透骨消痛颗粒干预膝骨性关节炎早期环氧合酶2和诱导型一氧化氮合酶的表达

背景:由巴戟天、杭白芍、肿节风、川芎等组成的透骨消痛颗粒能有效改善膝骨性关节炎的症状,但其具体机制尚不清楚.目的:观察透骨消痛颗粒对膝骨性关节炎早期环氧合酶2和诱导型一氧化氮合酶表达的影响.方法:将90只新西兰大白兔随机等分为6组:除正常组外均采用改良Hulth法建立骨性关节炎动物模型,透骨消痛颗粒低、中、高剂量组及壮骨关节丸组分别在造模的基础上每天灌胃给予含透骨消痛颗粒5,10,20 g或壮骨关节丸10 g的水溶液.4周后,检测兔关节滑液中一氧化氮、一氧化氮合酶、前列腺素E2及关节软骨和滑膜组织中诱导型一氧化氮合酶、环氧合酶2的水平.结果与结论:骨性关节炎早期,关节滑液中一氧化氮、一氧化氮合酶、前列腺素E2水平及滑膜和软骨中环氧合酶2、诱导型一氧化氮合酶表达均明显升高.透骨消痛颗粒及壮骨关节丸均可降低关节滑液中一氧化氮、一氧化氮合酶、前列腺素E2及滑膜和软骨中环氧合酶2、诱导型一氧化氮合酶的表达,以透骨消痛颗粒中、高剂量组作用更明显.提示透骨消痛颗粒可通过抑制环氧合酶2和诱导型一氧化氮合酶表达,从而抑制软骨降解,缓解临床症状,达到保护关节软骨、防治骨性关节炎的作用.     

滑膜成纤维细胞环氧化酶-2和诱导型一氧化氮合酶在类风湿性关节炎中的表达

目的　研究类风湿性关节炎 (RA)患者关节滑膜成纤维细胞环氧化酶 2 (COX 2 )、诱导型一氧化氮合酶 (iNOS)及其诱导产物前列腺素E2 (PGE2 )、一氧化氮 (NO)的表达水平 ,探讨它们在RA致病过程中的作用。方法　分离滑膜成纤维细胞 ;用酶联免疫吸附法和酶还原法检测正常人关节滑膜液和滑膜细胞培养上清液中PGE2 、NO的含量 ;采用半定量逆转录 聚合酶联反应法检测滑膜组织及滑膜细胞COX 2、iNOSmRNA表达 ;并用细胞酶联免疫法检测细胞COX 2、iNOS蛋白表达水平。结果　RA患者关节滑膜组织COX 2、iNOSmRNA表达及滑膜液PGE2 、NO的水平分别为 0 42 ,0 83和(4 82± 1 74) μg/L、(80 4± 2 3 2 ) μmol/L ,明显高于正常对照组的 0 0 8,0 1 4和 (0 64± 0 1 3) μg/L、(2 6 2± 1 4 4) μmol/L(P <0 0 1 ) ;在炎性细胞因子刺激下RA患者滑膜成纤维细胞COX 2、iNOSmRNA水平 (0 48,0 98)、蛋白表达水平 (0 92± 0 1 8,0 78± 0 1 3)以及培养上清液中PGE2 、NO含量 [(2 1 44±4 2 6) μg/L ,(1 2 6 6± 32 2 ) μmol/L]均显著高于正常对照组 (P <0 0 1 )。 结论　滑膜成纤维细胞COX 2、iNOS的过度表达及PGE2 、NO的大量合成 ,可能是RA关节病变的一个重要因素     

