组成型一氧化氮合酶的表达与胃癌的侵袭转移和预后的关系

目的　观察胃癌组织中cNOSmRNA表达分布情况 ,探讨其与胃癌侵袭转移和预后的关系。　方法　运用原位杂交技术 ,检测 119例原发性胃癌标本中cNOSmRNA的表达 ,并对所获得的 82份病例随访资料进行生存分析。　结果　胃癌组织中胃腺癌细胞、血管 淋巴管内皮细胞及巨噬细胞中均可见有cNOSmRNA表达 ;肿瘤细胞分化程度越低、恶性度越高 ,胃腺癌细胞和血管 淋巴管内皮细胞cNOSmRNA阳性表达率就越高 ;胃腺癌细胞和血管 淋巴管内皮细胞cNOSmRNA阳性表达率与胃癌侵袭深度、淋巴结转移以及TNM分期呈正相关 ;胃癌血管 淋巴管内皮细胞cNOSmRNA阳性表达者术后生存率较低 ;巨噬细胞中cNOSmRNA表达与胃癌组织学分型、侵袭深度、淋巴结转移、TNM分期及患者术后生存期无关。　结论　胃腺癌细胞和血管 淋巴管内皮细胞cNOSmRNA表达情况与肿瘤恶性程度、侵袭和转移潜能有关 ,血管 淋巴管内皮细胞上cNOSmRNA阳性表达提示患者预后不良     

益肾降压方对RPH大鼠内皮由来因子的调控作用

目的研究中药经验方“YSJY”对RPH大鼠肾组织cNOS与ET1蛋白及基因表达的作用,初步探讨其可能的作用机制。方法通过大鼠肾次全切除法制备RPH动物模型,采用免疫组化及RTPCR法检测肾组织cNOS与ET1蛋白及基因表达变化。结果YSJY治疗8wk后:①免疫组化显示模型组近曲小管cNOS呈弱阳性表达、ET1呈强阳性表达,而YSJY组近曲小管cNOS表达呈强阳性,ET1阳性表达明显减弱,且其近曲小管、肾小球已经接近正常组。②与模型组比较,YSJY组肾组织ET1mRNA表达减弱(P<0.01),cNOSmRNA表达上调(P<0.05)。结论YSJY可通过抑制肾组织ET1mRNA表达、促进cNOSmRNA表达,使肾组织局部ET1含量减少、NO含量增加,从而实现其降压、保护肾功能的作用。     

实验性微栓塞缺血豚鼠耳蜗cNOS的表达变化

目的 探究一氧化氮（nitricoxide，NO）在一过性微栓塞耳蜗缺血性损伤中的作用。方法 20只豚鼠随机分为实验组和对照组各10只，实验组动物通过磁微粒栓塞造成实验性耳蜗缺血模型；以扫描电镜观察缺血耳蜗听纤毛变化；以免疫组织化学方法观察原生型一氧化氮合酶（constitutive nitric oxide synthase，cNOS）在两组耳蜗的表达变化。结果 实验性耳蜗缺血造成内外毛细胞听纤毛散在的粘结、倒伏或缺失，内毛细胞听纤毛病变明显；内皮型一氧化氮合酶（endothelial NOS，eNOS）表达分布于蜗轴及内侧螺旋板内神经纤维、螺旋神经节、内侧螺旋缘、Corti器细胞及血管纹／螺旋韧带区；神经型一氧化氮合酶（neuron NOS，nNOS）主要分布于蜗轴及内侧螺旋板内神经纤维、螺旋神经节细胞，在Corti器细胞、血管纹细胞及内侧螺旋缘上皮细胞有较弱的表达；实验性缺血组豚鼠耳蜗与正常对照组耳蜗原生型一氧化氮合酶的表达无差异。结论 微栓塞耳蜗缺血引起散在听毛细胞损伤；两种原生型NOS在耳蜗固有表达，分布有交叉，功能存在相互代偿，但未发现因微栓塞缺血而引起变化。     

Dynamic physiological and molecular changes in gastric ulcer healing achieved by melatonin and its precursor L-tryptophan in rats

Abstract:  Following induction of gastric ulcer in rats by serosal application of acetic acid, local mucosal necrosis ensues accompanied by a reduction in mucosal microcirculation and by almost immediate expression of inducible nitric oxide (NO) synthase (iNOS) and proinflammatory cytokines. Daily application of melatonin (20 mg/kg) or l -tryptophan (100 mg/kg) accelerates ulcer healing by affecting the cyclooxygenase-2 (COX-2)–prostaglandin (PG) system with excessive production of protective PG, especially in later period of ulcer healing. Furthermore, expression of hypoxia inducible factor, vascular-endothelial growth factor, an activation of cNOS–NO system and the stimulation of sensory nerves with the expression and release of calcitonin gene related peptide (CGRP) appear to aid the restoration of mucosal repair and microcirculation in the ulcer bed. The enhanced expression of the melatonin MT₂ receptors (MT₂-R) combined with overexpression of key enzymes involved in biosynthesis of melatonin such as N -acetyltransferase and hydroxyindole- O -methyltransferase contribute to the acceleration of ulcer healing by this indole. Melatonin-induced acceleration of ulcer healing is also mediated by release of gastrin and ghrelin, the most potent stimulants of gastric mucosal cell proliferation and mucosal repair. These sequential steps in ulcer healing accelerated by melatonin can be interfered with by the blockade of MT₂R, COX-2/PG and cNOS/NO systems, and by reduction in the inflammatory iNOS/NO system. Thus, melatonin and its precursor l -tryptophan, trigger the cascade of molecular events leading to the functional improvement in ulcer healing.     

