甲醛足底致痛大鼠脊髓后角神经元内蛋白激酶C活性改变对一氧化氮合酶的影响

目的通过改变蛋白激酶C（protein kinaseC，PKC）的活性观察其对甲醛炎性痛及痛觉过敏时大鼠脊髓后角一氧化氮合酶（nitric oxide synthase，NOS）尤其是对诱生型NOS（induciable NOS,iNOS）的影响，以探讨在该过程中PKC的作用机制。方法将实验动物54只分为9组，每组6只，分别为正常组、甲醛12h组、甲醛24h组、甲醛12h加0．9％氯化钠溶液组、甲醛24h加0．9％氯化钠溶液组、甲醛12h加佛波醇脂（PMA）组、甲醛24h加PMA组、甲醛12h加灯盏花素乙（CH）组和甲醛24h加CH组。先进行疼痛行为学检测，然后分别于注射甲醛后12,24h将大鼠麻醉后取材，采用免疫组织化学方法观察脊髓后角iNOS阳性神经元数目及染色深度，以探讨PKC对NOS的影响。结果与甲醛12h组比较。甲醛12h加PMA组iNOS阳性细胞数明显增加（P〈0．01），神经细胞及神经纤维染色也明显加深（P〈0．01）；甲醛12h加CH组iNOS阳性细胞数明显减少（P〈0．01），神经细胞及神经纤维染色明显变浅（P〈0．01）。同时产生相应的行为痛觉过敏变化。结论鞘内注射PKC兴奋剂PMA能显著增加L5脊髓后角神经元NOS的活性；鞘内注射PKC抑制剂CH能显著抑制L5脊髓后角神经元iNOS的活性。在甲醛炎性痛及痛觉过敏中，脊髓后角iNOS活性在一定程度上受PKC调控。     

中缝背核注射L-NAME抑制炎性痛大鼠脊髓Fos的表达

目的 观察中缝背核(DRN)内一氧化氮合酶(NOS)对炎性痛大鼠脊髓伤害性信息传递的调控作用.方法 建立大鼠单侧足底甲醛炎性痛模型,采用行为学、c-Fos免疫组织化学及烟酰胺腺嘌呤二核苷酸磷酸黄递酶(NADPH-d)组织化学技术,观察甲醛诱发炎性痛后大鼠DRN内NADPH-d、NADPH-d/Fos双标阳性神经元数量的变化;同时观察DRN内预先给予NOS抑制剂L-NAME对甲醛致痛大鼠疼痛评分及脊髓Fos蛋白表达的影响.结果 单侧足底注射甲醛后,DRN内NADPH-d、NADPH-d/Fos双标阳性神经元的数量增加.DRN内预先注射L-NAME降低炎性痛大鼠的疼痛学评分及脊髓Fos阳性神经元的数量.结论 DRN内NOS可能促进甲醛炎性痛大鼠脊髓伤害性信息的传递.     

鞘内注射促皮质素抑制甲醛痛敏大鼠脊髓NOS阳性神经元增多

目的:研究鞘内注射促皮质素(Cor)对甲醛痛敏大鼠脊髓背角一氧化氮合酶(NOS)阳性神经元增多的影响。方法:采用痛级均数(PIR)测定、NADPH-d组织化学法、Fos免疫组织化学法染色,观察鞘内注射(ith)Cor对甲醛痛敏大鼠脊髓背角NOS阳性神经元、Fos免疫反应神经元、NOS/Fos双标记神经元及痛敏的影响。结果:ith Cor(0.5-1.5U)均能显著抑制甲醛引起的大鼠脊髓背角NOS、Fos、NOS/Fos阳性神经元的增多和痛敏反应,其作用为ith NOS底物左旋精氨酸(Arg,5-15nmol)部分翻转。结论:Cor通过抑制大鼠脊髓背角NOS阳性神经元的增多抑制痛敏。     

