强啡肽对脊髓原生型与诱生型一氧化氮合成酶活性,分布和基因表达的影响

蛛网膜下腔注射强啡肽Ａ１－１７引起剂量依赖性后肢和尾部瘫痪及甩尾甩足抑制。脊髓背角（侧）ＮＭＤＡ受体和ＮＯＳ／ＮＯ功能活性下降可能与Ｄｙｎ镇痛作用有关,脊髓腹角（）ＮＭＤＡ受体－Ｃａ＾２＋－ＮＯＳ／ＮＯ通路过度激活及ｃ－ｆｏｓ高表达可能与Ｄｙｎ致脊髓损伤作用有关。     

强啡肽A和CCK—8对大鼠脊髓突触小体摄取^45Ca的影响

为了探讨血管紧张素Ⅱ(AⅡ)和八肽胆囊收缩素(CCK-8)这两种肽的抗阿片作用机理,本实验中观察了三种阿片类物质(吗啡、强啡肽和 DPDPE)和两种抗阿片物质(AⅡ和 CCK-8)对大鼠脊髓突触小体摄取~(45) Ca 的影响。结果表明:(1)在脊髓腹柱突触小体上,10nmol/L—1μmol/L 的吗啡、强啡肽 A(Dyn A)和 DPDPE 对~(45)Ca 摄取均有较弱的抑制作用;(2)CCK-8在浓度高达lμmol/L 时对~(45)Ca 摄取有较弱的抑制作用;(3)AⅡ在浓度高达lμmol/L时也不影响腹柱突触小体摄取~(45)Ca;(4)在背柱的突触小体制备中,上述阿片物质中 Dyn A 对~(45)Ca 摄取有较强的抑制作用,并被 k 受体阻断剂 nor-BNI 所阻断。10和100nmol/L 的 CCK-8能翻转lμmol/L Dyn A 对~(45)Ca 摄取的抑制作用;(5)A Ⅱ不能翻转Dyn A 的抑制作用。以上结果提示,CCK-8阻断 Dyn A 抑制脊髓背柱突触小体摄取 Ca~(2+)的作用可能是其行为学中抗阿片作用的机理之一。AⅡ对脊髓 Ca~(2+)摄取和 Dyn A 抑制脊髓 Ca~(2+)摄取的作用皆无影响,与行为学中观察到的 AⅡ在脊髓内不能对抗阿片镇痛的现象一致,进一步说明 CCK-8和AⅡ拮抗阿片类物质对神经末梢 Ca~(2+)摄取的影响可能是其抗阿片作用的重要机理之一。     

眼镜蛇毒对大鼠脊髓和脊神经节一氧化氮合酶表达的影响

目的 观察眼镜蛇毒对脊髓和脊神经节一氧化氮合酶(NOS)表达的影响。方法 将眼镜蛇毒注入大鼠右侧大腿后部,采用还原型尼克酰胺嘌呤二核苷酸脱氢酶(NADPH．d)法显示NOS的表达。结果 在眼镜蛇毒注射组,脊髓和脊神经节内的NOS阳性神经元和深染NOS阳性神经元明显多于注入生理盐水组和正常对照组。结论 注入眼镜蛇毒能上调大鼠脊髓和脊神经节NOS表达。     

一氧化氮对植物细胞基因表达和代谢产物合成的影响机制

一氧化氮(NO)作为一种气体信号分子,在植物体内具有多种生理功能,许多研究逐步揭示了NO在植物发育、新陈代谢和疾病响应等方面的分子机制。内源和外源NO都可以使植物组织或悬浮细胞基因表达发生变化,高通量的基因表达的研究,如转录分析等,为信号网络通路分析提供了有力的证据。我们简要综述了NO的合成途径,并讨论了次生代谢产物及细胞程序性死亡过程中依赖NO调控的相应基因及蛋白的变化,揭示了NO是一种重要的植物信号分子,有较高的研究价值。     

