反复发热惊厥大鼠脑内NOS/NO体系的变化

目的研究一氧化氮合酶(NOS),一氧化氮(NO)体系与反复发热惊厥(febrile seizures,FS)的关系。方法采用热水浴诱导大鼠FS,隔日1次,每次大鼠进行热水浴的时间不超过5min,共10次。大鼠随机分为2组：正常对照组和发热组,后者又根据惊厥与否进一步分为发热对照组和反复FS组。用原位杂交法观察大脑皮层神经元型NOS(nNOS)mRNA的变化,用分光光度计检测大鼠脑组织及血浆中NO含量,用放射免疫法检测大鼠脑组织cGMP含量。结果在大脑皮层深层,FS组nNOS表达阳性的神经元明显增高,而发热对照组仅出现少量nNOS阳性神经元,正常对照组偶见nNOS阳性神经元;脑组织及血浆中NO含量各组间无统计学意义;FS组脑组织cGMP含量吸显高于正常对照组及高热对照组。结论大鼠反复FS后24h脑内nNOS mRNA表达增高,但此时NO不见增多,脑组织cGMP水平增高,可能由于其他途径调节所致。     

脑缺血预处理对大鼠海马CA1区一氧化氮合酶活性和一氧化氮含量的影响

目的：观察脑缺血预处理（CIP）后大鼠海马一氧化氮合酶（NOS）活性和一氧化氮（NO）含量的变化，探讨NO在脑缺血耐受（BIT）诱导中的作用。 方法： 将140只凝闭双侧椎动脉的Wistar大鼠分为sham、CIP组、损伤性缺血组和CIP＋损伤性缺血组。夹闭双侧颈总动脉致全脑缺血3 min作为CIP，10 min作为损伤性缺血，第4组中CIP与损伤性缺血之间间隔3 d。所有动物均于末次脑缺血恢复再灌注后0 h、2 h、16 h、24 h、36 h、72 h和7 d（每个时点n=5）取海马CA1区脑组织，分光光度法检测NOS活性，硝酸还原酶法检测NO2-/NO3-含量。 结果： CIP组NOS活性和NO2-/NO3-含量于再灌注后16 h开始升高，24 h达高峰，接近sham组的1.5倍，36 h降至基础水平，其升高的持续时间短于BIT诱导的时程（1-7 d）；损伤性缺血组NOS活性和NO2-/NO3-含量的变化趋势与CIP组类似，但其峰值（24 h）超过sham组的2倍，显著大于CIP组（P<0.05）；CIP+损伤性缺血组NOS活性和NO2-/NO3-含量亦有一定程度的升高，但其峰值（24 h）明显低于损伤性缺血组(P<0.05)。 结论： CIP引起NOS活性及NO2-/NO3-含量的适度增加，参与BIT的诱导；同时CIP阻止损伤性缺血后NO的过量生成所致的细胞毒性作用可能是其诱导BIT的另一途径。     

感染性休克下调大鼠血管内皮和血小板L-精氨酸/一氧化氮通路

目的：观察感染性休克对大鼠血小板及血管L-精氨酸(L-Arg)/一氧化氮合酶(NOS) /一氧化氮(NO)通路的影响及其相互联系,探讨感染性休克损伤的机制。方法:利用盲肠结扎并穿孔复制早期和晚期感染性休克大鼠模型,采用Greiss法测定血管各层及血小板孵育液亚硝酸盐(NO^-₂/NO^-₃)含量;以同位素示踪法检测其NOS活性及L -Arg转运。结果:早、晚期感染性休克大鼠血小板和主动脉内膜NO-2/NO-3水平、NOS 活性及低亲合L-Arg 转运量均显著低于假手术组(高亲合L- Arg 转运量在早期休克增加、晚期休克降低);而中膜和外膜的NO^-₂/NO^-₃水平、NOS 活性及L-Arg 转运量则显著高于假手术组,均以休克晚期改变为显著。血小板和主动脉内膜NO^-₂/NO^-₃生成、NOS 活性及高、低亲合L-Arg 转运的改变均呈正相关(均P< 0. 01)。结论:感染性休克下调血管内膜和血小板的L-Arg/NO通路,上调血管中膜和外膜L-Arg/ NO通路。提示检测血小板L-Arg/NO通路的变化可能反映休克时血管内皮功能的损伤。     