鞘内注射左旋布比卡因下调甲醛炎性痛大鼠远位触液神经元P物质的表达

目的观察鞘内注射左旋布比卡因对甲醛炎性痛大鼠P物质(substanceP,SP)在脊髓背角及远位触液神经元(thedistal cerebrospinal fluid contacting neuron,dCSF-CN)表达的影响。方法采用CB-HRP大鼠侧脑室注射示踪标记dCSF-CN;48h后先鞘内注射0.5%左旋布比卡因10μl(并以鞘内注射10μl人工脑脊液作对照),大鼠左后掌足跖部皮下注射2.5%福尔马林建立炎性痛模型,记录大鼠舔足时间,1h后测定机械缩足阈值(mechanical withdrawal threshold,MWT)评估机械痛敏;免疫组织化学光镜镜检及免疫电镜镜检P物质在脊髓背角(L4~5)及dCSF-CN的表达。结果鞘内注射左旋布比卡因减少大鼠福尔马林试验的舔足时间(P<0.01),MWT值无变化(与基础值对比,P>0.05),SP在脊髓背角及dCSF-CN的表达弱于对照组(P<0.01)。结论鞘内注射左旋布比卡因减低脊髓伤害性疼痛反应及下调SP在dCSF-CN的表达。     

海马内注射MK-801对甲醛炎性痛大鼠海马NO含量的影响

目的观察海马内给予N-甲基-D-天门冬氨酸(NMDA)受体拮抗剂MK-801对足底注射甲醛诱导的海马一氧化氮生成增加的影响。方法采用硝酸还原酶法测定海马组织NO含量。结果足底注射甲醛后大鼠即出现舔、咬注射侧脚掌等疼痛相关表现,注射甲醛后12h时,海马组织NO含量显著增加;预先海马内注射MK-801,可使甲醛炎性痛大鼠海马组织NO含量明显降低。结论海马内注射MK-801可逆转甲醛炎性痛诱导的海马NO产生的增加,海马内NO生成增加可能与NMDA受体的活动有关。     

乳铁蛋白对福尔马林致痛大鼠行为学及一氧化氮合酶表达的影响

目的通过观察鞘内注射乳铁蛋白对福尔马林致痛大鼠行为学及一氧化氮合酶（NOS）表达的影响，探讨乳铁蛋白可能的抗伤害机制。方法雄性SD大鼠80只，随机分为生理盐水组（NS组）和福尔马林组（F组）；实验组分为乳铁蛋白1ug-福尔马林组（F-R1组），乳铁蛋白10ug-福尔马林组（F—R10组）和乳铁蛋白100ug-福尔马林组（F—R100组）。五组鞘内分别给予NS 20ul，NS 20ul，乳铁蛋白1ug，乳铁蛋白10ug，乳铁蛋白100ug；10min后，除NS组大鼠组底注射NS 100ul外，其余各组均给予5％福尔马林100ul。经以上处理8只动物在福尔马林处理后30min取脊髓观察NOS的表达，另8只动物在福尔马林处理后0～1h观察行为学。结果F组的缩腿舔爪的时间和脊髓NOS表达显著长于，强于NS组；预先给予乳铁蛋白能抑制以上的作用，且呈剂量依赖性。结论乳铁蛋白明显抑制福尔马林致痛大鼠痛行为及NOS表达，可能是其产生抗伤害作用机制之一。     

鞘内注射可乐定对甲醛致痛大鼠脊髓磷酸化cAMP反应结合蛋白表达影响

目的研究鞘内注射可乐定对大鼠甲醛炎性痛脊髓磷酸化cAMP反应结合蛋白(pCREB)表达的影响。方法24只成年雄性SD大鼠,体质量230～270g,随机分为3组:空白对照组(NS组),致痛组(F组),可乐定组(CF组),每组8只鞘内注射可乐定或生理盐水后,在大鼠左足底部注射5%甲醛建立模型,记录大鼠1h内每5min的缩腿舔爪时间。待行为学观察结束,取脊髓L4~L5节段免疫组织化学染色,计数pCREB免疫反应阳性(pCREB-IR)神经细胞数量。结果CF组的缩腿舔爪时间显著短于F组(P<0.01)。F组pCREB-IR神经细胞数量多于NS组(P<0.01),CF组pCREB-IR神经细胞数量少于F组(P<0.01)。结论鞘内注射可乐定能明显抑制甲醛致痛大鼠的疼痛反应。甲醛致痛大鼠脊髓pCREB表达增加,可乐定可使甲醛致痛大鼠pCREB表达减少。pCREB可能是参与鞘内注射可乐定镇痛的重要分子物质。     

