isolated from the soluble pool. Depletion of intracellular tetrahydrobiopterin with an inhibitor of de novo pterin synthesis resulted in a predominance of monomeric iNOS without a greater relative distribution of iNOS to the peroxisomal pool. Thus, iNOS exists in a least two pools in hepatocytes: a soluble pool composed of both active dimer and monomer and a peroxisomal pool of monomeric iNOS. iNOS might localize to peroxisomes in long-lived cells such as hepatocytes as a protective mechanism to remove incompetent enzyme.     

Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats

AIM: To investigate the dynamic change and role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis (NEC) is associated with the levels of nitric oxide synthase (NOS) in the mucosa of the affected intestine tissue. METHODS: Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide (IPS). Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined. RESULTS: The LPS-injected pups showed a significant increase in injury scores versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury. CONCLUSION: nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.     

Responses to Leishmania donovani in mice deficient in both phagocyte oxidase and inducible nitric oxide synthase

Mice deficient in phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS), which are primary macrophage killing mechanisms, generated tissue granulomas but showed unrestrained Leishmania donovani visceral replication and suboptimal initial responsiveness to antimony treatment. Nevertheless, visceral infection was controlled post-treatment and did not recur. A phox/iNOS-independent macrophage mechanism, which was not triggered by L. donovani, emerges after chemotherapy.     

Experimental abdominal aortic aneurysms in mice lacking expression of inducible nitric oxide synthase

To determine if nitric oxide synthase (NOS) contributes to the pathophysiology of abdominal aortic aneurysms (AAAs), C57BL/6J mice underwent transient aortic injury to induce a chronic inflammatory response. Wild-type mice developed a significant increase in aortic diameter within 14 days of elastase perfusion (115+/-16%, 40% incidence of AAAs), along with intense and widespread staining for nitrotyrosine, mononuclear inflammation, and delayed destruction of the elastic lamellae. Expression of both endothelial and neuronal forms of NOS was substantially decreased within AAAs, whereas inducible NOS (iNOS) mRNA was increased 360%, and the enzyme was localized to infiltrating inflammatory cells. By using mice with targeted deletion of iNOS to evaluate the functional importance of this enzyme, male iNOS(-/-) mice developed the same extent of aneurysmal dilatation as congenic controls (121+/-22%, 40% incidence of AAAs) and exhibited similar structural features except for diminished nitrotyrosine staining. Aneurysmal dilatation was actually enhanced in female iNOS(-/-) mice (141+/-16%, 80% incidence of AAAs; P<0.05), but this effect was reversed by previous oophorectomy. Although extensive protein nitration and increased expression of iNOS accompany the development of elastase-induced experimental AAAs, iNOS is not required in this process and its absence may be deleterious.