肠三叶因子和环氧合酶2及诱导型一氧化氮合酶表达与胃黏膜适应性细胞保护的关系

目的研究肠三叶因子(ITF)、环氧合酶2(COX-2)、诱导型一氧化氮合酶(iNOS)在水浸束缚应激(WRS)大鼠胃黏膜基因表达变化,探讨其在应激胃黏膜适应性细胞保护中的相互关系及作用。方法采用重复WRS制作模型,Ⅲ型激光多谱勒血流仪(LDF-3)动态监测胃黏膜血流量(GMBF),大体及光镜下观察黏膜损伤程度(UI)及组织学变化,逆转录一多聚酶链反应(RT-PCR)和Western blot检测ITF、COX-2及iNOS表达变化。结果单次应激造成胃黏膜广泛损伤,重复应激后胃黏膜产生适应性,胃黏膜血流量上升,损伤逐渐减轻;4次应激后,损伤指数降低为单次应激时的21．99％,并且胃腺区细胞增殖。单次应激后,ITF、COX-2及iNOS均增强;重复应激后,ITF表达继续逐渐增强,而COX-2及iNOS表达则逐渐减弱;正常时,ITF、COX-2及iNOS几乎无表达;应激后,ITF不但在胃腺颈部黏膜增殖带表达增强,而且在胃腺基底部亦有表达,COX-2及iNOS在溃疡及其边缘存在。结论胃黏膜适应性细胞保护伴有细胞增殖,ITF表达逐渐增强,COX-2及iNOS表达逐渐减弱;表明三者在这一现象中具有重要的调节作用。     

诱导型一氧化氮合酶和血红素氧合酶-1在百草枯中毒大鼠肾脏中的表达

目的 观察百草枯中毒大鼠肾组织中诱导型一氧化氮合酶(iNOS)和血红素氧合酶-1(HO-1)的表达,探讨其可能的病理生理机制.方法 SD大鼠84只随机分为空白对照组(A组)42只和染毒组(B组)42只.B组百草枯(25 mg/kg)、A组等量盐水腹腔注射.观察2组肾组织病理学变化及iNOS、HO-1表达,并检测HO-1 mRNA.结果 ①A组组织结构清晰.未见充血、水肿及空泡变性等病理变化;B组组织结构清晰度下降,染毒3 h即可见充血、水肿及空泡变性等病理变化,1 d达顶峰,其后缓解趋势不明显.②iNOS:A组多不表达;B组染毒后3 h在皮质部肾小管上皮细胞及肾小球内皮细胞的胞质中出现表达,随时间延长明显增强,1 d达高峰,其后维持较强表达.③HO-1与HO-1 mRNA:A组多不表达;B组染毒3 h在皮质部肾d、管上皮细胞的胞膜及胞质HO-1呈阳性表达,免疫组织化学评分(IHS)升高,HO-1 mRNA的表达增强,与A组相比差异有统计学意义(P<0.05),1 d达顶峰,之后减弱,5 d仍有阳性表达,但与A组相比,IHS差异无统计学意义(P>0.05).结论 iNOS和HO-1参与百草枯中毒肾损伤的发病过程,但其作用方式、调节途径等尚需进一步研究.     

透骨消痛胶囊干预膝骨性关节炎软骨下骨重塑的分子机制

背景:前期研究表明透骨消痛胶囊能有效治疗膝骨性关节炎,且对膝关节软骨有保护作用,软骨下骨重塑在骨性关节炎病理进程中起重要作用。目的:观察透骨消痛胶囊干预骨性关节炎软骨下骨重塑的作用及并探讨其作用机制。方法:新西兰大白兔分为正常组、模型组、壮骨关节丸组、透骨消痛胶囊组4组。除正常组外动物均按改良Hulth法造模。正常组、模型组兔每天给予生理盐水灌胃;壮骨关节丸组兔每天给予壮骨关节丸水溶液灌胃;透骨消痛胶囊组兔每天给予透骨消痛胶囊水溶液灌胃。结果与结论:造模后6,9,12周,透骨消痛胶囊组软骨下骨CyclinD1基因、胰岛素样生长因子1、细胞核因子κB受体活化因子配体mRNA表达均显著高于正常组(P<0.05)。与模型组比较,造模后1周,透骨消痛胶囊组Cyclin D1 mRNA表达较低(P<0.05);造模后6周,透骨消痛胶囊组细胞核因子κB受体活化因子配体表达较低(P<0.05);造模后9,12周,透骨消痛胶囊组各项指标表达均较低(P<0.05)。与壮骨关节丸组比较,造模1周后,透骨消痛胶囊组Cyclin D1 mRNA表达较低(P<0.05);6周时透骨消痛胶囊组细胞核因子κB受体活化因子配体表达较低(P<0.05);9,12周后,透骨消痛胶囊组胰岛素样生长因子1mRNA表达较低(P<0.05)。提示透骨消痛胶囊可改变软骨下骨重塑的速率和模式,最终减轻软骨下骨硬化。     