吗啡依赖及戒断大鼠脊髓和脑干中一氧化氮合酶基因的表达

目的　观察吗啡依赖或吗啡戒断大鼠脊髓和脑干中一氧化氮合酶 (NOS)基因表达的变化。方法　以 β actin为内参照 ,用逆转录聚合酶链反应 (RT PCR)测定NOSmRNA的表达水平。结果　吗啡依赖大鼠脊髓和脑干NOS表达水平较正常对照大鼠降低 ,纳洛酮 ( 4mg·kg-1,ip)激发大鼠吗啡戒断症状 1h后脊髓和脑干中NOS表达水平明显升高 ,戒断 2h和 4h后NOS基因表达较 1h组减少。NOS抑制剂L N 硝基精氨酸甲酯 (L NAME ,10mg·kg-1)处理后大鼠吗啡戒断症状减少 ,同时脊髓和脑干的NOS基因表达水平较戒断 1h组明显降低。甲基东莨菪碱 ( 0 5mg·kg-1)处理组脊髓和脑干中NOS表达水平较戒断 1h组明显降低 ;选择性毒蕈碱受体M1拮抗剂 pirenzepine( 10mg·kg-1)处理组动物脊髓中NOS表达水平较戒断 1h组降低 ,而脑干中NOS表达水平没有改变 ;NMDA受体拮抗剂MK 80 1( 0 12 5mg·kg-1)处理后脊髓和脑干中NOS表达水平较戒断 1h组没有差异。结论　吗啡慢性处理后脊髓和脑干中NOSmR NA水平降低 ,抑制内源性NO生成和阻断毒蕈碱受体可以减少吗啡戒断所引起脊髓和脑干中NOS基因的表达     

糖皮质激素对双氯氛酸钠肝损伤大鼠一氧化氮合酶及一氧化氮的影响

目的探讨糖皮质激素地塞米松(Dex)对双氯氛酸钠(Dcf)肝损伤大鼠一氧化氮合酶(NOS)表达及一氧化氮(NO)的影响。方法大鼠随机分为正常组、Dcf组及Dex组。Dcf组给予Dcf 100 mg/kg腹腔注射,Dex组在Dcf注射前1 h予10 mg/kg腹腔注射,24 h后测血清ALT、AST、TBil水平观察肝组织病理学变化,化学法检测血清及肝组织NO含量,免疫组化法检测肝组织内皮型NOS(eNOS)和诱导型NOS(iNOS)的表达。结果 Dcf组血清ALT、AST、TBil水平明显升高(P<0.05),病理积分大幅度增高,血清和肝组织NO含量明显高于正常组(P<0.01),肝组织iNOS表达显著增强(P<0.01),eNOS表达减弱。Dex可明显改善升高的转氨酶及病理积分(P<0.05),并降低肝组织iNOS表达及NO含量(P<0.05)。结论 iNOS和NO在Dcf肝损伤中起促进作用,糖皮质激素可能通过抑制体内iNOS表达,减少NO合成起到肝保护作用。     

