肠缺血/再灌注致肺损伤时肺内HO-1/CO与iNOS/NO相互作用的研究

目的观察肠缺血/再灌注(I/R)致肺损伤时肺内HO-1/CO与iNOS/NO的相互作用。方法采用肠缺血/再灌注模型。32只Wistar大鼠随机分为假手术组(Sham组)、肠缺血1 h再灌注6 h组(I/R组)、氨基胍组(AG组)和血晶素组(hemin组)。检测肺组织中HO-1和iNOS的表达,观察肺组织丙二醛(MDA)、血清一氧化氮(NO)及动脉血中氧血红蛋白(Hb-CO)的含量,同时观察肺组织病理形态学改变。结果与Sham组比较,I/R组HO-1和iNOS表达显著增强(均P<0.01);AG组HO-1和iNOS表达较I/R组明显降低(均P<0.05);Hemin组iNOS表达较I/R组明显降低而HO-1表达明显升高(均P<0.05);I/R组肺组织MDA、血清NO、血中HbCO较Sham组显著增加(P<0.05或P<0.01);与I/R组比较,AG组、Hemin组肺组织MDA、血清NO显著降低(P<0.05或P<0.01)。AG组的HbCO明显降低而Hemin组的HbCO明显升高(P<0.05)。病理学检查显示,AG组与Hemin组肺组织损伤程度较I/R组明显减轻。结论NO及CO对肠I/R肺组织具有保护作用,两者之间存在着相互作用,肺内HO-1/CO的大量生成具有使NO产生减少的作用,同时iNOS/NO的过量生成具有上调HO-1表达使CO产生增多的作用。     

Expression of differential nitric oxide synthase isoforms in human gastric mucosa infected with Helicobacter pylori

Objective: To study the relationship between nitric oxide synthase ( NOS) expression in human gastric mu-cosa and Helicobacter pylori (H. pylori) infection. Methods: Gastric mucosa samples were obtained from antrum of 33 patients received gastroendoscopy. H. pylori infection was confirmed by Giems staining and bacteria culture under mi-croaerophilic conditions. Expression of iNOS, eNOS and nitrotyrosine were detected by immunohistochemistry. Results: (1) The positive rate of H. pylori infection was 66. 7% (22/33) . (2) iNOS positive staining in inflammatory cells was detected in 77. 3% (17/22) of samples with H. pylori and 27. 3% (3/11) without H. pylori infection (P < 0.01). (3) eNOS expression in inflammatory cells was found in 77. 3% (17/22) of samples with H. pylori and 18.2% (2/11) without H. pylori infection ( P < 0. 01) . (4) Nitrotyrosine expression in inflammatory cells was observed in 59. 1% ( 13/22) of samples with H. pylori and 54.5%(6/11) without H. pylori infection (P>0.05). (5) Moderate a     

大鼠上丘一氧化氮合酶阳性神经元的发育

目的：研究大鼠胚胎时期及生后早期一氧化氮合酶(NOS)阳性神经元在上丘的分布，进一步探讨一氧化氮(NO)在上丘发育过程中的作用。方法：应用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶(NADPH—d)组织化学方法观察孕17d起至生后14d大鼠上丘NOS阳性神经元的发育。结果：孕20d上丘深白层与深灰层出现NOS阳性神经元。P0上丘中层也观察到NOS阳性神经元。随着发育，阳性神经元增多，胞体增大，染色加深。到P14，视神经层出现少许：NOS阳性神经元，浅灰层观察到大量的NOS阳性神经元。结论：胚胎20天上丘深层首先出现NOS阳性神经元并沿腹背方向发生，提示NO在上丘发育过程中起着重要作用。     

Effect of curcumin on nitric oxide synthase expression in Iipopolysaccharide-activated microglia cells and the anti-oxidative effect of curcumin