内源性H2S在CCK-8减轻脂多糖所致急性肺损伤中的作用

目的：探讨内源性硫化氢(H₂S)在八肽胆囊收缩素(CCK-8)减轻脂多糖（LPS）所致急性肺损伤(ALI)中的作用。方法: 将84只SD大鼠随机分为正常对照组、LPS组（经气管内滴注LPS复制ALI）、NaHS(H₂S供体)+LPS组、炔丙基甘氨酸［胱硫醚-γ-裂解酶（CSE）抑制剂，PPG］+LPS组、CCK-8+LPS组、PPG+CCK-8+LPS组和CCK- 8组。给药后分别于4 h和8 h处死动物，测定肺湿/干比值；光镜观察肺组织形态学改变；化学法检测血浆H₂S含量，肺组织MDA含量、MPO活性和CSE活性；放免法检测肺组织P-selectin含量；RT-PCR检测肺组织CSE mRNA的表达；并行支气管肺泡灌洗，检测支气管肺泡灌洗液(BALF)中蛋白含量。结果: 气管内滴注LPS可引起肺组织明显的形态学改变；肺湿/干比值、BALF中蛋白含量及肺组织MDA、MPO活性和P-selectin水平增高；血浆H2S含量、肺组织CSE活性及CSE mRNA表达下降。预先给予NaHS或CCK-8可显著减轻LPS所致的上述改变，且血浆H₂S含量、肺组织CSE活性及CSE mRNA表达高于相应的LPS组；预先给予PPG可加重LPS所致的肺损伤，而血浆H₂S含量、肺组织CSE活性及CSE mRNA表达分别低于相应的LPS组和CCK-8+LPS组。结论: CCK-8可通过内源性H₂S介导的抗氧化、抑制PMN黏附聚集等效应发挥减轻LPS所致肺损伤的作用。     

睡眠剥夺对大鼠一氧化氮和一氧化氮合酶的影响

目的：探讨睡眠剥夺对大鼠脑组织一氧化氮（NO）及一氧化氮合酶（NOS）影响。方法：采用小平台水环境法（Flower Pot)制作大鼠睡眠剥夺模型，采用化学法和酶法观察不同时间睡眠剥夺后大鼠额叶、海马、中脑和下丘脑NO含量及NOS活性变化。结果：与正常对照组及大平台组比较，大鼠在SD后额叶和海马的NO含量及NOS活性增高，有显著性差异（P＜0．01－0．05），其余脑区无显著性差异（P＞0．05）。随着剥夺时间的延长，额叶和海马NO含量及NOS活性增高更加明显。结论：睡眠剥夺可致NO及NOS升高，可能与其学习障碍有关，NO可能参与大鼠的睡眠调节。     

参麦注射液对大鼠急性心肌缺血及eNOS mRNA表达的影响

目的：探讨参麦注射液(SMI)预处理可能对 大鼠急性心肌缺血及心肌内皮型一氧化氮合酶(eNOS)mRNA表达的影响。方法:大鼠随机分为对照组、模型组、处理组1和处理组2，应用异丙肾上腺素建立大鼠心肌 缺血模型,以心电图ST段抬高值作为心肌缺血指标，硝酸还原酶法测定血清和心肌的NO^-₂/NO^-₃，RT-PCR检测心肌eNOS mRNA表达。结果：模型组各时点的心 电图ST段均显著高于对照组，缺血20 min时达高峰，血清和心肌NO^-₂/NO^-₃含量及 心肌eNOS mRNA表达均低于对照组；两个处理组于缺血20、30、40 min时的心电图ST段抬高 幅度均显著低于模型组，血清和心肌NO^-₂/NO^-₃含量均显著高于模型组，心肌eNOS mRNA表达强于模型组；处理组1和处理组2比较，心电图ST段抬高幅度、血清和心肌的NO^-₂/NO^-₃含量及心肌eNOS mRNA表达差异均无显著。结论：参麦注 射液可能是通过促进心肌组织eNOS mRNA表达、增强eNOS活性而提高NO水平，达到抗心肌缺 血的作用。     