PTEN、环氧合酶-2及诱导型一氧化氮合酶在子宫内膜癌中的表达及其意义

目的：研究子宫内膜癌组织中PTEN、诱导型一氧化氮合酶（iNOS）及环氧合酶-2（COX-2）蛋白的表达,以及PTEN、iNOS及COX-2表达与临床病理的关系,探讨三者之间的相关性。方法：采用免疫组化EnVisionTM法检测55例子宫内膜腺癌,24例子宫内膜不典型增生和20例正常子宫内膜石蜡切片中PTEN、iNOS及COX-2的表达。结果：（1）子宫内膜癌组织与子宫内膜不典型增生、正常子宫内膜组织比较,PTEN阳性表达率明显降低,差异有显著性（P〈0.05）;iNOS、COX-2表达率明显增加,差异有显著性（P〈0.05）。（2）PTEN表达与病理分级、肌层浸润程度、淋巴结转移有关（P〈0.05）;随肿瘤子宫肌层浸润深度的增加,iNOS的表达显示逐渐增强（P〈0.05）。（3）在子宫内膜癌组织中,iNOS与PTEN表达呈负相关（r=-0.212,P〈0.05）,与COX-2表达呈正相关（r=0.513,P〈0.05）。结论：PTEN的失表达与iNOS、COX-2的过表达在子宫内膜癌的发生发展中起着一定的作用,PTEN与iNOS、COX-2的异常表达密切相关,三者的联合检测有助于子宫内膜癌的早期诊断。     

环氧合酶-2及诱导型一氧化氮合酶与喉癌

喉癌是危害广大人民健康的主要恶性肿瘤之一.既往的实验研究和临床资料表明环氧合酶-2(COX-2)和诱导型一氧化氮合酶(iNOS)在肿瘤细胞(包括喉癌)中的表达均有增高.二者之间存在一定的相关性.对二者在喉癌中表达的进一步研究可能对喉癌的发病机制以及治疗提供新的线索.     

骨性关节炎模型大鼠关节软骨变化及透骨消痛胶囊干预的作用

背景：透骨消痛胶囊是治疗骨性关节炎的临床验方,作用机制尚未完全阐明。尿激酶型纤溶酶原激活系统参与关节软骨的细胞外基质降解及关节滑膜增生,在骨性关节炎的病理过程中起着重要作用。目的：观察透骨消痛胶囊对膝骨性关节炎模型大鼠软骨中尿激酶型纤溶酶原激活系统的影响。方法：SD大鼠144只,随机取120只采用关节腔注射木瓜蛋白酶复制大鼠膝骨性关节炎模型,并随机分为模型组、壮骨关节丸组[1.2 g/（kg·d）]、透骨消痛胶囊低剂量组[0.092 g/（kg·d）]、透骨消痛胶囊中剂量组[0.184 g/（kg·d）]和透骨消痛胶囊高剂量组[0.368 g/（kg·d）],每组24只,每2周为1个疗程,中间休息2 d,共4个疗程。另取24只正常大鼠为空白组。每2个疗程后,处死一批实验动物,苏木精-伊红染色观察软骨组织病理改变;免疫组织化学反应观察尿激酶型纤溶酶原激活剂、尿激酶型纤溶酶原激活剂受体、纤溶酶原激活物抑制因子阳性表达情况;Western blot检测尿激酶型纤溶酶原激活剂、尿激酶型纤溶酶原激活剂受体、纤溶酶原激活物抑制因子蛋白表达情况。结果与结论：透骨消痛胶囊组和壮骨关节丸组的骨性关节炎大鼠关节软骨 Mankin’s 评分较模型组明显降低（P 〉0.01）,具有时间依赖;免疫组织化学反应显示,透骨消痛胶囊组和壮骨关节丸组尿激酶型纤溶酶原激活剂、尿激酶型纤溶酶原激活剂受体的阳性率明显降低,而纤溶酶原激活物抑制因子明显升高,具有时间依赖。Western blot检测结果与免疫组织化学具有相同的趋势。提示透骨消痛胶囊可能通过调控尿激酶型纤溶酶原激活剂系统对骨性关节炎发挥防治作用。     

