内质网应激蛋白BiP在姜黄素抗2型糖尿病神经病理性疼痛大鼠的作用

目的: 探讨姜黄素对2型糖尿病诱导的神经病理性疼痛(DNP)大鼠机械痛敏(MWT)、热痛敏(TWL)、脊髓背角和背根神经节(DRG)的保护作用及机制。方法: 高脂高糖饲料喂养8周诱导胰岛素抵抗后,继以单次小剂量链脲佐菌素(STZ)35 mg/kg腹腔注射,在注射STZ前和14 d后采用Electronic Von Fery触觉测痛仪和甩尾/足底测试仪测大鼠MWT和TWL,注射STZ 3 d后血糖≥16.7 mmol/L且在注射STZ 14 d后痛阈下降至基础值85%以下者入选为D组(81只)。将D组随机分为3组(n=27): DNP组、DNP+姜黄素100 mg·kg^-1·d^-1组(DCur组)和DNP+溶剂组(DSC组),另取27只为正常对照组(C组),给予普通饲料喂养。给姜黄素3、7、14 d后测血糖、MWT与TWL,并在同时点取大鼠L4~L6脊髓和DRG,用免疫组化和免疫印迹法测定脊髓背角和DRG中免疫球蛋白重链结合蛋白(BiP)的表达。结果: 与C组相比,DNP组各时点MWT降低,TWL缩短,血糖值升高,脊髓背角和DRG中BiP均出现表达上调(P<0.05)。与DNP组相比,DCur组在给姜黄素7 d后MWT升高,TWL延长;给姜黄素14 d后脊髓背角和DRG中BiP表达下调(P<0.05)。DSC组和DNP组相比差异无统计学意义。结论: BiP参与了2型糖尿病神经病理性疼痛的发病机制。姜黄素减轻2型糖尿病大鼠神经病理性疼痛的机制可能与抑制BiP表达有关。     

p-ERK1/2-AP-1通路在姜黄素抗大鼠糖尿病神经病理性痛中的作用

目的: 观察p-ERK1/2-AP-1通路在姜黄素(Cur)抗大鼠糖尿病神经病理性痛(DNP)中的作用。方法: 雄性SD大鼠96只,随机分为4组(n=24):正常对照组、DNP组、DNP+溶剂组(DNP+Sol组)和DNP+Cur 100 mg/kg组(DNP+Cur组)。除正常对照组外,其余各组采用腹腔注射链唑霉素75 mg/kg的方法制备DNP模型,造模成功后每天1次腹腔注射相应的溶剂或Cur,持续2周。于造模前2 d、造模后14 d、腹腔给药后3、7、14 d时测定机械缩足痛阈(MWT)、热缩足潜伏期(TWL)和非空腹尾静脉血糖值,取脊髓腰膨大及L₄/L₅背根神经节(DRG),采用免疫组化及Western blotting法测定脊髓背角和DRG p-ERK1/2的表达,电泳迁移率变动分析AP-1的表达。结果: 与正常对照组相比,DNP组各时点MWT降低、TWL缩短;血糖值升高;脊髓背角及DRG p-ERK1/2均出现表达上调(P<0.05);脊髓背角AP-1表达上调(P<0.05)。与DNP组相比,DNP+Cur组在给药后7 d MWT回升、TWL延长;在给药后14 d脊髓背角及DRG p-ERK1/2、脊髓背角AP-1表达下调(P<0.05),但并不影响血糖水平。结论: Cur可减轻大鼠糖尿病神经病理性痛,其机制可能与抑制脊髓背角和DRG神经元p-ERK1/2-AP-1通路激活有关。     

脉冲电磁刺激对糖尿病大鼠神经病理性疼痛的影响

目的:系统研究外源性的脉冲电磁干预法对糖尿病神经病理性疼痛(DNP)大鼠的作用效果。方法:30只成熟的雄性SD大鼠,随机等分成3组:空白对照(Control)组,DNP组以及DNP协同电磁刺激(DNP+EMF)组。DNP模型使用腹腔注射链脲佐菌素方法构建。对DNP+EMF组大鼠行外源性的全身性电磁刺激干预(脉冲波形,频率15 Hz,强度30 Gs),每天刺激6 h。在电磁刺激的第0、2、4、6、8周对各组大鼠进行足底机械痛阈值、足底热痛阈值、感觉神经传导速率和运动神经传导速率检测。结果:DNP+EMF组大鼠的足底机械痛阈值和热痛阈值在第4、6、8周显著高于DNP组大鼠(P0.05);脉冲电磁刺激从第2周开始显著提升了DNP大鼠足底的感觉神经传导速率(P0.05),并从第4周起显著提高了DNP大鼠足底的运动神经传导速率(P0.05)。结论:外源性的脉冲电磁刺激能够显著改善糖尿病大鼠症状,表现为痛觉敏感性和神经传导速率的提升,脉冲电磁刺激有望成为临床治疗DNP的一种新的物理治疗手段。     

