Protective effect of inducible nitric oxide synthase inhibitor on pancreas transplantation in rats

AIM： To investigate the effect of inducible nitric oxide synthase inhibitor, aminoguanidine, on pancreas transplantation in rats. METHODS： A model of pancreas transplantation was established in rats. Streptozotocin-induced diabetic male Wistar rats were randomly assigned to sham-operation control group （n = 6）, transplant control group （n = 6）, and aminoguanidine （AG） treatment group （n = 18）. In the AG group, aminoguanidine was added to intravascular infusion as the onset of reperfusion at the dose of 60 mg/kg, 80 mg/kg, 100 mg/kg body weight, respectively. Serum nitric oxide （NO） level, blood sugar and amylase activity were detected. Nitric oxide synthase （NOS） test kit was used to detect the pancreas cNOS and inducible NOS （iNOS） activity. Pancreas sections stained with HE and immunohistochemistry were evaluated under a light microscope. RESULTS： As compared with the transplant control group, the serum NO level and amylase activity decreased obviously and the evidence for pancreas injury was much less in the AG group. The AG （80 mg/kg body weight） group showed the most significant difference in NO and amylase （NO： 66.0 ± 16.6 vs 192.3 ± 60.0, P 〈 0.01 and amylase： 1426 ± 177 vs 4477± 630, P 〈 0.01）. The expression and activity of tissue iNOS, and blood sugar in the AG （80 mg/kg body weight） group were much lower than those in the transplant control group （iNOS： 2.01 ± 0.23 vs 26.59 ±5.78, P 〈 0.01 and blood sugar： 14.2 ±0.9 vs 16.8 ± 1.1, P 〈 0.01）. CONCLUSION： Selective iNOS inhibitor, aminoguanidine as a free radical, has a protective effect on pancreas transplantation in rats by inhibiting NO and reducing its toxicity.     

Subcellular distribution of nitric oxide synthase isoforms in the rat duodenum

AIM:To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase(NOS) in the rat duodenum.METHODS:Postembedding immunoelectronmicroscopy was performed,in which primary antibodies for neuronal NOS(nNOS),endothelial NOS(eNOS),and inducible NOS(iNOS),were visualized with protein A-gold-conjugated secondary antibodies.Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera.The specificity of t...     

Early effects of diabetes on inducible nitric oxide synthase in the kidney

NO may be responsible for the glomerular hyperfiltration observed in diabetic kidney by inducing vasodilation of the afferent arteriole. The aim of this study was to evaluate which isoform of nitric oxide synthase (NOS) is responsible for increased renal production of NO in diabetic kidney. Thirty male WKY rats were divided into 6 groups. Five rats were sacrificed immediately, five after 20 days. In the other rats, diabetes was induced by streptozotocin. The four diabetic groups were sacrificed respectively after 5, 10, 15 and 20 days. Urine excretion of NO metabolites was assayed; immunochemistry showed the presence of inducible (iNOS) and endothelial constitutive (ecNOS) synthases in the kidney. Urinary excretion of NO metabolites increased significantly in diabetic rats five days after the induction of diabetes and at the end of the study whereas it was unchanged in the control group. Renal ecNOS remained unchanged throughout the study in all rats whereas iNOS increased significantly in diabetic rats from the fifth day until the end of the study. The results demonstrate that iNOS is activated in the kidney of rats, soon after the induction of diabetes, thus suggesting its involvement in the increased production of NO observed immediately after the onset of diabetes. Received: 19 September 2001 / Accepted in revised form: 31 January 2002     

诱导型一氧化氮合酶在创伤性脑损伤后肠黏膜屏障损伤中表达的变化

目的观察创伤性脑损伤（TBI）后肠黏膜屏障损伤时诱导型一氧化氮合酶（iNOS）表达的变化及其可能的作用。方法 48只健康雄性W istar大鼠随机分为TBI组（n=24）和对照组（n=24）,各组动物分别在手术后3、6、12、24 h处死,每个时间点6只。动物处死后,抽门静脉血测血清内毒素、二胺氧化酶（DAO）,取脑组织和回肠黏膜,观察组织形态学改变,并测定肠黏膜组织DAO的含量和iNOS的表达情况。结果 TBI组肠黏膜受损,血中内毒素含量增加（P〈0.05）;肠黏膜DAO活性下降（P〈0.01）,而血中DAO活性则升高（P〈0.05）;iNOS的表达3h已经增高,12h达到最高,而后逐渐降低,但仍然高于对照组（P〈0.05）。结论 iNOS在TBI后肠黏膜屏障损伤时表达明显增加,参与了TBI后肠黏膜屏障损伤。     

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.     

