大鼠额叶损伤后诱导型一氧化氮合酶的表达变化

为了探讨大鼠额叶损伤后不同时间诱导型一氧化氮合酶(iNOS)的表达变化及意义,本研究采用大鼠额叶锐器损伤模型,经Nissl和H.E.染色观察伤后的病理变化过程,RT-PCR及免疫组织化学染色方法检测伤后iNOSmRNA和iNOS阳性细胞的表达变化。结果显示:伤后12、24h创伤区炎症细胞大量浸润;创伤区及周边iNOSmRNA的表达3h开始上升,24h达到高峰;伤后iNOS阳性细胞数量也增多,伤后3、6h主要由神经细胞表达iNOS,在12、24h主要由巨噬细胞表达iNOS,而72、120h则主要由胶质细胞表达iNOS。上述结果说明大鼠额叶锐器伤后iNOS的表达增加,iNOS阳性细胞的种类和数量变化与伤后时程有关。     

大鼠额叶皮质损害的脑电功率谱和形态学研究

３５只ＳＤ大鼠随机分成三组：正常组、假损害组和损害组。采用机械抽吸法制作大鼠右侧额叶皮质损害模型。对各组大鼠分别进行脑电功率谱分析和比较．结果发现：正常大鼠和假损害大鼠两侧大脑半球δ、θ、α和β频段相对功率值相近似（Ｐ＞０．０５），ＤＴ／ＡＢ值（即δ＋θ／α＋β的绝对功率值之比）也相似（Ｐ＞０．０５），功率谱曲线两侧基本对称。额叶皮质损害大鼠损害侧δ、θ频段相对功率值较健例显著增高（Ｐ＜０．０５），α、β段相对功率值较健侧显著降低（Ｐ＜０．０５）；ＤＴ／ＡＢ值较健侧显著增加（Ｐ＜０．０５），功率谱曲线两侧明显不对称。额叶皮质损害后３天、２周、４用及８周功率谱分析结果无明显不同。形态学观察证实额叶皮质存在缺损区不因时间延长而自行修复．额叶皮质损害出现的脑功能障碍及损害区形态变化均不能自行修复，表明功能效应和形态结果相一致，提示脑电功率谱分析可作为临床和实验研究中评价脑功能的有效手段。     

运动对大鼠额叶和纹状体一氧化氮合酶阳性细胞的影响

目的　研究运动对运动中枢神经型一氧化氮合酶阳性细胞的影响。方法　将大鼠分为对照组和运动组 ,运动组大鼠游泳 5d ,用免疫组织化学方法检测额叶、纹状体一氧化氮合酶阳性细胞的变化。结果　运动组额叶、纹状体一氧化氮合酶阳性细胞的染色比对照组深 ,运动组额叶阳性细胞数量平均每张切片为 (7.2± 1.9)个 ,比对照组 (2 .4± 0 .6 )个明显增加 (P <0 .0 5 ) ,运动组纹状体阳性细胞数量平均每张切片为 (35 .0± 7.2 )个 ,也比对照组 (8.2± 1.3)个增加显著 (P <0 .0 1)。结论　运动增加了运动中枢一氧化氮合酶阳性细胞的表达     

大鼠额叶皮质损害后胚脑移植适宜时期的探讨

目的:探讨脑外伤后进行脑组织移植的适宜时期。方法:机械抽吸法制作大鼠右侧额叶皮质损害模型。在大鼠额叶皮质损害后立即、2周及2月时采用铺片法将E15-17天胚脑额叶皮质移植入皮质损害腔内。动物存活2月再进行脑功能测试和形态学观察。结果:额叶皮质损害后立即移植鼠移植侧δ、0频段相对功率值显著高于健侧(P<0.05),α、β频段相对功率值显著低于健侧(P<0.05),脑电功率谱曲线两侧明显不对称,表明受损的脑功能也没有恢复。额叶皮质损害后2周及2月移植鼠移植侧δ、0、α、β频段相对功率值均与健侧近似(P>0.05),脑电功率谱曲线两侧基本对称,脑电功率谱改善。形态学观察可见损害后立即移植鼠少见有移植组织存活,损害后2周及2月移植鼠,绝大多数见有良好的移植组织存活。结论:大鼠额叶皮质损害后2周至2月均为胚脑组织移植适宜时期。     