透骨消痛胶囊对体外关节软骨细胞Caspase-9和Caspase-3表达的影响

目的:观察透骨消痛胶囊对大鼠体外培养关节软骨细胞凋亡的影响。方法:采用4周龄SD大鼠膝关节软骨建立稳定的软骨细胞体外培养体系,采用Ⅱ型胶原对第3代软骨细胞进行鉴定。用20 ng/ml TNF-α诱导体外培养软骨细胞的凋亡,诱导成功后,给予不同浓度的透骨消痛胶囊干预24 h后,镜下观察软骨细胞的生长情况;采用MTT法检测各组软骨细胞的存活率;DAPI染色观察各组软骨细胞的凋亡情况,分光光度法检测凋亡软骨细胞中Caspase-9和Caspase-3活性。结果:透骨消痛胶囊能够减轻软骨细胞的核固缩,减少软骨细胞凋亡,提高细胞的存活率,同时能够下调Caspase-9和Caspase-3活性。结论:透骨消痛胶囊可以通过下调Caspase-9和Caspase-3活性,抑制软骨细胞的凋亡,从而起到治疗骨性关节炎的作用。     

脂多糖诱导巨噬细胞产生诱导型一氧化氮合酶的实验研究

目的观察脂多糖(Lipopolysaccharide,LPS)对小鼠腹腔巨噬细胞诱导型一氧化氮合酶(in-ducible nitric oxide synthase,iNOS)的诱导作用。方法常规方法获取小鼠腹腔巨噬细胞随机分成两组:空白对照组(培养基中不含LPS)及LPS组(分别含LPS0.01,0.1,1.0,10mg/L的培养基培养24h)。采用逆转录-多聚酶链式反应(RT-PCR)和Western Blotting方法测定诱导型一氧化氮合酶的基因及蛋白表达水平。结果RT-PCR和Western Blotting结果表明,经0.1mg/LLPS刺激后细胞iNOS mRNA及蛋白表达水平均明显升高,与空白对照组比较有显著性差异(P<0.05),并且在一定范围内呈剂量-效应关系。结论LPS可诱导小鼠巨噬细胞产生诱导型一氧化氮合酶。     

幽门螺杆菌相关胃炎和胃癌组织中COX和iNOS的表达及意义

胃癌是我国最常见的恶性肿瘤之一，幽门螺杆菌（Hp）感染是慢性胃炎的主要致病因子和胃癌的重要始动因素，Hp感染可导致胃黏膜环氧合酶-2（COX-2），诱导型一氧化氮合酶（iNOS）表达升高，且与Hp感染密度，炎症程度密切相关，而COX-2和iNOS的表达与胃癌发生密切相关。本比较在Hp阳性胃炎及胃癌与Hp阴性慢性胃炎中COX和iNOS表达情况，探讨Hp的致癌机制。     

骨延长患者诱导型一氧化氮合酶的表达

背景：骨延长的牵张刺激如何转变为骨质再生的生物学信息至今不明确。目的：观察骨延长患者诱导型一氧化氮合酶的动态表达。方法：单侧胫腓骨延长组患者8例,分别于手术前第3天、术后第3,7,8,11,30天、停止延长时、停止延长第3,15,30天、拆除外固定器时、拆除外固定器后30d,检测血清诱导型一氧化氮合酶水平。并设立年龄相匹配的对照组8例。结果与结论：骨延长组牵张操作开始第1天时（术后第8天）,血清诱导型一氧化氮合酶即开始升高,停止延长时达高峰,这一时段各个时相骨延长组血清诱导型一氧化氮合酶均显著高于对照组（P〈0.01）,停止延长3d时的血清诱导型一氧化氮合酶亦高于对照组（P〈0.05）。提示诱导型一氧化氮合酶的高表达是骨延长的分子生物学机制之一。     

骨延长患者诱导型一氧化氮合酶的表达

背景:骨延长的牵张刺激如何转变为骨质再生的生物学信息至今不明确。目的:观察骨延长患者诱导型一氧化氮合酶的动态表达。方法:单侧胫腓骨延长组患者8例,分别于手术前第3天、术后第3,7,8,11,30天、停止延长时、停止延长第3,15,30天、拆除外固定器时、拆除外固定器后30d,检测血清诱导型一氧化氮合酶水平。并设立年龄相匹配的对照组8例。结果与结论:骨延长组牵张操作开始第1天时(术后第8天),血清诱导型一氧化氮合酶即开始升高,停止延长时达高峰,这一时段各个时相骨延长组血清诱导型一氧化氮合酶均显著高于对照组(P<0.01),停止延长3d时的血清诱导型一氧化氮合酶亦高于对照组(P<0.05)。提示诱导型一氧化氮合酶的高表达是骨延长的分子生物学机制之一。     