Expression of nitric oxide synthase in macula densa in streptozotocin diabetic rats

Summary Renal haemodynamic changes are suggested to be an early sign of diabetic glomerulopathy. The juxtaglomerular apparatus relevant to the renin-angiotensin system, known to be the site of nitric oxide (NO) production, is considered to play a role in the regulation of glomerular blood flow. This study was therefore designed to clarify whether in situ expression of nitric oxide synthase (NOS) is altered in the kidney of diabetic rats. Streptozotocin-induced diabetic rats with 6, 8, 12 and 32 weeks' diabetes duration and age-matched normal control rats were used. The expression of a constitutive form of NOS (cNOS, neural type) and NADPH diaphorase activity in the renal cortex were studied immunohistochemically and histochemically. Diabetic rats had lower body weight and heavier kidney mass compared to control rats at each time point examined. Mean glomerular surface area was greater in 6, 8 and 12-week diabetic rats compared to age-matched control rats. cNOS reaction was localized in the macula densa and appeared less intense in diabetic rats compared to age-matched control rats. The mean number of macula densa cells positive for cNOS in each glomerulus and in each glomerular area was significantly lower in diabetic rats compared to control rats at any time examined. In contrast, NADPH diaphorase activity was detected in both juxtaglomerular arterioles and macula densa cells. The staining reaction of NADPH diaphorase in the arterioles remained positive but appeared less intense in macula densa cells in diabetic rats. These results suggest that NO production in macula densa cells may be reduced in diabetic rats, modulating the vasodilatory function of afferent arterioles. Further investigation on the changes in inducible NOS as well as endothelial cNOS are necessary to clarify mechanisms of haemodynamic changes in the diabetic kidney. [Diabetologia (1996) 39: 793–799] Received: 27 September 1995 and in final revised form: 13 February 1996     

法舒地尔对肾盂输尿管连接梗阻部位cNOS表达的影响

目的探讨法舒地尔对肾盂输尿管连接梗阻（UPJO）部位cNOS表达的影响。方法成年新西兰兔24只,随机挑选16只行套叠法建立UPJO动物模型,模型成功后,平均分为两组：A组8只,经原生型一氧化氮合酶（cNOS）耳缘静脉给予法舒地尔1-5mg,1次／d,共10周。B组8只,仅予饲料喂养10周。C组8只,行假手术。均喂养至第10周后处死。H-E染色观察UPJO段病理变化、免疫组织化学法检测cNOS的表达。结果cNOS主要表达于管壁的肌层及黏膜层。B组表达明显低于A、C两组（P〈0．05）,A组低于C组（P〈0．05）。结论梗阻后的肾孟输尿管连接部cNOS表达减少,镜下示纤维组织增生,平滑肌断裂,提示cNOS在梗阻的发病过程中起重要作用。法舒地尔可能通过增强cNOS的表达,起到减轻梗阻部位的纤维化、阻碍连接部梗阻形成的作用。     

Involvement of interleukin-4 in down-regulation of endothelin-1 during gastric ulcer healing: Effect of ebrotidine

Aims: Healing gastric mucosal injury implies an orderly involvement of the signaling molecules that exert their influence on the processes leading to the restoration of the mucosal integrity. In this study, we investigated the effect of antiulcer agent, ebrotidine, on the course of events during gastric ulcer healing by analyzing the expression of interleukin-4 (IL-4), endothelin-1 (ET-1), tumor necrosis factor- (TNF-), and the mucosal activity of caspase-3, and constitutive (cNOS) and inducible (NOS-2) nitric oxide synthase. Methods: Rats with experimentally induced chronic gastric ulcers were administered twice daily for 14 days with either ebrotidine at 100 mg/kg or vehicle, and at different stages of treatment their stomachs were used for macroscopic and biochemical assessments. Results: The ulcer onset was characterized by a massive epithelial apoptosis associated with a significant increase in caspase-3, NOS-2, TNF- and ET-1, and a decline in the mucosal expression of cNOS and IL-4. The healing was reflected in a rapid recovery in IL-4, decrease in apoptosis, caspase-3, TNF-, ET-1 and NOS-2, and a slow recovery in cNOS, and the process was accelerated in the ebrotidine-treated group. In the absence of ebrotidine the expression of IL-4 returned to the level of normal mucosa by the seventh day of healing, and that of ET-1 and TNF- by the fourteenth day. An accelerated ulcer healing (7 days) with ebrotidine treatment was associated with IL-4 recovery by the fourth day, ET-1 by the seventh day, and TNF- and cNOS by the 10th day. However, in both groups of animals the apoptotic DNA fragmentation rate, and the expression of caspase-3 and NOS-2 remained significantly elevated even after the ulcer healed. Conclusions: Our findings suggest that a drop in the mucosal expression of IL-4 at the ulcer onset causes dysregulation of ET-1 production, induction of TNF- and triggers the apoptotic events that affect the efficiency of the repair process. Ebrotidine, by eliciting rapid recovery in IL-4, is capable of suppression the mucosal apoptotic events and accelerate the ulcer healing.     