一氧化氮合酶抑制剂抑制吗啡依赖及戒断大鼠脊髓神经元ERK的表达

目的探讨鞘内分别注射非选择性一氧化氮合酶(NOS)抑制剂L-NAME、选择性神经元型一氧化氮合酶(nNOS)抑制剂7-硝基吲哚(7-NI)对吗啡戒断大鼠戒断症状和痛敏行为以及脊髓神经元p-ERK表达的影响。方法采用吗啡依赖及戒断模型,分为正常对照组、依赖组、戒断组、L-NAME组(L-NAME)、7-NI组(7-NI),分别作行为学评分(n=8)、免疫组织化学(n=6)和免疫印迹检测(n=4)。结果实验结果表明,①鞘内注射L-NAME、7-NI可明显减轻吗啡依赖大鼠戒断症状,戒断组戒断症状评分为28.6±4.89,L-NAME组、7-NI组分别为22.1±4.52(P<0.05)、16.2±3.99(P<0.01);戒断组促诱发痛评分(touch evoked agitationscores,TEA score)为13.5±2.55,L-NAME组、7-NI组分别为9.8±3.11(P<0.05)、7.5±2.56(P<0.01)。②鞘内注射L-NAME、7-NI可明显减少胸腰段脊髓背角Fos阳性神经元的数目,L-NAME组、7-NI组分别为293±47、267±52,均低于戒断组(380±71,P<0.05,P<0.01)。③L-NAME、7-NI组p-ERK阳性神经元的数目分别为46.8±11.58、40.5±8.55,均低于戒断组(66.6±11.6,P<0.05,P<0.01),两给药组脊髓p-ERK蛋白的表达也减少。④W estern b lot显示:鞘内注射NOS明显抑制吗啡戒断大鼠脊髓p-ERK蛋白表达增加。结论脊髓水平NO参与吗啡依赖和戒断反应,ERK信号通路可能介导NO的上述作用。     

Involvement of spinal nitric oxide (NO) in rat pain-related behaviors induced by the venom of scorpion Buthus martensi Karsch

In the present study, we investigated the role of spinal nitric oxide (NO) in rat pain-related behaviors induced by the venom of scorpion Buthus martensi Karsch (BmK). The results showed that the number of neuronal NO synthase (nNOS) positive neurons significantly increased in superficial (I-II), deep (V-VI) dorsal horn laminae and the ventral gray laminae (VII-X), but not in the nucleus proprius (III and IV) of bilateral L4-L5 lumbar spinal cord after unilateral intraplantar injection of BmK venom from 2h to 7d. This increase on the ipsilateral side to BmK venom injection was always greater than that on the contralateral side. Western blotting analysis confirmed that spinal nNOS expression was significantly up-regulated following BmK venom administration. In addition, intrathecal delivery of N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME; a NOS inhibitor) before intraplantar injection of BmK venom by 10 min significantly attenuated spontaneous nociceptive responses and prevented the development of primary thermal hyperalgesia as well as bilateral mechanical hyperalgesia. Intrathecal injection of l-NAME could also partially inhibit BmK venom-induced c-Fos expression in lumbar spinal cord at 2 h. Thus, the results suggest that spinal NO as a critical mediator is involved in various pain-related behaviors and c-Fos expression induced by BmK venom in rats.     

戊四唑癫痫大鼠脑组织一氧化氮含量和一氧化氮合酶活性变化

探讨一氧化氮在戊四唑癫病发机制中的作用。方法每天注射戊四唑建立在鼠癫痫模型,测定癫病发作后大鼠大脑皮质,海马一氧化氮和一氧化氮合酶活性变化,结果癫痫发作后海马ＮＯ含量和ＮＯＳ活性显著升高,结 戊四唑诱导的癫痫中具有致痫性。     

一氧化氮合酶抑制剂的研究进展

一氧化氮（ｎｉｔｒｉｃｏｘｉｄｅ,ＮＯ）是一种能调节细胞多种功能的信息分子,它参与心血管、外周和中枢神经以及免疫等系统生理过程和生物信号的调节。体内组织中的ＮＯ由ＮＯ合酶（Ｎｉｔｒｉｃｏｘｉｄｅｓｙｎｔｈａｓｅ,ＮＯＳ）催化左旋精氨酸而合成,合成后的ＮＯ迅速跨膜扩散释放。各种调节ＮＯ释放的因素均作用于ＮＯＳ催化的化学反应过程,而体内影响该反应的ＮＯＳ在各组织的表达不同。特异性ＮＯＳ抑制剂通过调控ＮＯ的合成,对ＮＯＳ表达相关的各种疾病的预防和治疗具有重要的临床意义。本文对近年来ＮＯＳ抑制剂的研究进展作一概述。     