阿霉素对大鼠心肌诱导型一氧化氮合酶mRNA表达的影响

目的研究阿霉素(ADM)对大鼠心肌诱导型一氧化氮合酶(iNOS)mRNA表达的影响。方法将雄性wistar大鼠24只,随机分为ADM组和对照组,每组12只,ADM组按每次给ADM 2 mg/kg腹腔注射,隔日一次共6次;对照组给等体积的生理盐水腹腔注射。于实验第30 天,应用生化方法测定心肌组织中的一氧化氮(NO)水平及iNOS的活性;用TUNEL法检测心肌细胞的凋亡指数;用RT—PCR方法检测心肌组织iNOS mRNA的表达。结果与对照组比较ADM组大鼠心肌组织NO、iNOS活性增加(P< 0.01),心肌细胞凋亡指数及iNOS mRNA的表达均显著增高(P< 0.01)。结论ADM能诱导大鼠心肌细胞iNOS mRNA表达增加,使NO合成增多,引起细胞凋亡而参与对心肌的损害。     

应激对大鼠结肠神经系统nNOS表达的影响

目的:探讨应激对大鼠结肠神经系统nNOS表达的影响. 方法:SD大鼠30只随机分为对照组,应激组和L-NAME 组,采用水浸-束缚应激(WRS)动物模型,用免疫组织化学ABC法检测nNOS在大鼠结肠黏膜下神经丛和肌间神经丛的表达,应用计算机图像分析系统对其表达进行定量分析．结果:与对照组比较,应激组黏膜下神经丛和肌间神经丛的nNOS阳性神经元的灰度值明显减少(P=0．02或P =0．005),阳性神经元细胞数的平均密度增加(P=0．04 或P=0．01),表达增强,且在黏膜上皮细胞、固有层淋巴细胞也有nNOS表达．L-NAME组黏膜下神经丛和肌间神经丛的nNOS阳性神经元的灰度值较应激组增加 (P=0．04),平均密度下降(P=0．04或P=0．03),表达减弱,而与对照组比较均无明显差异(P>0．05)．结论:应激可引起大鼠结肠神经系统nNOS表达增强, 提示一氧化氮(NO)在应激所致的结肠功能失调中可能起重要作用．     

Expression of inducible nitric oxide synthase in human gastric cancer

INTRODUCTIONInduciblenitricoxidesynthase(iNOS)isanenzymethatcatalyzestheformationofnitric0xide(N0)fromL-arginine.iNOSexpressionandactivityresultsintheproduction0fhighlevelsofNO[1].ThegenerationofphysiologicallevelsofNOisimp0rtantformucosalfunctionanditalsoexertsacytoprotectiveeffectonthegastr0intestinalmucosa.However,increasediNOSexpressionhasbeenobservedinpatientswithchronicinflammatorydiseasesofthegastr0intestinaltract,suchasulcerativec0litis[2'3],andgastritis['Jandithasbeenspecul…     