大鼠创伤性脑损伤后细胞凋亡及NOS阳性细胞的变化

目的：探讨大鼠创伤性脑损伤后不同时相皮质、海马、隔区细胞凋亡及NOS、ChAT阳性细胞的变化。方法：采用大鼠自由落体脑损伤模型，伤后1、2、3、4、5、7、10d取脑切片，经Nissl染色，用TUNEL法检测细胞凋亡，NADPH—d组化染色观察NOS阳性细胞，ChAT免疫组化染色观察隔区ChAT阳性细胞。结果：Nissl染色可见损伤侧海马CA2、CA3区锥体细胞层细胞消失或紊乱。损伤区周围皮质凋亡细胞伤后3d达到高峰；损伤侧海马凋亡细胞伤后5d达到高峰；损伤侧隔区凋亡细胞7d达到高峰。正常侧上述脑区各时相点均未见到凋亡细胞。损伤区周围皮质、损伤侧海马和隔区iNOS阳性细胞数量明显增加。损伤侧隔区ChAT阳性神经元也明显减少。结论：大鼠创伤性脑损伤后损伤区周围皮质和损伤侧海马、隔区细胞凋亡数量的变化与伤后时程有关。伤后细胞iNOS表达增加是导致细胞凋亡的因素。     

创伤后应激障碍大鼠前额叶皮质PKMζ表达的变化

目的探讨创伤后应激障碍(PTSD)大鼠前额叶皮质PKMζ的表达变化情况。方法采用单程长时应激(SPS)制备PTSD大鼠模型,旷场试验、高架十字迷宫试验检测造模效果。将大鼠分为:SPS1d、SPS3d、SPS7d和SPS14d组,正常饲养大鼠作为对照组。利用Western blot和real-time RT-PCR检测PKMζ蛋白及m RNA在大鼠前额叶皮质表达。结果 SPS模型大鼠表现出明显的焦虑症状,SPS7d、SPS14d组大鼠前额叶皮质PKMζ蛋白和m RNA表达水平显著高于对照组。结论在应激后7d和第14d,PTSD大鼠前额叶皮质PKMζ表达显著增高。     

大鼠坐骨神经损伤后诱导型一氧化氮合酶表达的变化

为了探讨大鼠坐骨神经损伤后再生过程中诱导型一氧化氮合酶(iNOS)表达的变化及意义,本研究采用大鼠坐骨神经切断缝合模型,分别于术后1、3、7、14、21及28d取吻合口远端的神经,采用免疫组织化学和实时荧光定量聚合酶链反应(RT-PCR)方法检测损伤神经远端iNOSmRNA及其蛋白的表达水平。结果显示:假手术对照组坐骨神经中未见明显的iNOS阳性产物,iNOSmRNA表达极低。实验组神经损伤后iNOSmRNA及其蛋白的表达水平均明显增高(P<0.01),iNOS阳性产物的吸光度(A)值在术后7d达高峰。iNOSmRNA表达在术后1、3、7d维持较高水平,此后则明显下降。上述结果说明大鼠坐骨神经损伤后神经纤维中iNOS的表达增加,iNOS可能在周围神经损伤后的再生过程中起着一定的作用。     

大鼠坐骨神经结扎后脊髓内诱导型一氧化氮合酶的表达

目的:研究坐骨神经结扎损伤后诱导型一氧化氮合酶(iNOS)在大鼠脊髓内的表达变化规律.方法:健康成年SD大鼠随机分为正常组、假手术组和坐骨神经结扎组.存活1、3、5、7、14、21、28 d后取腰4～6脊髓节段冷冻切片,用免疫组织化学方法结合图像分析技术检测脊髓内iNOS的表达变化,同时用免疫荧光双标染色技术检测14 d组中央管周围iNOS和神经肽Y(NPY)的共表达.结果:根据免疫阳性产物灰度值,损伤侧脊髓前角iNOS表达1 d明显升高,21 d达高峰,28 d接近正常,损伤后1～21 d损伤侧脊髓前角与对侧和正常组免疫阳性产物相比有统计学意义;损伤侧脊髓后角1 d iNOS表达增强,其他各组未见明显变化;损伤后中央管周围免疫阳性细胞数增多,3 d尤为明显,28 d恢复正常,免疫荧光双标染色显示14 d组iNOS和NPY在中央管周围共表达.结论:iNOS可能参与神经损伤后的再生过程及伤后神经性痛的凋节.     