BACKGROUND: It has been demonstrated that curcumin can increase the activities of various anti-oxidase in blood and tissue, effectively eliminate various free radicals, reduce the production of peroxisome, and alleviate oxidative stress reaction. Whether it has the same effect on microglia? OBJECTIVE: To observe the effects of curcumin on the expressions of inducible nitric oxide synthase (iNOS), nuclear factor-κB (NF-κB), and superoxide dismutase (SOD) in microglial cell line BV stimulated by lipopolysaccharide (LPS). DESIGN: An observational comparative study. SETTING: Research Room of Biochemistry, Medical College of Nantong University. MATERIALS: Mice microglia cell line BV, iNOS and NF-κB reporter gene plasmids were presented by Dr. Bhat.NR. from the Medical University of South Carolina (USA). Curcumin was produced by the Xi'an Branch of China Chengdu Scholar Bio-Tech. Co.,Ltd.; LPS (E.Coli O26:B6), anti-mice iNOS monoclonal antibody, horseradish peroxidase labeled goat-anti-mice IgG were the products of Sigma Company (USA). METHODS: The experiments were carried out in the Research Room of Biochemistry, Medical College of Nantong University from May 2006 to April 2007. ① Detection of iNOS: The cells were seeded onto 24-well plate at the density of 1×105, After the cells had adhered to the cover glasses, the cells were grouped as negative control group (the primary antibody was replaced by phosphate buffered solution PBS); normal control group (the cells were normally cultured); LPS-treated group (the cells were treated with LPS for 24 hours); curcumin+LPS group (the cells were treated with curcumin for 1 hour and LPS for 24 hours). The expressions of iNOS protein were detected with immunocytochemical staining. ② Determination of iNOS and NF-κB gene activities: According to the introduction of the kit for transfection, iNOS or NF-κB report gene plasmids were transiently transfected with LipofectamineTM2000 liposomes into the cells in the 24-well plate for 24 hours. The cells were divided into normal control group (the cells were normally cultured after transfected with report gene plasmids); blank plasmid group (the cells were normally cultured after transfected with blank plasmids); LPS-treated group (the cells were treated with LPS for 4 hours after transfected with report gene plasmids); curcumin+LPS group (the cells were treated with curcumin for 1 hour and LPS for 24 hours after transfected with report gene plasmids). The content of luciferase in the cell lysis buffer was determined after cell lysis. ③ Determination of SOD activity: The cells were seeded into culture bottle at the density of 1×106, and the divided into four groups, including normal control group (the cells were normally cultured); LPS-treated group (the cells were treated with LPS for 24 hours); curcumin+LPS group (the cells were treated with curcumin for 1 hour and LPS for 24 hours); vitamin C+LPS group (the cells were treated with vitamin C for 1 hour and LPS for 24 hours). The SOD activity was determined with xanthine oxidase and quantitative colorimetric assay. MAIN OUTCOME MEASURES: The expressions of iNOS protein, iNOS and NF-κB, and the activity of SOD were observed. RESULTS: ① Expression of iNOS protein in microglia: The expression of iNOS protein in the LPS-treated group was obviously higher than that in the negative control group (P < 0.01); Those in the curcumin+LPS group were significantly decreased as compared with that in the LPS-treated group (P < 0.01). ② Expressions of iNOS and NF-κB genes: The expressions of iNOS and NF-κB genes in the LPS-treated group were significantly higher than those in the normal control group (P < 0.01); Those in the curcumin+LPS group were significantly lower than those in the LPS-treated group (P < 0.01). ③ SOD activity: The activity of SOD in the LPS-treated group was significantly lower than those in the normal control group (P < 0.01). It in the curcumin+LPS group and vitamin C +LPS group was significantly higher than that in the LPS-treated group (P < 0.01). CONCLUSION: Curcumin could inhibit the expression of iNOS in the activated microglia, and it also has the abilities in eliminating free radicals and antagonizing lipid peroxidation.     

Induction of NADPH-diaphorase activity in the hippocampus in a rat model of cerebral ischemia and ischemic tolerance

Preconditioning of the brain with sublethal ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). In this study, we used NADPH-diaphorase histochemistry to investigate the postischemic changes of nitric oxide synthase (NOS) in the hippocampus in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by 4-vessel occlusion for 3 min as an ischemic preconditioning. Three days after the preconditioning or sham operation, second ischemia was induced for 6 min. A transient increase in NADPH-diaphorase activity, beginning after 2 h and maximal after 1 day, was observed in CA1 pyramidal neurons of rats subjected to 3 min of preconditioning ischemia as well as 6 min of subsequent ischemia both with and without preconditioning. In addition, expression of NADPH-diaphorase activity was seen in reactive glial cells in the damaged CA1 region of animals subjected to 6 min of ischemia without preconditioning. Thus, direct involvement of increased NADPH-diaphorase activity in ischemic tolerance was not suggested because the increased NADPH-diaphorase activity preceded the induction of ischemic tolerance which takes place 1–7 days after preconditioning. However, the present findings suggest that the induction of neuronal NADPH-diaphorase activity occurs in response to cerebral ischemia.     