一氧化氮合酶在N-乙酰半胱氨酸调控大鼠心肌冷缺血-再灌注损伤中的变化

目的:观察一氧化氮合酶(NOS)在N-乙酰半胱氨酸(NAC)调控大鼠心肌冷缺血-再灌注损伤中的变化。方法:将健康雄性Lewis大鼠60只随机分为3组。(1)对照组:摘取供心前30 min,经供体大鼠下腔静脉注射生理盐水0.5 mL;(2)供体预处理组:摘取供心前30 min,经供体大鼠下腔静脉注射NAC300 mg/kg,受体大鼠不作预处理;(3)受体预处理组:移植前30 min,经受体大鼠下腔静脉注射NAC300 mg/kg,供体大鼠不作预处理。将冷藏于4℃HTK液18 h的供心移植至受体大鼠腹腔,建立同种异体心脏移植模型。于再灌注24 h后取供心采用免疫组化方法检测诱导型一氧化氮合酶/内皮型一氧化氮合酶(iNOS/eNOS)蛋白表达水平,以免疫组化评分(IHS)表示。采用Real time-PCR法检测iNOS/eNOS mRNA表达。结果:与对照组相比,供、受体预处理组的iNOS蛋白表达降低,IHS评分分别为3.00±0.15、1.50±0.22、1.63±0.26,P<0.05;而eNOS蛋白表达升高,IHS评分分别为2.00±0.21,3.60±0.16,3.40±0.26,P<0.05。与对照组相比,受体预处理组iNOS mRNA表达降低(0.43±0.17对1.00±0.41,P<0.05),供体预处理组eNOS mRNA表达升高(3.06±1.47对1.00±0.65,P<0.05)。结论:NOS参与了NAC预处理减轻移植大鼠心肌缺血-再灌注损伤的过程。     

Estrogen downregulates neuronal nitric oxide synthase in rat anterior pituitary cells and GH3 tumors

Mammary cell mitogenic responsiveness was evaluated with an established nontransformed caprine mammary epithelial cell line (CMEC). As expected, the cells responded to insulin-like growth factor 1 (IGF-1), insulin, and hydrocortisone with an increased number of cells after 3 and 5 d in culture. In combination, insulin and hydrocortisone augmented each other. A proliferation response was also observed for transforming growth factor-alpha (TGF-alpha), and epidermal growth factor (EGF), but not for fibroblast growth factor a (FGFa), FGFb, IGF-2, bovine somatotropin, or prolactin. Comparison of mitogenic potential for these growth factors with cells grown on plastic substratum indicated that hydrocortisone was most potent, followed by TGF-alpha in inducing a proliferative response measured by 3H-thymidine incorporation assay. Hydrocortisone augmented proliferation by 149% and TGF-alpha stimulated proliferation by 126% relative to the media control (p < 0.01). EGF, which binds to the same receptor as TGF-alpha in other species, induced a modest 35% increase in proliferation. Comparison of culture conditions with plastic, fibronectin, and type I collagen suggests that extracellular matrix/stroma influences the magnitude and effective concentration for cytokine-mediated growth response. Studies on responsiveness to ovarian steroids estradiol 17-beta (E2) and progesterone (P4) showed a modest proliferation response to E2 only in combination with triiodio-L-thyronine (T3), and no response to P4 or T3 either alone or in combination when grown on plastic.     

香菇多糖对巨噬细胞一氧化氮合酶活性和还原型谷胱甘肽的影响

目的　研究香菇多糖 (LTN)对巨噬细胞诱导型一氧化氮合酶 (i NOS)活性和细胞内还原型谷胱甘肽 (GSH)水平的影响及其关系 ,探讨 L TN的免疫调节作用机制。方法　 1观察 LTN诱导小鼠腹腔巨噬细胞 i NOS活性和细胞内 (GSH)水平的影响 ;2观察 m RNA转录抑制剂、蛋白质合成抑制剂和 NOS抑制剂对巨噬细胞 i NOS活性和细胞内 GSH水平的影响 ;3观察 GSH去除剂对巨噬细胞 i NOS活性的影响。结果　 1 LTN诱导小鼠腹腔巨噬细胞 i NOS活性增加和细胞内 GSH减少 ;2 3种抑制剂抑制 L TN诱导的小鼠腹腔巨噬细胞 i NOS活性增加和细胞内 GSH减少 ;3GSH去除剂抑制 LTN诱导的巨噬细胞 i NOS活性。结论　 LTN能增加小鼠腹腔巨噬细胞 i NOS的活性 ,并同时消耗细胞内的 GSH,提示细胞内GSH可能起到调节巨噬细胞 i NOS活性和保护宿主细胞免受 NO介导的细胞毒作用。     