大鼠创伤性脑损伤后星形胶质细胞的变化

目的：探讨大鼠创伤性脑损伤后星形胶质细胞的形态学变化及GFAP和NOS的表达情况。方法：采用大鼠自由落体脑损伤模型，伤后1、3、7d取脑切片，行Nissl染色以及GFAP免疫组化和NADPH—d组化单标记及双标记染色。结果：损伤区周围皮质GFAP阳性细胞胞体增大、突起增粗增长，GFAP阳性细胞数量与正常侧及对照组相比，伤后1d即有明显增加，伤后3d、7d数量持续增加；损伤侧海马CAI～3区和DG各层GFAP阳性细胞排列紊乱，胞体增大、突起增粗增长，GFAP阳性细胞数量与正常侧及对照组相比则无明显变化。损伤区周围皮质、损伤侧海马NOS阳性细胞数量明显增加。伤后3d损伤区周围皮质和损伤侧海马中GFAP与NOS双标细胞分别占GFAP阳性细胞的14．2％和13．4％左右。结论：大鼠创伤性脑损伤后大量的星形胶质细胞活化、GFAP表达增加并且部分转化为NOS阳性细胞，提示其参与了脑组织的损伤与修复过程。     

诱导型一氧化氮合酶在精索静脉曲张大鼠睾丸中的表达

目的：探讨精索静脉曲张大鼠睾丸组织中诱导型一氧化氮合酶(iNOS)的表达与不育的关系。方法：选择30只成年SD大鼠,随机分为精索静脉曲张组20只,假手术组10只,应用免疫组织化学EnVision法检测iNOS在睾丸组织中的表达并测量曲细精管的直径,比较两者之间差异。结果：精索静脉iNOS的表达明显高于假手术组,差异有非常显著性意义,曲张组曲细精管的直径明显小于假手术组,差异有非常显著性意义。结论：精索静脉曲张大鼠睾丸组织中iNOS过度表达是导致不育的原因之一。     

诱导型一氧化氮合酶与环氧合酶-2在肿瘤组织中的共表达

诱导型一氧化氮合酶(iNOS)和环氧合酶-2(COX-2)均与肿瘤发生、生长、血管形成及转移有关。在正常生理条件下,两者在多数组织内均检测不到,但在受到各种病理因素的刺激后,两者均能被迅速诱导表达。在许多恶性肿瘤组织中,两者的表达一致上调,呈正相关,其联合表达与肿瘤的生物学行为密切相关。研究发现iNOS与COX-2有可能通过Wnt-β-catenin信号实现了信息交流和相互调控,并通过这种机制,共同在肿瘤的各种生物学行为中发挥作用。     

Anti-inflammatory effect of Mentha longifolia in lipopolysaccharide-stimulated macrophages: Reduction of nitric oxide production through inhibition of inducible nitric oxide synthase

Abstract

Mentha longifolia is an aromatic plant used in flavoring and preserving foods and as an anti-inflammatory folk medicine remedy. The present study assessed the effects of M. longifolia extracts, including essential oil and crude methanol extract and its fractions (ethyl acetate, butanol and hexane), on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression in lipopolysaccharide (LPS)-stimulated J774A.1 cells using real-time polymerase chain reaction (PCR). The cytotoxic effects of the extracts on the cells were examined and non-cytotoxic concentrations (<0.2?mg/ml) were used to examine their effects on NO production and iNOS mRNA expression. Only the hexane fraction that contained high levels of phenolic and flavonoid compounds at concentrations from 0.05–0.20?mg/ml significantly reduced NO production in LPS-stimulated cells (p?<?0.001). Real-time PCR analysis indicated the ability of this fraction at the same concentrations to significantly decrease iNOS as well as TNFα mRNA expression in the cells (p?<?0.001). All extracts were able to scavenge NO radicals in a concentration-dependent manner. At concentrations greater than 0.2?mg/ml, total radicals were 100% scavenged. In conclusion, M. longifolia possibly reduces NO secretion in macrophages by scavenging NO and inhibiting iNOS mRNA expression, and also decreases TNFα pro-inflammatory cytokine expression, thus showing its usefulness in the inflammatory disease process.     

巨噬细胞诱导型一氧化氮合酶的表达调节机制

一氧化氮是一种重要的巨噬细胞免疫效应分子,它参与免疫调节和宿主防御反应.一氧化氮的生成主要由诱导型一氧化氮合酶调节,然而诱导型一氧化氮合酶表达的调节机制及信号通路尚不完全清楚.     