晚妊人胎脊髓内一氧化氮合酶阳性神经元和阳性纤维的分布及其生物学意义

取７例人胎脊髓标本,用还原型辅酶Ⅱ（β－ＮＡＤＰＨ）组织化学方法对人胎脊髓内一氧化氮合酶（ＮＯＳ）阳性神经元和阳性纤维的分布进行了观察。ＮＯＳ阳性神经元在妊娠３２周至３９周胎龄人胎脊髓内的分布和细胞形态无明显差异,主要位于后角深层（Ⅲ、Ⅳ层）、中央管周围灰质和中间带外侧核（ＩＭＬ）;前角内可见少数散在的ＮＯＳ阳性神经元;在脊髓白质内有密集的ＮＯＳ阳性的胶质样细胞分布。ＮＯＳ阳性纤维主要见于后角浅层（Ⅰ、Ⅱ层）和中间带。脊髓内ＮＯＮ阳性神经元和阳性纤维的分布,提示脊髓内ＮＯＳ可能与内脏活动的调节和躯体感觉传入的调制有关;ＮＯＳ阳性的胶质样细胞可能参与白质内神经纤维的髓化过程。     

大鼠实验性乙醇胃粘膜损伤中一氧化氮合成酶和内皮素合量的变化及意义

目的 :探讨一氧化氮和内皮素在急性乙醇胃粘膜损伤中的作用及其相互关系。方法 :采用大鼠乙醇胃粘膜损伤模型 ,测定其胃粘膜内一氧化氮合成酶 (NOS)和内皮素 (ET)含量并观察其胃粘膜病理变化。结果 :随着乙醇作用时间延长和胃粘膜损伤的加重 ,胃粘膜内ET含量显著上升 (P <0 .0 5 ) ,而NOS的含量显著下降 (P <0 .0 5 ) ,两者呈负相关。结论 :胃粘膜内ET释放增加和NOS活性下降参与了急性乙醇胃粘膜损伤的病理生理过程。     

大鼠脊髓诱发电位与脊髓损伤的关系——Ⅱ、皮肤、椎板和硬膜上诱发电位的比较

从大鼠的背侧皮肤表面和椎板分别记录刺激坐骨神经诱发的脊髓电位,并与硬膜上电位进行了比较。结果表明:皮肤表面电位与硬膜上直接记录具有相同的节段性特征。从硬膜上经椎板至皮肤表面、反应潜伏时延长、电位幅度递减。各波峰潜伏时也相应增加。电位的波形、幅度与记录方式有关,但反应潜伏时不受影响。     

姜黄素对大鼠脊髓损伤后微生物多样性及脊髓转录组学的影响

【背景】脊髓损伤是一种中枢神经系统的严重创伤,除了导致运动、感觉、自主神经功能障碍外,还会引起胃肠道功能障碍,不利于后续功能恢复。先前的研究表明,姜黄素对脊髓损伤具有一定的治疗作用,但姜黄素对脊髓损伤后肠道菌群是否具有调节作用及治疗相关通路鲜有报道。【目的】观察姜黄素对脊髓损伤大鼠行为学和组织形态学的影响,并利用高通量测序技术探讨姜黄素对盲肠组织微生物及其代谢产物的影响,探究姜黄素对脊髓损伤大鼠微生物多样性及脊髓转录组学的影响。【方法】将50只6−8周、220−240 g左右雌性SD大鼠随机分为假手术组、脊髓损伤模型组、脊髓损伤后姜黄素低、中、高剂量给药组。假手术组无脊髓损伤,模型组和姜黄素组建立脊髓损伤模型,在脊髓损伤模型建立30 min后分别腹腔注射姜黄素50、100和200 mg/kg,每天1次,连续治疗1周。术后采用大鼠脊髓损伤评分(basso, beattie & bresnahan locomotor rating scale, BBB scale)量表评定运动功能;苏木精-伊红染色观察大鼠脊髓组织形态学变化;通过对16S rRNA基因的V3+V4可变区进行高通量测序,分析α及β多样性、菌群的相对丰度以及短链脂肪酸含量,评估治疗前后对肠道菌群的影响。【结果】中等剂量姜黄素100 mg/kg可使脊髓损伤后肢体运动功能明显改善,对受损脊髓有一定的保护和修复作用;姜黄素给药可改善脊髓损伤后微生物群多样性,提高短链脂肪酸水平;脊髓损伤后大鼠肠道菌群信号转导通路发生变化,在应用姜黄素后部分信号通路恢复表达。【结论】姜黄素可以影响脊髓损伤大鼠肠道微生物多样性并提高短链脂肪酸水平,对脊髓损伤大鼠具有治疗作用。     