Handling of asymmetrical dimethylarginine and symmetrical dimethylarginine by the rat kidney under basal conditions and during endotoxaemia.

BACKGROUND: Asymmetrical dimethylarginine (ADMA) is capable of inhibiting nitric oxide synthase enzymes, whereas symmetrical dimethylarginine (SDMA) competes with arginine transport. The potential role of inflammation in the metabolism of ADMA has been elucidated in an in vitro model using tumour necrosis factor-alpha, resulting in a decreased activity of the ADMA-degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH). The kidney probably plays a crucial role in the metabolism of ADMA by both urinary excretion and degradation by DDAH. We aimed to further elucidate the role of the kidney in a rat model under basal conditions and during endotoxaemia. METHODS: Twenty-five male Wistar rats weighing 275-300 g were used for this study. The combination of arteriovenous concentration differences and kidney blood flow allowed calculation of net organ fluxes. Blood flow was measured using radiolabelled microspheres according to the reference sample method. Concentrations of ADMA, SDMA and arginine were measured by high-performance liquid chromatography. RESULTS: The kidney showed net uptake of both ADMA and SDMA and fractional extraction rates were 35% and 31%, respectively. Endotoxaemia resulted in a lower systemic ADMA concentration (P = 0.01), which was not explained by an increased net renal uptake. Systemic SDMA concentrations increased during endotoxaemia (P = 0.007), which was accompanied by increased creatinine concentrations. CONCLUSIONS: The rat kidney plays a crucial role in the regulation of concentrations of dimethylarginines, as both ADMA and SDMA were eliminated from the systemic circulation in substantial amounts. Furthermore, evidence for the role of endotoxaemia in the metabolism of dimethylarginines was obtained as plasma levels of ADMA were significantly lower in endotoxaemic rats.     

一氧化氮与胚胎异常发育的相关性研究

为了解开一氧化氮（ＮＯ）是否与畸胎发生有关这一谜团和进一步阐明砷致畸作用机理，本实验应用诱生型ＮＯ合成酶（ｉＮＯＳ）组织化学、扫描电镜（ＳＥＭ）及体内致畸试验等方法研究了砷对小鼠卵黄囊胎盘（ＹＳＰ）和胚胎发育的影响。结果表明ＹＳＰ细胞ｉＮＯＳ表达与砷浓度之间存在明显的剂量—反应关系（Ｐ＜０．０５）；ＳＥＭ观察可见ＹＳＰ内皮层和间皮层细胞受损；光镜下可见ＹＳＰ变小、萎缩和微血管分化不良；随着染毒剂量的升高，畸胎率和死胎率亦逐步增加，最高分别达到５６．８％和２４．７％；畸胎的主要表现是神经管未闭，心包积液和体位异常等。研究结果率先提示过量ＮＯ与畸胎发生及致畸机理关系密切；推荐在致畸研究中ｉＮＯＳ可作为一种有效的生物标志物。     

Inhibition of nitric oxide synthase attenuates blood-brain barrier disruption during experimental meningitis