哮喘模型大鼠肺组织一氧化氮合酶的分布

研究一氧化氮 (NO)在哮喘大鼠肺组织中的作用。采用组化法观察一氧化氮合酶 (NOS)在大鼠哮喘模型肺组织中的分布 ,应用免疫组化法观察大鼠哮喘模型气道mIL 2R+ 细胞变化。结果显示 ,哮喘大鼠肺NADPH染色呈强阳性 ,并波及肺泡膈。肺组织中NOS含量明显高于对照组 [哮喘组 (37 44± 0 77)pmol/mg,对照组 (8 73± 0 79)pmol/mg],气道炎性细胞增多 ,特别是mIL 2R+ 细胞 [哮喘组mIL 2R+ 细胞为 (2 3 8± 7 9)个 ,对照组为 0个 ],而NOS抑制剂DMA组气道炎性细胞少 ,NADPH呈阴性。提示NO是哮喘大鼠的炎性效应分子。     

败血症休克大鼠血管外膜L-精氨酸/一氧化氮合酶/一氧化氮通路的变化

目的：观察败血症休克大鼠主动脉外膜L-精氨酸（L-Arg）转运，一氧化氮合酶（NOS）活性和一氧化氮(NO)生成的变化。方法:雄性Wistar大鼠盲肠结扎并穿孔复制败血症休克模型。测定大鼠主动脉外膜亚硝酸盐(NOx)含量、一氧化氮合酶(NOS)活性及L-精氨酸(L-Arg)转运；RT-PCR方法测定诱导型一氧化氮合酶(iNOS) mRNA水平。结果:严重感染休克大鼠呈现严重的血流动力学紊乱, 心功能抑制。败血症休克大鼠表现为严重的低血糖和高乳酸血症。血管外膜iNOS的mRNA水平均明显高于假手术组（均P<0.01），主动脉外膜NOx生成、NOS活性及L-Arg转运速率显著高于假手术组（P<0.01）。结论:败血症休克时血管外膜L-Arg/NOS/NO系统激活在败血症休克发病中可能起重要作用。     

木犀草素对H2O2氧化损伤的血管内皮细胞的影响

目的： 探讨木犀草素（luteolin）对氧化损伤的血管内皮细胞（endothelial cells）的影响。方法： 体外培养内皮细胞，将细胞分为7组，即空白对照组（control）、溶剂对照组（DMSO）、氧化损伤组（H₂O₂）、氧化损伤加入槲皮素对照组(quercetin+H₂O₂)、氧化损伤加入木犀草素低、中、高浓度组(luteolin-L+H₂O₂、luteolin-M+H₂O₂、luteolin-H+H₂O₂)。将750 μmol/L H₂O₂作用于加入槲皮素及不同浓度木犀草素预培养24h的内皮细胞，继续培养18h（半胱氨酸蛋白酶表达测定时间为14h），然后观察木犀草素对细胞培养液内乳酸脱氢酶（LDH）、一氧化氮（NO）、乳酸脱氢酶（LDH）丙二醛（MDA）含量和细胞活力的影响, 并对细胞进行流式细胞和免疫组化分析，观察该药对细胞凋亡和半胱氨酸蛋白酶-3（caspase-3）表达的影响。 结果： 木犀草素呈剂量依赖性降低H₂O₂对内皮细胞生长抑制率，降低MDA、LDH量，增加培养液中 NO^-₂/NO^-₃含量，并显著抑制半胱氨酸蛋白酶-3阳性表达，减少细胞凋亡数量，各指标差异显著（P<0.01）。结论： 木犀草素可拮抗和修复过氧化氢诱导的血管内皮细胞的损伤，其作用可能与抗氧化、促进NO释放，抑制caspase-3表达有关。     