大肠癌组织中COX-2，iNOS，VEGF的表达及其临床意义

目的探讨COX-2,iNOS,VEGF在大肠癌组织中的表达及其临床意义。方法采用免疫组化方法检测58例大肠癌组织中COX-2,iNOS,VEGF的表达,并分析其临床意义。结果58例大肠癌组织中COX-2,iNOS,VEGF的阳性表达率分别为79．31％（46／58）、84．48％（49／58）和79．31％（46／58）,显著高于正常大肠组织（P〈0．01）,与患者年龄、有无淋巴结转移、肿瘤体积及组织学类型差异无统计学意义（P〉0．05）,COX-2与VEGF的表达存在一定相关性（P〈0．05）,COX-2与iNOS的表达也存在一定相关性（P〈0．05）。结论大肠癌组织COX-2、iNOS的表达与肿瘤血管生成密切相关,二者可能与VEGF协同促进肿瘤血管生成,是判断肿瘤恶性程度及预后的的重要生物学指标。     

Immune dysfunction in endotoxicosis: role of nitric oxide produced by inducible nitric oxide synthase

OBJECTIVE: To investigate whether stimulation with lipopolysaccharide (LPS) alters cytokine production by splenocytes in mice and whether it changes the T-helper 1 (Th1)/Th2 balance. The role of nitric oxide in such immunologic changes was also explored using mice with genetic lack of inducible nitric oxide synthase (iNOS). DESIGN:: Prospective animal study with concurrent controls. SETTING: University research laboratory. SUBJECTS: iNOS knockout mice and wild-type littermates. INTERVENTIONS: iNOS knockout mice or wild-type mice were injected with LPS or saline with or without anti-interleukin (IL)-6 antibody, and survival was monitored for 7 days. MEASUREMENTS AND MAIN RESULTS: At 24 and 48 hrs after administration, blood samples and splenocytes were obtained to examine immunologic variables. Cell surface markers and cytokine expression of splenocytes were used to characterize the Th1/Th2 balance and were measured by flow cytometry. At 48 hrs after LPS administration, the Th1/Th2 balance shifted toward Th2 predominance in wild-type mice, irrespective of the IL-6 level, whereas it showed Th1 predominance in iNOS knockout mice, and the increase of IL-6 and IL-10 in response to LPS persisted in these animals. After LPS administration, the mortality rate was significantly higher in iNOS knockout mice than in wild-type mice, irrespective of the IL-6 level. CONCLUSIONS: These findings suggest that nitric oxide produced by iNOS during endotoxemia may be involved in down-regulation of Th1 cytokines and up-regulation of Th2 cytokines, whereas IL-6 has no such role. The increased lethality of LPS in iNOS knockout mice suggests that nitric oxide may be protective against proinflammatory cytokine-induced damage. Nitric oxide excess may increase susceptibility to nosocomial infections, so-called immunoparalysis.     

大鼠小肠移植术后血清一氧化氮及小肠组织一氧化氮合酶和诱导型一氧化氮合酶活性的变化

目的:研究同种大鼠小肠移植后内源性一氧化氮、一氧化氮合酶(nitricoxidesynthase,NOS)及诱导型一氧化氮合酶(induciblenitricoxidesyn-thase,iNOS)的变化及一氧化氮与急性排斥反应的关系。方法:以大鼠小肠移植为研究对象,16只SD大鼠进行同系移植作为对照组,8只SD大鼠和8只Wistar大鼠进行同种移植作为实验组,两组移植后分别于第3,5,7天同时取血液及小肠组织,病理为常规苏木精-伊红染色观察,血清一氧化氮采用硝酸还原酶法测定,NOS和iNOS采用分光光度法测定。结果:在急性排斥反应发生的早期实验组血清一氧化氮水平与对照组比较显著升高(术后第3,5,7天t值分别为9.7900,9.0073,6.3159,P<0.01),小肠组织NOS及iNOS活性亦显著高于对照组(NOS术后第3,5,7天t值分别为5.9318,9.1237,3.0457,iNOS术后第3,5,7天t值分别为3.2008,5.4930,4.8170,P<0.01)。结论:大鼠小肠移植后NOS及iNOS变化与排斥反应相关,血清一氧化氮水平的检测可作为干预移植措施始动的指标之一。     