肝癌组织中一氧化氮合酶及其基因表达的研究

目的研究肝癌组织中一氧化氮合酶(iNOS)及其基因表达与肝癌发生发展的关系。方法用免疫组化和原位杂交的方法对21例肝癌及癌旁组织中的诱导型一氯化氮合酶(iNOS)及其基因表达进行原位检测和观察。结果:NOS 阳性反应物质呈黄色或棕黄色,位于细胞浆中。非癌殖织(肉眼观距癌组织边缘>1.5)多呈阴性或弥漫弱阳性,但部分非癌组织中可见 iNOS 呈阳性的细胞呈点状分布;癌旁组织多呈阳性,提示 iNOS 表达与肝组织癌变有关。癌组织核心多呈阴性或弥漫弱阳性,但分化中和差的癌组织核心也分别有一例 NOS 呈强阳性;周边癌组织呈局灶阳性,侵入纤维组织中的弥敢癌细胞星强阳性,提示 NOS 的表达与肝癌组织的侵润能力有关。肝癌组织 iNOSmRNA 阳性细胞的分布与 iN-OS 蛋白的表达基本相似。结论 iNOS 蛋白及其基因表达与肝组织癌变及肝癌侵润能力有关。     

Inhibition of matrix metalloproteinases and inducible nitric oxide synthase by andrographolide in human osteoarthritic chondrocytes

Objective

The aim of this study was to investigate the effects of andrographolide on matrix metalloproteinases (MMP) 1, 3, and 13 and inducible nitric oxide synthase (iNOS) in human articular chondrocytes from osteoarthritic cartilage.

Methods

Passaged chondrocytes were pretreated with or without andrographolide for 2 h, followed by coincubation with interleukin-1 beta (IL-1β) 1 ng/ml for 24 h. Expression levels of MMP-1, 3, and 13, tissue inhibitor of metalloproteinase-1 (TIMP-1), and iNOS were evaluated using real-time-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting. Nitric oxide (NO) was analyzed using the Griess reaction assay. Involvement of nuclear factor kappa B (NF-κB) was assessed by Western blotting, transient transfection, and luciferase reporter assay.

Results

Andrographolide tested in these in vitro studies was found be an effective antiarthritic agent, as evidenced by potent inhibition of MMP-1, 3, and 13 and iNOS expression, as well as upregulation of TIMP-1 in IL-1β-stimulated human articular chondrocytes (p < 0.05). The mechanism of andrographolide’s inhibitory effects was mediated by attenuating the activation of NF-κB in human chondrocytes in the presence of IL-1β.

Conclusions

Andrographolide was a potent inhibitor of the production of inflammatory and catabolic mediators by chondrocytes, suggesting that this natural compound may merit consideration as a therapeutic agent for treating and preventing osteoarthritis.     

复元胶囊对大鼠膝骨关节炎软骨iNOS及血清PGE2、NO的影响

目的 观察复元胶囊(FYJN)对实验性大鼠膝骨关节炎软骨诱导型一氧化氮合酶(iNOS)及血清一氧化氮(NO)、前列腺素E2(PGE2)的影响.方法 Hulth法建立骨关节炎模型,随机分成正常组、模型组、塞来昔布(CE)组、FYJN低、中、高剂量组,各组分别予相应药物灌胃12 w.HE染色并光镜观察股骨内侧髁软骨病理形态,免疫组化检测软骨中iNOS表达,硝酸基还原酶法及酶联免疫吸附法分别测定血清NO、PGE2含量.结果 FYJN低、中、高剂量组软骨iNOS及血清NO、PGE2均低于模型组(P<0.05,P<0.01,P<0.001).结论 FYJN呈剂量依赖关系降低骨关节炎软骨中iNOS表达及血清中NO、PGE2含量,对减轻关节炎症有一定的作用.     