大鼠严重烧伤后体内一氧化氮水平变化与预后的关系

目的:研究一氧化氮(NO)及一氧化氮合酶(NOS)在严重烧伤早期大鼠体内的变化规律及其与预后的可能联系。方法:检测严重烧伤前后大鼠血液中NO代谢产物NO^-₂/NO^-₃及脑、肺脏和十二指肠组织中神经型(nNOS)和诱生型一氧化氮合酶(iNOS)蛋白的水平,同时统计各组大鼠的存活率。结果:烧伤后大鼠血液中NO^-₂/NO^-₃水平显著增高,非选择性NOS抑制剂L-NAME和选择性iNOS抑制剂氨基胍(AG)对其均有抑制作用,以L-NAME为甚;nNOS蛋白在伤后部分升高,L-NAME和AG均轻度上调nNOS水平;iNOS在正常组织中不表达,烧伤后表达异常增高,L-NAME和AG对此均无影响;与对照组比较,AG组大鼠存活时间延长,L-NAME组存活时间缩短。结论:严重烧伤后的血管扩张、血压降低和血管反应性低下与iNOS蛋白水平过度增高及其释放的大量NO关系密切。     

Pseudorabies virus-induced expression of nitric oxide synthase isoforms

In addition to its role as a neurotransmitter, studies have postulated both neuroprotective and neurotoxic roles for nitric oxide (NO) generated in response to infections with neurotropic viruses. This study examined the expression of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) isoforms of NOS induced by neuronal infection with virulent and attenuated strains of pseudorabies virus (PRV). Caudal brainstem neurons infected by peripheral inoculation of the viscera served as the model system. Neuronal infection induced the expression of nNOS and iNOS, but the timing and the apparent magnitude of NOS expression varied according to the virulence of the infecting strain of virus. Expression of nNOS was observed in infected neurons that did not express this enzyme in control animals, and the onset of expression was earlier in animals infected with virulent PRV. Expression of iNOS was largely restricted to monocytes and macrophages that invaded the brain in response to PRV infection. These iNOS-expressing cells were observed earlier in animals infected with the virulent virus, and were differentially concentrated in areas exhibiting virus-induced neuropathology. Collectively, these data suggest functionally diverse roles for NO in the brain response to PRV neuronal infection.     

硫化氢供体对阿霉素心肌病大鼠氧化应激作用的影响

目的： 探讨外源性给予硫化氢供体硫氢化钠(NaHS)对阿霉素(adriamycin, ADR)心肌病大鼠氧化应激作用的影响。方法：雄性Wistar大鼠54只，随机分为5组：(1) ADR组(n=12)：腹腔注射ADR，每次2.5 mg/kg，每周1次，共用药10周；(2)ADR+小剂量NaHS组（n=12）：ADR用药方法同ADR组，同时经腹腔注射NaHS，2.8 μmol·kg-1·d-1，连续用药10周；(3)ADR+大剂量NaHS组（n=12）：ADR用药方法同ADR组，同时经腹腔注射NaHS，14 μmol·kg-1·d-1，连续用药10周；(4)对照组（n=9）：用相同容量的生理盐水代替ADR，给药方法同ADR组；(5)NaHS组（n=9）：经腹腔注射NaHS，14 μmol·kg-1·d-1，连续用药10周。于第10周检测大鼠心功能和血流动力学指标,并测定其血清及心肌组织中H2S、脂质过氧化物丙二醛(MDA) 的含量以及超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性，比较其中的差异。结果：ADR组大鼠心功能较对照组明显降低(均P<0.01)，且血清及心肌组织H2S含量均明显低于对照组(P<0.01），MDA含量明显较对照组升高(均P<0.01)，SOD的活性明显降低(均P<0.01)，GSH-Px的活性亦明显降低(血清及心肌组织：P<0.05，P<0.01)；经外源性补充H2S供体NaHS后，大鼠心功能较前明显改善，ADR+大剂量NaHS组心肌组织中过氧化产物MDA含量明显降低(均P<0.01)，而血清MDA含量无明显差异(P>0.05)；ADR+大剂量NaHS组血清SOD活性明显升高(P<0.01)，而心肌组织SOD活性无明显差异(P>0.05)；ADR+大剂量NaHS组心肌组织GSH-Px活性明显增高(均P<0.05)，而血清GSH-Px活性无明显差异(P>0.05)。结论：硫化氢参与了大鼠阿霉素心肌病的发病过程,外源性补充硫化氢供体NaHS可以改善阿霉素心肌病大鼠心功能，降低心肌组织中脂质过氧化物的含量，显著提高抗氧化酶体系的活性，减轻氧化应激损伤，从而参与对大鼠心肌的保护机制。     