The regional distribution of nitric oxide synthase activity in the spinal cord of the dog

The aim of this study was to examine the distribution of calcium-dependent nitric oxide synthase activity (cNOS) in the white and gray matter in cervical, thoracic, lumbar and sacral segments of the spinal cord and cauda equina of the dog. The enzyme's activity, measured by the conversion of [3H]arginine to [3H]citrulline revealed considerable region-dependent differences along the rostrocaudal axis of the spinal cord in general and in cervical (C1, C2, C4, C6 and C8) and lumbar (L1-L3, L4-L7) segments in particular. In the non-compartmentalized spinal cord, the cNOS activity was lowest in the thoracic and highest in the sacral segments. No significant differences were noted in the gray matter regions (dorsal horn, intermediate zone and ventral horn) and the white matter columns (dorsal, lateral and ventral) in the upper cervical segments (C1-C4), except for a significant increase in the ventral horn of C4 segment. In C6 segment, the enzyme's activity displayed significant differences in the intermediate zone, ventral and lateral columns. Surprisingly, extremely high cNOS activity was noted in the dorsal horn and dorsal column of the lowest cervical segment. Comparing the enzyme's activity in upper and lower lumbar segments of the spinal cord, cNOS activity prevailed in L4-L7 segments in the dorsal horn and in all the above mentioned white matter columns.     

Nitric Oxide Synthase Inhibition Delays Axonal Degeneration and Promotes the Survival of Axotomized Retinal Ganglion Cells

Nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been implicated in neuronal cytotoxicity following trauma to the central nervous system. The aim of the present study was to examine the role of NO in mediating axotomy-induced retinal ganglion cell (RGC) death. We observed increases in iNOS expression by microglia and Müller cells in the retina after optic nerve transection. This was paralleled by the induced expression of constitutive NOS (cNOS) in RGCs which do not normally express this enzyme. In order to determine if NO is cytotoxic to axotomized RGCs, the nonspecific NOS inhibitors Nomega-nitro-L-arginine (NOLA) or N-nitro-L-arginine methyl ester (L-NAME) were delivered to the vitreous chamber by intraocular injections. Both NOLA and L-NAME significantly enhanced RGC survival at 7, 10, and 14 days postaxotomy. The separate contributions of iNOS and cNOS to RGC degeneration were examined with intraocular injections of the specific iNOS inhibitor L-N(6)-(I-iminoethyl)lysine hydrochloride or the specific cNOS inhibitor L-thiocitrulline. Our results suggest that cNOS plays a greater role in RGC degeneration than iNOS. In addition to enhancing RGC survival, NOS inhibitors delayed the retrograde degeneration of RGC axons after axotomy. We conclude that NO synthesized by retinal iNOS and cNOS plays a major role in RGC death and retrograde axonal degeneration following axotomy.     

Chitooligosaccharides in combination with interferon-gamma increase nitric oxide production via nuclear factor-kappaB activation in murine RAW264.7 macrophages.

A low-molecular weight chitosan (LMWC) with a molecular mass of 20 kDa and a chitooligosaccharide mixture (oligomixture) which is composed of sugars with a degree of polymerization (DP) of 1-6 were isolated from the chitosan hydrolysate. The effects of the chitosan hydrolysate, LMWC and oligomixture on the production of nitric oxide (NO) in RAW 264.7 macrophages were evaluated, and their effects on NF-kappaB activation and the gene expression of inducible NO synthase (iNOS) were further investigated. None of the tested 3 samples of hydrolysate, LMWC and oligomixture alone affected the NO production in RAW 264.7 macrophages. However, treatment of macrophages with a combination of hydrolysate/oligomixture and interferon-gamma (IFN-gamma) significantly induced NO production in a dose-dependent manner, whereas a combination of LMWC and IFN-gamma inhibited NO production. These effects on NO synthesis were evidenced via regulating the iNOS gene expression. Both hydrolysate and oligomixture promoted the migration of NF-kappaB into the nucleus and enhanced its DNA binding activity. MG132, a specific inhibitor of NF-kappaB, eliminated the NO synthesis in IFN-gamma plus hydrolysate/oligomixture-induced RAW264.7 macrophages. The treatment of RAW264.7 macrophages with anti-CD14, anti-TLR4, and anti-CR3 antibodies significantly blocked NO production induced by IFN-gamma plus hydrolysate/oligomixture. These results demonstrated that the oligomixture, which is the main functional component in the chitosan hydrolysate, in combination with IFN-gamma, synergistically induced NF-kappaB activation and NO production through binding with the receptors of CD14, TLR4 and CR3 in RAW264.7 macrophages.