Increased permeability of the blood-brain (B-B) barrier is observed during meningitis. Preventing B-B barrier alterations is important because adverse neurological outcomes are correlated with breeches in barrier integrity. It was hypothesized that pathological production of nitric oxide (NO) contributes to B-B barrier disruption during meningitis in the rat. Experimental meningitis was induced by intracisternal (i.c.) administration of lipopolysaccharides (LPS) or vehicle. Groups of rats were concomitantly infused intravenously (i.v.) with saline or the NO synthase inhibitor, aminoguanidine (AG). Eight h after i.c. dosing, B-B barrier alterations were quantitated pharmacokinetically using [¹⁴C]sucrose. Serum and regional brain tissues were obtained 0–30 min after tracer dosing and sucrose influx transfer coefficients ( K_{in (app)}) were calculated from the brain tissue data. Compared to the control groups (i.c. vehicle/i.v. saline), the K_{in (app)} of the i.c. LPS/i.v. saline group increased 1.6–2.1-fold in various brain regions, thus confirming previous observations of increased [¹⁴C]sucrose barrier penetration during meningeal inflammation. Remarkably, i.v. administration of AG to i.c. LPS-treated rats significantly inhibited meningeal NO synthesis and decreased K_{in (app)} permeability alterations in the B-B barrier, compared to i.c. LPS/i.v. saline-treated rats. Regional brain K_{in (app)} estimates in the i.c. LPS/i.v. AG group were similar to control groups (i.c. vehicle/i.v. AG and i.c. vehicle/i.v. saline). In conclusion, these data suggest the general concept that excessive NO production during neuroinflammatory diseases contributes to disruption of the blood-brain barrier.     

Identification of a Thymidylate Synthase Gene within the Genome of Chilo Iridescent Virus

The thymidylate synthase (TS, EC 2.1.1.45) is essential for the de novo synthesis of dTMP in pro- and eucaryotic organisms. Consequently it plays a major role in the replication of the DNA genome of a cell or a DNA virus. The gene encoding the TS of Chilo iridescent virus (CIV) was identified by nucleotide sequence analysis of the viral genome and was mapped within the EcoRI CIV DNA fragments G and R. Computer assisted analysis of the DNA nucleotide sequence between the genome coordinates 0.482 and 0.489 revealed an open reading frame (ORF) of 885 nucleotides. This ORF was found to encode a polypeptide of 295 amino acid residues (33.9 kDa) that showed significant homologies to known TS of different species including mammals, plants, fungi, protozoa, bacteria, and DNA viruses. The highest amino acid homologies were found between the CIV-TS and the TS of herpesvirus ateles (54.0%), Saccharomyces cerevisiae (51.8%), herpesvirus saimiri (51.0%), rhesus monkey rhadinovirus (50.7%), mouse (50.5%), rat (50.2%), varicella-zoster virus (50.2%), equine herpesvirus 2 (50.0%), and the human TS (48.4%). The CIV-TS contains six amino acid domains that are highly conserved in the TS of other species. Within these domains the major amino acid residues are present for which a functional role has been reported. The CIV-TS was found to be more closely related to the TS of eucaryotes than to the TS of procaryotes indicating the phylogenetic origin of the CIV-TS gene. The identification of a TS gene in the genome of CIV is the first report of a viral TS that is not encoded by a herpesvirus or a bacteriophage.     

Effect of different types of high carbohydrate diets on glycogen metabolism in liver and skeletal muscle of endurance-trained rats

Male Wistar rats were fed ad libitum four different diets containing fructose, sucrose, maltodextrins or starch as the source of carbohydrate (CH). One group was subjected to moderate physical training on a motor-driven treadmill for 10 weeks (trained rats). A second group received no training and acted as a control (sedentary rats). Glycogen metabolism was studied in the liver and skeletal muscle of these animals. In the sedentary rats, liver glycogen concentrations increased by 60%–90% with the administration of simple CH diets compared with complex CH diets, whereas skeletal muscle glycogen stores were not significantly affected by the diet. Physical training induced a marked decrease in the glycogen content in liver (20%–30% of the sedentary rats) and skeletal muscle (50%–80% of the sedentary rats) in animals fed simple (but not complex) CH diets. In liver this was accompanied by a two-fold increase of triacylglycerol concentrations. Compared with simple CH diets, complex CH feeding increased by 50%–150% glycogen synthase (GS) activity in liver, whereas only a slight increase in GS activity was observed in skeletal muscle. In all the animal groups, a direct relationship existed between tissue glucose 6-phosphate concentration and glycogen content (r = 0.9911 in liver, r = 0.7177 in skeletal muscle). In contrast, no relationship was evident between glycogen concentrations and either glycogen phosphorylase activity or adenosine 5-monophosphate tissue concentration. The results from this study thus suggest that for trained rats diets containing complex CH (compared with diets containing simple CH) improve the glycogenic capacity of liver and skeletal muscle, thus enabling the adequate regeneration of glycogen stores in these two tissues.