Expression of inducible nitric oxide synthase and nitrotyrosineduring the evolution of experimental pulmonary tuberculosis

Nitric oxide (NO) is a relevant antimycobacterial factor in mouse macrophages. NO is a product of inducible nitric oxide synthase (iNOS). NO toxicity is greatly enhanced by reacting with superoxide to form peroxynitrite that reacts with many biological molecules. Tyrosine is one of the molecules with which NO reacts and the product is nitrotyrosine (NT). The production of peroxynitrite and the nitrosylation of proteins might play a role in bacterial killing and also in mediating host injury. In this study, we used a well-characterized mouse model of pulmonary tuberculosis to examine the local kinetics of expression and cellular distribution of iNOS and NT at the cellular and subcellular level. The histopathological study showed two phases of the disease: early and late. The early phase was characterized by mononuclear inflammation and granuloma formation. During this phase, high percentages of activated macrophages were observed that were immunostained for iNOS and NT. Immuno-electronmicroscopy showed NT immunoreactivity in lysosomes and mycobacterial wall and cytoplasm. The concentration of iNOS mRNA and NO metabolites were also elevated. The late phase was characterized by progressive pneumonia with focal necrosis and a decrease of iNOS mRNA and NO metabolites. The strongest NT immunostained areas were the necrotic tissue. Macrophages became foamy cells with scarce iNOS immunostaining but strong NT immunoreactivity. At the ultrastructural level, these cells showed NT immunolabeling in cytoskeleton, mitochondria, lysosomes and cell membrane. NT was also located in bronchial epithelial cell mitochondria, in cell membranes and cytoplasm of endothelial cells and in actin bundles within smooth muscle cells. These results suggest an important role of NO in mycobacterial killing, particularly during the early phase of the infection. They also suggest an important participation by NO in tissue damage during the late phase of the disease.     

诱导型一氧化氮合酶在糖尿病性阴茎勃起功能障碍大鼠阴茎的表达

目的:探讨诱导型一氧化氮合酶(iNOS)在糖尿病性阴茎勃起功能障碍(ED)发病进程中的可能作用.方法:注射链脲佐菌素建立糖尿病大鼠模型,分别在注射8周和12周后观察阴茎勃起次数,取大鼠阴茎,用ABC免疫组织化学方法检测阴茎iNOS的分布与表达,免疫印迹检测阴茎iNOS蛋白量的变化.结果:糖尿病大鼠的阴茎勃起次数低于对照组,并随病程延长降低;与对照组比较,糖尿病组阴茎内iNOS阳性细胞数和平均光密度、iNOS蛋白量升高,并随病程延长而进行性升高.结论:糖尿病组大鼠阴茎iNOS表达的升高与ED相关.     

High nitric oxide production,secondary to inducible nitric oxide synthase expression,is essential for regulation of the tumour‐initiating properties of colon cancer stem cells

Chronic inflammation is a leading cause of neoplastic transformation in many human cancers and especially in colon cancer (CC), in part due to tumour promotion by nitric oxide (NO) generated at inflammatory sites. It has also been suggested that high NO synthesis, secondary to inducible NO synthase (iNOS) expression, is a distinctive feature of cancer stem cells (CSCs), a small subset of tumour cells with self‐renewal capacity. In this study we explored the contribution of NO to the development of colon CSC features and evaluated potential strategies to treat CC by modulating NO production. Our data show an integral role for endogenous NO and iNOS activity in the biology of colon CSCs. Indeed, colon CSCs with high endogenous NO production (NO^high) displayed higher tumourigenic abilities than NO^low fractions. The blockade of endogenous NO availability, using either a specific iNOS inhibitor or a genetic knock‐down of iNOS, resulted in a significant reduction of colon CSC tumourigenic capacities in vitro and in vivo. Interestingly, analysis of genes altered by iNOS‐directed shRNA showed that the knockdown of iNOS expression was associated with a significant down‐regulation of signalling pathways involved in stemness and tumour progression in colon CSCs. These findings confirm that endogenous NO plays an important role in defining the stemness properties of colon CSCs through cross‐regulation of several cellular signalling pathways. This discovery could shed light on the mechanisms by which NO induces the growth and invasiveness of CC, providing new insights into the link between inflammation and colon tumourigenesis. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.