一氧化氮对豆科植物结瘤及固氮的影响机制

豆科植物-根瘤菌共生过程受双方基因复杂且精细的调控, 能够产生特异的根瘤结构并可将大气中的惰性氮气(N₂)转化为可被植物直接利用的氨态氮。结瘤与固氮受多种因素影响, 其中, 一氧化氮(NO)作为一种自由基反应性气体信号分子, 可参与调节植物的许多生长发育过程, 如植物的呼吸、光形态建成、种子萌发、组织和器官发育、衰老以及响应各种生物及非生物胁迫。在豆科植物中, NO不仅影响寄主与菌共生关系的建立, 还参与调控根瘤菌对氮气的固定并提高植株氮素营养利用效率。该文主要从豆科植物及共生菌内NO的产生、降解及其对结瘤、共生固氮的影响和对环境胁迫的响应, 阐述了NO调控豆科植物共生体系中根瘤形成和共生固氮过程的作用机制, 展望了NO信号分子在豆科植物共生固氮体系中的研究前景。     

TNF-α、IL-1β及LPS对血管平滑肌细胞一氧化氮合酶基因表达的影响及作用机制研究

通过ＲＮＡ印迹分析和亚硝酸盐含量测定检查ＴＮＦ－α、ＩＬ－１β和ＬＰＳ对大鼠血管平滑肌细胞（ＶＳＭＣ）诱导型一氧化氮合酶（ｉＮＯＳ）基因表达及ＮＯ生成的影响．结果表明,ＴＮＦ－α、ＩＬ－１β和ＬＰＳ均能显著诱导ＶＳＭＣｉＮＯＳ基因表达和促进ＮＯ生成,其作用强度与浓度和作用时间有关;双因素（ＴＮＦ－α＋ＬＰＳ,ＬＰＳ＋ＩＬ－１β）对诱导ｉＮＯＳ基因表达及ＮＯ生成产生协同作用．ＰｏｌｙｍｙｘｉｎＢ和地塞米松可部分抑制ＴＮＦ－α对ｉＮＯＳ基因表达的诱导作用及ＮＯ生成     

脊髓细胞外调节激酶1/2在大鼠后足切割痛中的表达和作用

目的：探讨大鼠后足切割后脊髓ERK的表达情况。方法：以大鼠右后足切割作为急性疼痛模型;用免疫组织化学法测试脊髓磷酸化ERK（pERK）表达情况。ERK抑制剂U0126（1μg）在切割前20min或切割后20min鞘内注射。用von Frey纤维测试大鼠机械性痛敏。结果：大鼠后足切割后1min,在切割侧L4-L5脊髓浅层背侧角（板层Ⅰ和板层Ⅱ）ERK被迅速地激活,并在5min达到峰值,随后恢复到基础值。切割前鞘内给予U0126能显著减轻机械性痛敏,然而,切割后鞘内给予U0126对机械性痛敏的作用并不明显。结论：脊髓ERK在大鼠后足切割痛中产生机械性痛敏发挥了重要的作用。     

大鼠坐骨神经结扎后脊髓钙结合蛋白PV的表达变化

目的:研究大鼠坐骨神经结扎模型钙结合蛋白Parvalbumin(PV)在脊髓的时空变化规律,为探讨其在神经再生中的作用与机制提供实验依据。方法:SD大鼠随机分为假手术对照组和坐骨神经结扎组,实验组结扎后分别存活1,3,7,14或21d,采用免疫组化结合图像分析技术观察PV在脊髓的表达变化。结果:在对照组,PV免疫阳性神经元主要分布于腰髓背角Ⅱ层,Ⅲ～Ⅵ层只观察到少量散在分布的PV样阳性神经元,脊髓前角Ⅷ层和Ⅸ层内也可见少量多极的大型阳性神经元。术后各时间点PV样阳性神经元表达下降,14d下降最显著,21d表达有所上升,但还是低于7d组。脊髓后角PV免疫阳性产物灰度值测定结果显示:术后14d后角PV表达最低,与对侧和对照组以及1、3d组相比有统计学意义(P<0.05)。结论:坐骨神经结扎后PV表达变化呈现一定的时空模式,为进一步揭示PV在神经系统疾病中的作用提供实验依据。     