氨基胍等对严重烧伤大鼠一氧化氮表达及烧伤休克的影响

目的:研究一氧化氮合酶(NOS)抑制剂与严重烧伤大鼠体内NO产量、NOS表达以及平均动脉压(MAP)变化的关系。方法:复制大鼠重症烧伤模型,检测应用非选择性NOS抑制剂L-NAME和选择性诱生型NOS(iNOS)抑制剂氨基胍(AG)后大鼠血液中NO代谢产物(NO₂^-／NO₃^-)以及肺和十二指肠组织中神经型NOS(nNOS)mRNA的表达水平,同时测定各组大鼠的MAP。结果:烧伤后大鼠血液中NO₂^-／NO₃^-含量显著增高,L-NAME和AG都能抑制NO₂^-／NO₃^-的升高,P<0.01;烧伤后nNOS的mRNA表达在肺和十二指肠中均有不同程度升高,AG和L-NAME使nNOS表达增加,L-NAME作用更为显著,P<0.01;烧伤后大鼠MAP略有上升,然后进行性下降,L-NAME组大鼠MAP显著升高,但于3h后急剧下降,AG组大鼠MAP下降速度明显低于对照组。结论:结构型NOS(cNOS)与iNOS在烧伤休克病理生理过程中的作用明显不同,iNOS活性过度增高与烧伤休克发病关系密切。     

mRNA and protein expression and activities of nitric oxide synthases in the lumbar spinal cord of neonatal rats after sciatic nerve transection and melatonin administration

Sciatic axotomy in 2-day-old rats (P2) causes lumbar motoneuron loss, which could be associated with nitric oxide (NO) production. NO may be produced by three isoforms of synthase (NOS): neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). We investigated NOS expression and NO synthesis in the lumbar enlargement of rats after sciatic nerve transection at P2 and treatment with the antioxidant melatonin (sc; 1 mg/kg). At time points ranging from P2 to P7, expression of each isoform was assessed by RT-PCR and immunohistochemistry; catalytic rates of calcium-dependent (nNOS, eNOS) and independent (iNOS) NOS were measured by the conversion of [3H]L-arginine to [3H]L-citrulline. All NOS isoforms were expressed and active in unlesioned animals. nNOS and iNOS were detected in some small cells in the parenchyma. Only endothelial cells were positive for eNOS. No NOS isoform was detected in motoneurons. Axotomy did not change these immunohistochemical findings, nNOS and iNOS mRNA expression and calcium-independent activity at all survival times. However, sciatic nerve transection reduced eNOS mRNA levels at P7 and increased calcium-dependent activity at 1 and 6 h. Melatonin did not alter NOS expression. Despite having no action on NOS activity in unlesioned controls the neurohormone enhanced calcium-dependent activity at 1 and 72 h and reduced calcium-independent catalysis at 72 h in lesioned rats. These results suggest that NOS isoforms are constitutive in the neonatal lumbar enlargement and are not overexpressed after sciatic axotomy. Changes in NO synthesis induced by axotomy and melatonin administration in the current model are discussed considering some beneficial and deleterious effects that NO may have.     

Modulating effect of hydrogen sulfide on gamma-aminobutyric acid B receptor in recurrent febrile seizures in rats

Hydrogen sulfide (H2S) is recognized as a new neuromodulator in regulating various brain functions. Some of our recent studies showed that H2S alleviates the hippocampal damage induced by recurrent febrile seizures (FS). In the present study, we used a rat model of recurrent FS and found that sodium sulfhydrate (NaHS, a donor of H2S) down-regulated the expression of c-fos and increased the expression of gamma-aminobutyric acid B receptor subunits 1 (GABA(B)R1) and 2 (GABA(B)R2). Hydroxylamine (an inhibitor of cystathionine b-synthase) up-regulated the expression of c-fos and down-regulated the expression of GABA(B)R2, but did not change the expression of GABA(B)R1. These results suggest that H2S plays a regulatory role through modulating GABA(B)R function in the pathogenesis of recurrent FS.     