花色素苷诱导小鼠巨噬细胞一氧化氮生成及其机制

目的研究花色素苷对小鼠腹腔巨噬细胞一氧化氮(NO)合成的诱导作用及其机制。方法用CCK-8试剂检测花色素苷对小鼠脾细胞增殖的影响;硝酸盐还原酶法检测小鼠腹腔巨噬细胞NO含量;荧光法检测一氧化氮合酶(NOS)活性;RT-PCR检测iNOS mRNA的表达。结果花色素苷可促进小鼠脾细胞增殖,诱导小鼠腹腔巨噬细胞合成NO,提高NOS活性,其中矢车菊素-3-葡萄糖苷能够诱导iNOS mRNA的表达。结论花色素苷能够促进脾细胞的增殖,诱导小鼠腹腔巨噬细胞合成NO,使巨噬细胞激活,具有一定的免疫调节活性。     

Evidence that inhibition of spinal nitric oxide production contributes to the antinociceptive effects of emulsified isoflurane on formalin-induced pain in rats

The aim of the present study was to investigate the contribution of spinal nitric oxide (NO) to the antinociceptive effects of emulsified isofluane in rats. The formalin test was used to assess nociceptive responses. Immunocytochemistry and histochemistry were performed to determine the effects of emulsified isoflurane on formalin-induced changes in Fos-like immunoreactive (Fos-LI)- and nicotinamide adenine dinucleotide phosphatediaphorase (NADPH-d)-positive neurons, respectively. The results showed that emulsified isofluane, administered intraperitoneally, significantly decreased the formalin-induced paw licking time and that this was attenuated by pretreatment with intrathecal injection of the NO precursor L-arginine. Furthermore, Fos-LI- and NADPH-d-positive neurons were mainly found in the ipsilateral dorsal horn after injection of formalin, some of which were Fos-LI/NADPH-d double-labelled neurons. Administration of emulsified isofluane significantly decreased Fos-LI- and NADPH-d-positive, as well as Fos-LI/NADPH-d double-labelled, neurons. Finally, emulsified isofluane produced a significant reduction of NOS activity and a decrease of NO production in the spinal cord of formalin-treated rats. In conclusion, the results suggest that inhibition of spinal NO production contributes to the antinociceptive effects of emulsified isofluane on formalin-induced pain in rats.     

The role of nitric oxide and prostaglandin E2 on the hyperalgesia induced by excitatory amino acids in rats

The present study was designed to investigate the role of nitric oxide (NO), N-methyl-D-aspartate (NMDA) receptor and prostaglandins on hyperalgesia induced in rats by excitatory amino acids and the possibility that prostaglandins may act as the retrograde messenger in the spinal cord like NO. Nomega-nitro-L-arginine methyl ester (L-NAME; 500 microg/paw, intraplantarly (i.pl.)), MK-801 (10 microg/paw, i.pl.) or indomethacin (300 microg/paw, i.pl.) reduced the duration of phase 2 of the biting/licking and scratching (B/L + S) response induced by formalin injection from 255.6+/-16.7 s to 155.6+/-16.9, 172.25+/-33.3 or 205.6+/-16.7 s, respectively. L-NAME (0.3 mg, i.th.), MK-801 (8 microg, i.th.) or indomethacin (20 microg, i.th) reduced the duration of phase 2 of the B/L + S response induced by saline injection from 288.5+/-7.7s to 207.7+/-19.2, 184.6+/-7.7 or 1923+/-38.5 s, respectively. L-NAME or indomethacin injected into the spinal cord of the rat significantly reduced the hyperalgesia induced by NMDA (1 microg, i.th.) from 43.8+/-4.6% to 12.3+/-3.1 and 19.2+/-2.3%, respectively. It is assumed that NO produced by excitatory amino acids may increase prostaglandin production by cyclooxygenase activation. L-NAME, MK-801 or indomethacin injected into the rat spinal cord significantly reduced the hyperalgesia induced by prostaglandin E2 (PGE2, 25 ng, i.th.) in the tail-flick test from 40.6+/-3.5% to 18.2+/-3.2, 18.8+/-1.8 or 17.6+/-4.1%, respectively, but had little effect on hyperalgesia in the paw pressure test (except for indomethacin). In conclusion, NO and PGE2 affect the hyperalgesia induced by excitatory amino acids. It is suggested that PGE2, like NO, may act as a retrograde messenger in the spinal cord.     