大麻素CB_1受体活化对神经病理性疼痛大鼠脊髓背角嘌呤能P2X_2受体表达的抑制作用

目的:观察脊髓背角大麻素CB_1受体(CB_1R)在坐骨神经缩窄性损伤(CCI)所致的神经病理性疼痛中的作用及其对嘌呤能P2X_2受体表达的调节。方法:7~8周龄SD大鼠分为4组:(1)sham组;(2)CCI组;(3)CP55940+CCI组;(4)AM251+CP55940+CCI组。分别于CCI术前1 d,术后1、3、5、7、10、14 d测定热缩足反射潜伏期(TWL);免疫印迹技术检测各组大鼠损伤侧L_4~L_6段脊髓背角P2X_2受体表达。结果:CCI术后大鼠出现热痛敏,TWL明显缩短;鞘内给予非选择性大麻素受体激动剂CP55940可明显延长CCI大鼠TWL(P0.05);预先鞘内注射CB_1R拮抗剂AM251(0.05 mg/kg)可显著降低CP55940的镇痛效果(P0.05)。免疫印迹实验结果显示:CCI大鼠脊髓背角P2X_2受体在术后7、14 d表达明显增加(P0.05);鞘内给予CP55940可显著降低P2X_2受体表达(P0.05),而预先给予AM251可降低CP55940抑制P2X_2受体表达的效应(P0.05)。结论:脊髓背角CB_1受体激活对CCI所致的神经病理性疼痛具有良好的镇痛作用,其镇痛效应可能与抑制CCI大鼠嘌呤能P2X_2受体表达有关。     

脊髓背角MCP-1-JAK2/STAT3信号转导参与大鼠2型糖尿病神经病理性痛的机制研究

目的:探讨脊髓背角MCP-1-JAK2/STAT3信号转导是否参与大鼠2型糖尿病神经病理性疼痛(DNP)的形成与发展。方法:雄性SD大鼠高糖高脂饲养8周,通过腹腔单次注射链脲佐菌素(STZ)制备大鼠2型DNP模型。将2型DNP大鼠随机分为4组,每组16只:DNP组、DNP+MCP-1抑制剂组(DM组)、DNP+JAK2抑制剂AG490组(DA组)和溶剂对照组(SC组)。DA、DM和SC组分别于注射STZ 14 d后蛛网膜下腔置管,3 d后DM、DA和SC组分别给予MCP-1中和抗体10μL(0.1 mg/L)、AG490 10μL(1 mmol/L)和3.5%DMSO 10μL,每天1次,连续14 d。DNP组不做任何处理。另取16只大鼠为对照(control,C)组,普通饲料喂养。蛛网膜下腔给药后第1、3、7和14天时测定机械缩足阈值(MWT)和热缩足反射潜伏期(TWL),各组随机取4只大鼠处死,取L4-6脊髓膨大,采用Western blot法检测p-JAK2和p-STAT3的表达。结果:与C组比较,DNP和SC组第1、3、7和14天时MWT降低,TWL缩短,脊髓背角p-JAK2和p-STAT3表达上调(P0.05);与DNP组比较,DM和DA组第1、3、7和14天时MWT升高和TWL延长,脊髓背角p-JAK2和p-STAT3表达下调(P0.05);DNP组与SC组各指标比较差异无统计学意义。结论:脊髓背角MCP-1-JAK2/STAT3信号转导参与大鼠2型DNP的产生和维持。     