Fluorocitrate,an Inhibitor of Glial Metabolism,Inhibits the Up-Regulation of NOS Expression,Activity and NO Production in the Spinal Cord Induced by Formalin Test in Rats

Previous experiments have suggested that nitric oxide plays an important role in nociceptive transmission in the spinal cord. In order to explore the involvement of glia in the NO-mediated nociceptive transmission, the present study was undertaken to investigate the effect of fluorocitrate (FC), an inhibitor of glial metabolism, on NOS expression and activity and NO production in the spinal cord during the process of peripheral inflammatory pain and hyperalgesia induced by formalin test in rats. Sixty adult male Sprague–Dawley rats were randomly assigned into sham, formalin, formalin + normal saline (NS), and formalin + FC groups. The NOS expression, NOS activity and NO production was detected by NADPH-d histochemistry staining, NOS and NO assay kit, respectively. It was found that formalin test significantly up-regulated NOS expression and activity and NO production in the laminae I–II of the dorsal horn and the grey matter around the central canal in the lumbar spinal cord at 1 h after the formalin test. Selective inhibition of glia metabolism with intrathecal administration of FC (1 nmol) significantly inhibited the up-regulation in NOS expression and activity and NO production normally induced by the formalin test, which was represented with decreases in the number and density of the NADPH-d positive cells in the dorsal horn and grey matter around the central canal, and decrease in density of NADPH-d positive neuropil in the dorsal horn in formalin + FC group compared with formalin group. The results suggested that glia may be involved in the NO-mediated nociceptive transmission in the spinal cord. X.-C. Sun, W.-N. Chen and S.-Q. Li contributed equally to this work.     

Effects of Peripheral Axotomy on Cholecystokinin Neurotransmission in the Rat Spinal Cord

Abstract : Because cholecystokinin (CCK) acts as a "functional" endogenous opioid antagonist, it has been proposed that changes in central CCKergic neurotransmission might account for the relative resistance of neuropathic pain to the analgesic action of morphine. This hypothesis was addressed by measuring CCK-related parameters 2 weeks after unilateral sciatic nerve section in rats. As expected, significant decreases (-25-38%) in the tissue concentrations and in vitro release of both substance P and calcitonin gene-related peptide were noted in the dorsal quadrant of the lumbar spinal cord on the lesioned side. In contrast, the tissue levels and in vitro release of CCK were unchanged in the same area in lesioned rats. Measurements in dorsal root ganglia at L4-L6 levels revealed no significant changes in proCCK mRNA after the lesion. However, sciatic nerve section was associated with a marked ipsilateral increase in both CCK-B receptor mRNA levels in these ganglia (+70%) and the autoradiographic labeling of CCK-B receptors by [³H]pBC 264 (+160%) in the superficial layers of the lumbar dorsal horn. Up-regulation of CCK-B receptors rather than CCK synthesis and release probably contributes to increased spinal CCKergic neurotransmission in neuropathic pain.     

大鼠坐骨神经结扎后脊髓钙结合蛋白Calbindin D-28k的表达变化

目的:研究大鼠坐骨神经结扎模型的钙结合蛋白(Calbindin D-28k,CB)在脊髓的时空变化规律,为探讨其在神经再生中的作用与机制提供实验依据。方法:SD大鼠随机分为假手术对照组和坐骨神经结扎组,实验组结扎后分别存活1,3,7,14或21d,免疫组化结合图像分析技术观察CB在脊髓的表达变化。结果:在对照组,CB阳性神经元主要分布于腰髓背角Ⅰ、Ⅱ层,Ⅲ～Ⅵ层只观察到少量散在分布的CB样阳性神经元,脊髓前角Ⅷ层和Ⅸ层内也可见少量多极的大型阳性神经元。术后各时间点CB样阳性神经元表达下降,14d下降最显著,21d表达有所上升,但还是低于7d组。脊髓后角CB免疫阳性产物灰度值测定结果显示:术后14d后角CB表达最低,与对侧和对照组以及1、3d组相比有统计学意义(P<0.05)。结论:坐骨神经结扎后CB表达变化呈现一定的时空模式,为进一步揭示CB在神经系统疾病中的作用提供实验依据。     