In vitro modulation of inducible nitric oxide synthase gene expression and nitric oxide synthesis by procalcitonin

OBJECTIVE: Serum procalcitonin (PCT) concentration was recently introduced as valuable diagnostic marker for systemic bacterial infection and sepsis. At present, the cellular sources and biological properties of PCT are unclear. During sepsis and septic shock, inducible nitric oxide synthase (iNOS) gene expression is stimulated followed by the release of large amounts of nitric oxide (NO). We investigated the possible association between PCT and iNOS gene expression in an in vitro cell culture model. DESIGN: Prospective, controlled in vitro cell culture study. SETTING: University research laboratories. INTERVENTIONS: Confluent rat vascular smooth muscle cells (VSMC) were incubated for 24 hrs and 48 hrs with PCT (1 ng/mL, 10 ng/mL, 100 ng/mL, 1,000 ng/mL, 5,000 ng/mL) alone or with the combination of tumor necrosis factor-alpha (TNF-alpha, 500 U/mL) plus interferon-gamma (IFN-gamma, 100 U/mL). iNOS gene expression was measured by qualitative as well as quantitative polymerase chain reaction analysis, NO release was estimated by the modified Griess method. MEASUREMENTS AND MAIN RESULTS: PCT in increasing concentrations had no effect on iNOS gene expression and nitrite/nitrate release for 24 hrs and 48 hrs, respectively. However, PCT ameliorated TNF-alpha/IFN-gamma-induced iNOS gene expression in a dose-dependent manner (maximal inhibition at PCT 100 ng/mL by -66% for 24 hrs and -80% for 48 hrs). This was accompanied by a significantly reduced release of nitrite/nitrate into the cell culture supernatant (maximal reduction at PCT 100 ng/mL by -56% and -45% for 24 hrs and 48 hrs, respectively). CONCLUSIONS: We conclude that recombinant PCT inhibits the iNOS-inducing effects of the proinflammatory cytokines TNF-alpha/ IFN-gamma in a dose-dependent manner. This might be a counter-regulatory mechanism directed against the large production of NO and the concomitant systemic hypotension in severe sepsis and septic shock.     

Ameliorative effect of combined administration of inducible nitric oxide synthase inhibitor with cyclooxygenase-2 inhibitors in neuropathic pain in rats.

OBJECTIVES: The objective of this study was to examine the effects of rofecoxib, meloxicam, both cyclooxygenase-2 (COX-2) inhibitors and aminoguanidine hydrochloride, an inducible nitric oxide synthase (iNOS) inhibitor and their combinations in neuropathic pain in rats. METHODS: Neuropathy was induced by chronic constriction injury (CCI) of right sciatic nerve under ketamine anesthesia in rats. Effect of ED(50) of aminoguanidine hydrochloride, rofecoxib and meloxicam administered orally was investigated using behavioral tests. Effect of combinations of aminoguanidine hydrochloride with rofecoxib and meloxicam was also investigated in neuropathic pain employing behavioral tests. RESULTS: Behavioral tests, mechanical, thermal and cold stimuli confirmed the development of neuropathic pain after CCI. Aminoguanidine hydrochloride, rofecoxib and meloxicam when administered alone, produced significant increase in paw withdrawal threshold to mechanical stimuli at 6 h in ipsilateral hind paw after CCI. Co-administration of aminoguanidine hydrochloride (30 mg/kg) with rofecoxib (1.31 mg/kg) and meloxicam (1.34 mg/kg) was also found to produce significant increase in paw withdrawal latencies to mechanical stimuli at 6 h. Combined administration of aminoguanidine hydrochloride with meloxicam and rofecoxib produced significant rise in pain threshold for mechanical hyperalgesia in ipsilateral hind paw when compared with the groups treated with aminoguanidine hydrochloride, meloxicam and rofecoxib alone. CONCLUSION: Co-administration of meloxicam and rofecoxib with aminoguanidine hydrochloride may be an alternative approach for the treatment of neuropathic pain.