乙型肝炎病毒前-S2蛋白下调诱导型一氧化氮合酶基因启动子的转录活性

目的探讨乙型肝炎病毒(HBV)前-S2(pre-S2)蛋白对诱导型一氧化氮合酶(iNOS)基因启动子转录的调节作用。方法利用生物信息学技术确定iNOS基因的启动子区域(iNOSp)和3个缺失突变体的基因序列,聚合酶链反应(PCR)分别扩增iNOSp和3个缺失突变体的基因序列,分别克隆至报告基因表达载体pCAT3-Basic中,构建pCAT3-iNOSp载体;以构建的这4种报告基因表达载体,分别转染人肝母细胞瘤细胞系HepG2,用酶联免疫吸附法(ELISA)检测氯霉素乙酰转移酶(CAT)的表达活性;并与真核表达载体pcDNA3.1(-)-HBV pre-S2共转染HepG2细胞系,用ELISA法检测CAT的表达活性。结果成功获得iNOS基因启动子和3个缺失突变体的正确克隆,p1-iNOSp、p3-iNOSp启动子和pcDNA3.1 (-)-HBV pre-S2瞬时共转染HepG2细胞时,iNOS启动子的转录活性明显下降,HBV pre-S2蛋白对p1- iNOSp、p3-iNOSp表达活性的抑制率分别是54.7%和79.5%,p2-iNOSp、p4-iNOSp与pcDNA3.1(-)-HBV pre-S2共转染HepG2细胞后,HBV pre-S2蛋白对p2-iNOSp、p4-iNOSp表达活性没有明显的调节作用。重复试验得到了相似的结果。结论HBV pre-S2蛋白在细胞内的表达对iNOS启动子的转录活性具有明显的下调作用。     

New progress in understanding roles of nitric oxide during hepatic ischemia-reperfusion injury

Hepatic ischemia-reperfusion injury (HIRI) is a major clinical cause of morbidity and mortality in liver surgery and transplantation. Many studies have found that nitric oxide (NO) plays an important role in the HIRI and its increase or decrease can affect the progression and outcome of HIRI. However, the role of NO in HIRI is controversial and complicated. NO derived by endothelial NO synthase (eNOS) shows a protective role in HIRI, while excessive NO derived by inducible NO synthase (iNOS) accelerates inflammation and increases oxidative stress, further aggravating HIRI. Nevertheless, the overexpression of eNOS may exacerbate HIRI and iNOS-derived NO in some cases reduces HIRI. Here we review the new progress in the understanding of the roles of NO during HIRI: (1) NO possesses different roles in HIRI by increasing NO bioavailability, down-regulating leukotriene C4 synthase, inhibiting the activation of the nuclear factorκB (NFκB) pathway, enhancing cell autophagy, and reducing inflammatory cytokines and reactive oxygen species (ROS). And NO has both protective and deleterious effects by regulating apoptotic factors; (2) eNOS promotes NO production and suppresses its own overexpression, exerting a hepatoprotective effect reversely. Its activation is regulated by the PI3K/Akt and KLF2/AMPK pathways; and (3) iNOS derived NO mainly has deteriorating effects on HIRI, while it may have a protective function under some conditions. Their expression should reach a balance to reduce the adverse side and make NO protective in the treatment of HIRI. Thus, it can be inferred that NO modulating drugs may be a new direction in the treatment of HIRI or may be used as an adjunct to mitigate HIRI for the purpose of protecting the liver.     

Role of inducible nitric oxide synthase in trinitrobenzene sulphonic acid induced colitis in mice

BACKGROUND: Studies using inhibitors of nitric oxide synthase (NOS) to date are inconclusive regarding the role of inducible NOS (iNOS) in intestinal inflammation. AIMS: (1) To examine the role of iNOS in the development of chronic intestinal inflammation; (2) to identify the cellular source(s) of iNOS. METHODS: Colitis was induced by an intrarectal instillation of trinitrobenzene sulphonic acid (TNBS, 60 mg/ml, 30% ethanol), in wild type (control) or iNOS deficient mice. Mice were studied over 14 days; the colons were scored for injury and granulocyte infiltration was quantified. Blood to lumen leakage of (51)Cr-EDTA was measured as a quantitative index of mucosal damage. RESULTS: At 24 and 72 hours, iNOS deficient mice had significantly increased macroscopic inflammation compared with wild type mice. Granulocyte infiltration increased significantly at 24 hours and remained elevated in iNOS deficient mice at 72 hours, but significantly decreased in controls. However, by seven days post-TNBS macroscopic damage, microscopic histology, granulocyte infiltration, and mucosal permeability did not differ between wild type and iNOS deficient mice. A four- to fivefold increase in iNOS mRNA was observed in wild type mice at 72 hours and seven days post-TNBS and was absent in iNOS deficient mice. Immunohistochemistry techniques showed that iNOS expression was predominantly localised in neutrophils, with some staining also in macrophages. CONCLUSIONS: These results suggest that leucocyte derived iNOS ameliorates the early phase, but does not impact on the chronic phase of TNBS induced colitis despite the presence of iNOS.