Localization of Endomorphin-2-Like Immunoreactivity in the Rat Medulla and Spinal Cord

Martin-Schild, S., J. E. Zadina, A. A. Gerall, S. Vigh and A. J. Kastin. Localization of endomorphin-2-like immunoreactivity in the rat medulla and spinal cord. Peptides 18(10) 1641–1649, 1997.—Endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂) are endogenous ligands that have greater affinity and selectivity for the μ-opiate receptor than any other known mammalian peptide. A polyclonal antiserum, screened for specificity to endomorphin-2 by immunodot-blot assay and preabsorption controls, was used for localization of this peptide. Immunocytochemistry performed on the brainstem, spinal cord, and sensory ganglia of rats by the avidin–biotin–peroxidase method revealed a continuous dense aggregation of endomorphin-2-like immunoreactive varicose fibers in the superficial laminae of the dorsal horn of the medulla and spinal cord. Immunoreactive fibers were detected in the dorsal root as well as within the dorsal root ganglia. The results suggest that endomorphin-2 is synthesized in primary sensory neurons in ganglia, transported to the superficial dorsal horn, and released near neurons expressing μ receptors. Its distribution appears to represent a functional unit likely to be associated with modulation of nociceptive stimuli.     

5-Hydroxytryptamine-Facilitated Release of Substance P from Rat Spinal Cord Slices Is Mediated by Nitric Oxide and Cyclic GMP

Abstract: The role of nitric oxide (NO) in the control of 5-hydroxytryptamine (5-HT)-induced release of substance P was investigated in rat spinal cord in vitro. 5-HT facilitated the 60 m M K⁺-evoked release of substance P-like immunoreactive materials (SPLI) from the superfused rat dorsal spinal cord slices without affecting spontaneous SPLI release. The facilitatory effect of 5-HT was significantly inhibited by ICS 205-930 or granisetron (potent and specific 5-HT₃ receptor antagonists), by N ^G-monomethyl- l -arginine (NMMA, a NO synthase inhibitor), and by methylene blue or 1 H -[1,2,4]oxadiazolo[4,3- a ]quinoxaline-1-one (MB or ODQ, respectively; both are inhibitors of soluble guanylyl cyclase) and was mimicked by 2-methylserotonin (2-m-5-HT, a selective 5-HT₃ receptor agonist), l -arginine (a precursor of NO), or 8-bromo-cyclic GMP. NMMA, MB, or ODQ inhibited the 2-m-5-HT-induced increase of cyclic GMP levels in the rat dorsal spinal cord slices. These data suggest that the facilitatory effect of 5-HT on the release of SPLI is mediated by the 5-HT₃ receptor and that the intracellular signaling is mediated via NO by an increase in cyclic GMP production.     

Spatiotemporal Expression of Dexras1 After Spinal Cord Transection in Rats

Dexras1, a brain-enriched member of the Ras subfamily of GTPases, as a novel physiologic nitric oxide (NO) effector, anchor neuronal nitric oxide synthase (nNOS) that increased after spinal cord injury (SCI), to specific targets to enhance NO signaling, and is strongly and rapidly induced during treatment with dexamethasone. It is unknown how the central nervous system (CNS) trauma affects the expression of Dexras1. Here we used spinal cord transection (SCT) model to detect expression of Dexras1 at mRNA and protein level in spinal cord homogenates by real-time PCR and Western blot analysis. The results showed that Dexras1 mRNA upregulated at 3 day, 5 day, and 7 day significantly (P < 0.05) that was consistent with the protein level except at 7 day. Immunofluorescence revealed that both neurons and glial cells showed Dexras1 immunoreactivivty (IR) around SCT site, but the proportion is different. Importantly, injury-induced expression of Dexras1 was co-labeled by caspase-3 (apoptotic marker) and Tau-1 (marker for pathological oligodendrocyte). Furthermore, colocalization of Dexras1, carboxy-terminal PSD95/DLG/ZO-1 (PDZ) ligand of nNOS (CAPON) and nNOS was observed in neurons and glial cells, supporting the existence of ternary complexes in this model. Thus, the results that the transient high expression of Dexras1 which localized in apoptotic neurons and pathological oligodendrocytes might provide new insight into the secondary response after SCT. Xin Li, Chun Cheng, and Min Fei contributed equally to this work.