Age-related changes in nitric oxide synthase and arginase in the rat prefrontal cortex

Increasing evidence suggests that nitric oxide (NO), generated by nitric oxide synthase (NOS) from l-arginine, plays an important role in the ageing process. The present study, for the first time, investigates age-related changes in NOS and arginase, an enzyme that shares a common substrate with NOS, in the prefrontal cortex of rats assessed with and without prior behavioural testing. A significant increase in total NOS activity was found in the prefrontal cortex in aged (24-month-old) as compared with young (4-month-old) rats. Western blotting revealed that there were no significant differences between young and aged rats in neuronal NOS (nNOS) and endothelial NOS (eNOS) protein expression. Inducible isoform of NOS (iNOS), in terms of activity and protein expression, was not detected in either group. Total arginase activity and arginase I and II protein expression did not differ between the young and aged groups. The present findings support the contribution of NOS/NO to ageing but question the importance of iNOS in the normal ageing process.     

Neuronal nitric oxide synthase is the dominant nitric oxide supplier for the survival of dorsal root ganglia after peripheral nerve axotomy

This study was designed to determine whether nitric oxide supply may be a major factor in the survival of dorsal root ganglia in a sciatic nerve injury model. Wild-type (WT) mice were compared with knockout (KO) mice lacking neuronal nitric oxide synthase (nNOS) or endothelial (eNOS). The NO-generating capacities were analysed by NOS immunohistochemistry and NADPH-diaphorase staining 1, 2, 6, and 12 weeks after nerve transection. The occurrence and morphological type of neuronal death were determined by TUNEL reaction and ultrastructural examination. Cell loss following nerve section, whist dependent on the availability of NO, as shown by its marked elevation in nNOS KO mice, did not correlate well with nNOS expression in WT animals. Whereas a lack of eNOS was tolerated, deficiency of nNOS led to an enhanced cell loss. The results suggest a crucial role of NO supply after transection of peripheral nerves with a particular significance of the nNOS isoform.     

Localization of nitric oxide synthase in human trophoblast cells: role of nitric oxide in trophoblast proliferation and differentiation

PROBLEM: There are conflicting reports about the isoform of nitric oxide synthase (NOS) present in trophoblast cells. In this study, we have examined the presence of different NOS isoforms in trophoblast cells. In addition, the role of nitric oxide (NO) in trophoblast function has also been studied by investigating the possible role of nitric oxide in trophoblast proliferation and differentiation. METHOD OF STUDY: NOS isoforms in primary-term trophoblast and JEG-3 cells were identified by immunocytochemistry. The intracellular localization of this enzyme was determined by confocal laser scanning microscopy. Trophoblast proliferation was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasolium bromide (MTT) conversion assay and cellular differentiation was monitored by human chorionic gonodotropin (hCG) and progesterone secretion, measured by radioimmunoassay. RESULTS: The immunoreactive NOS was present in human trophoblast cells of normal term placenta and JEG-3 cells (a choriocarcinoma cell line) maintained in culture. Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase activity overlapped with the immunostaining of NOS. Specific antibodies against the different isoforms of NOS detected the presence of neuronal-type NOS (nNOS) only. The other two isoforms, i.e., eNOS (endothelial) and iNOS (macrophage specific) were completely absent. The nNOS was localized in cell cytoplasm. In culture, JEG-3 cells normally undergo proliferation and cytotrophoblast cells in primary culture differentiate to form hormone-secreting syncytial cells. Sodium nitroprusside (SNP), a nitric oxide donor, when added to the culture, significantly increased proliferation of JEG-3 cells and inhibited the differentiation of cytotrophoblast cells. The arrest by SNP in the formation of syncytial cells was further evidenced by the low secretion profile of hCG and progesterone. CONCLUSIONS: Our findings suggest for the first time the presence of nNOS in the human trophoblast cells and a previously unrecognized role of NO in trophoblast proliferation and differentiation.