Altered L-arginine/nitric oxide synthase/nitric oxide pathway in the vascular adventitia of rats with sepsis

1. In recent studies, the vascular adventitia has been established as an important source of inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) production, even more powerful than the media in response to certain inflammatory factors, such as lipopolysaccharide (LPS). The adventitia has an independent L-arginine (L-Arg)/NOS/NO pathway and is involved in the regulation of vascular function. In the present study, we explored the changes in and the pathophysiological significance of the L-Arg/NOS/NO pathway in the adventitia of rats with sepsis. 2. Sepsis was induced by caecal ligation and puncture in order to observe changes in L-Arg transport, NOS gene expression and activity and NO generation in the vascular adventitia to determine the mechanism of activation of the L-Arg/NOS/NO pathway. 3. Severe sepsis resulted in severe disturbance of haemodynamic features, with decreased mean arterial blood pressure, brachycardia and inhibited cardiac function (decreased left ventricular +/-dP/dt(max)). Left ventricular end-diastolic pressure was elevated threefold (P < 0.01) under anaesthesia. Rats with sepsis showed severe glucopenia and lacticaemia. Plasma levels of the inflammatory factors macrophage chemoattractant protein-1 and interleukin-8 were increased five- and 29-fold, respectively (P < 0.01). 4. In the adventitia of the thoracic and abdominal aortas, the L-Arg/NO pathway was similarly characterized: the uptake of [(3)H]-L-Arg was Na(+) independent, with the peak occurring at approximately 40 min incubation. Total NOS activity was largely calcium independent (> 90%). The V(max) of L-Arg transport in the sepsis group was increased by 83.5% (P < 0.01), but the K(m) value was not significantly different compared with controls. 5. The mRNA levels of cationic amino acid transporter (CAT)-1 and CAT-2B in the sepsis group were increased by 86 and 62%, respectively (both P < 0.01). Inducible NOS activity was increased 2.8-fold compared with controls (P < 0.01) and iNOS mRNA levels were elevated approximately sixfold (P < 0.01). The NO levels in the plasma and incubation media (incubation for 40 min) in the sepsis group were increased by 144 and 273%, respectively (both P < 0.01). 6. The Arg/NOS/NO pathway was activated in the vascular adventitia of rats with sepsis shock. The L-Arg/NOS/NO pathway in the aortic adventitia may play an important role in the pathogenesis of sepsis and septic shock.     

细胞因子对膀胱平滑肌细胞内诱导性一氧化氮合酶的激活作用(英文)

探讨肿瘤坏死因子α (TNFα)和γ干扰素(INFγ)对大鼠膀胱平滑肌细胞诱导性一氧化氮合酶(iNOS )的影响 .将TNFα (1nmol·L- 1)或INFγ(5 0kU·L- 1)分别或同时加入膀胱平滑肌细胞培养液 ,2 4h后测定细胞培养液中一氧化氮 (NO)水平 ,并用Western印迹方法检测iNOS的表达 .结果显示 ,TNFα或INFγ单独不能诱导iNOS表达 ,也不能引起NO水平显著提高 .但当TNFα和INFγ联合诱导细胞 2 4h ,则细胞培养液中NO水平明显升高 ,用Western印迹分析可见iNOS表达 ,说明TNFα和INFγ具有协同诱导作用 .在TNFα和INFγ加入膀胱平滑肌细胞前 30min ,加入NOS抑制剂L 氮 精氨酸甲酯 (L NAME) ,可显著抑制TNFα和INFγ对NO的生成诱导 .结果提示 ,TNFα和INFγ联合应用可激活膀胱平滑肌细胞iNOS .