鞘内注射p38MAPK抑制剂SB203580对CCI大鼠脊髓背角多种嘌呤受体表达的影响

目的:观察鞘内注射p38MAPK磷酸化抑制剂SB203580对慢性坐骨神经结扎(chronic constriction injury,CCI)大鼠热痛阈及脊髓背角P2X_4、P2X_7、P2Y_(12)、P2Y_(13)受体mRNA表达变化的影响。方法:成年SD大鼠随机分为5组(n=8):假手术组(sham组)、CCI模型组(CCI+鞘内注射0.005%DMSO生理盐水)、CCI+鞘内注射SB203580 1μmol/L组、CCI+鞘内注射SB203580 10μmol/L组、CCI+鞘内注射SB203580 50μmol/L组。CCI模型组及CCI+SB203580处理组大鼠均于鞘内置管7 d后行慢性坐骨神经结扎,连续14 d鞘内注射0.005%DMSO生理盐水或SB203580(1,10,50μmol/L),2次/d,分别在术前1 d、术后第1,3,5,7,10,14 d测定给药1 h后热缩足潜伏期(TWL);并在第3,7,14 d取大鼠脊髓背角进行荧光定量PCR,观察脊髓背角P2X_4、P2X_7、P2Y_(12)、P2Y_(13)受体mRNA的表达变化。结果:CCI术后大鼠即形成稳定的热痛敏,与sham组相比,CCI模型组大鼠术后TWL明显缩短(P0.05);与CCI模型组相比,CCI+SB203580 10,50μmol/L处理组大鼠随药物剂量增加,TWL延长更为明显(P0.05)。荧光定量PCR结果显示:与sham组相比,CCI模型组第3,7,14 d,P2X_4、P2X_7、P2Y_(12)、P2Y_(13)受体mRNA的表达上调(P0.05);给予SB203580 50μmoL/L后,大鼠脊髓背角P2X_7、P2Y_(13)受体mRNA表达有所下调(P0.05),但P2X_4和P2Y_(12)受体mRNA表达没有明显变化。结论:鞘内注射p38MAPK磷酸化抑制剂SB203580可以明显抑制CCI大鼠热痛敏行为学表现,其机制与抑制脊髓背角P2X_7、P2Y_(13)受体mRNA表达有关。这提示脊髓背角小胶质细胞p38MAPK活化后上调P2X_7和P2Y_(13)受体基因转录水平可能是p38MAPK在脊髓水平参与伤害性信息传递的机制之一。     

JNK/MCP-1信号通路在姜黄素抗糖尿病神经病理性疼痛中的作用

 目的:观察JNK/MCP-1通路在姜黄素抗大鼠糖尿病神经病理性疼痛（DNP）中的作用及机制。方法:雄性SD大鼠诱导为2型糖尿病神经病理性痛大鼠（DNP）模型,将其随机分为6组（n=27）： DNP组、姜黄素组（Cur组）、溶剂对照组（DSC组）、JNK抑制剂组 (DJ组)、JNK抑制剂溶剂对照组(DJS组)、姜黄素+单核细胞趋化蛋白 1 (MCP-1)激动剂组（DM组）。另取27只正常大鼠为正常对照组（C组）,给药后3 d、7 d、14 d时测定机械缩足痛阈和热缩足潜伏期,并在同一时点取脊髓腰膨大及L4~6背根神经节（DRG）,用免疫印迹法测定脊髓和DRG中p-JNK水平,用ELISA测定脊髓和DRG中的MCP-1含量。结果:与DNP组相比,在给药后的7 d、14 d Cur组、DJ组、DM组p-JNK的表达明显下降（P<0.05）;与C组相比,链脲佐菌素给药后其它6组MCP-1含量出现明显下降;与DNP组相比,在给药后的7 d、14 d Cur组、DJ组MCP-1出现明显上升,而DM组出现进一步下降（P<0.05）。结论: DNP大鼠脊髓和DRG中的p-JNK、MCP-1表达明显升高,姜黄素减轻2型糖尿病大鼠神经病理性疼痛的机制可能与JNK/MCP-1信号通路有关。     

嘌呤受体P2X7抑制剂BBG对异氟烷麻醉致老年大鼠认知功能障碍的影响

目的观察嘌呤受体P2X7阻断剂BBG对异氟烷麻醉致大鼠认知功能障碍的影响,初步探讨P2X7受体在其中的作用机制。方法雄性18月龄SD大鼠100只随机分为C组(对照组)、BBG组、ISO组(异氟烷组)和BBG+ISO组,每组25只。C组和ISO组腹腔注射0.9%生理盐水,BBG组和BBG+ISO组腹腔注射BBG溶液50 mg·kg~(-1)·d~(-1),连续3 d。第4 d,C组和BBG组持续吸入100%O26 h;ISO组和BBG+ISO组持续吸入1.4%异氟烷麻醉6 h,以100%O2作为载气。24 h后行Moris水迷宫实验观察大鼠认知功能变化,免疫荧光染色法观察Iba-1染色评估大鼠海马区小胶质细胞激活程度,酶联免疫吸附(ELISA)法检测大鼠海马组织中白细胞介素-1β(IL-1β)和IL-6浓度。Sigmaplot 12.0统计学软件进行分析,组间比较采用单因素方差分析,组内比较采用重复测量设计的方差分析,P0.05为差异有统计学意义。结果与C组比较,ISO组行为学训练第3~5天逃避潜伏期明显延长,探索时间明显缩短(P0.05),海马CA3区Iba-1阳性染色面积明显更高(P0.01),海马IL-1β和IL-6含量明显高于其它3组(P0.01)。与ISO组比较,BBG+ISO组训练第3~5天逃避潜伏期缩短,探索时间延长(P0.05)。海马CA3区Iba-1阳性面积染色明显更低(P0.05),海马IL-1β和IL-6含量更低(P0.01)。结论P2X7受体阻断剂BBG可改善异氟烷麻醉引起的老年大鼠认知功能障碍,其作用机制可能是BBG抑制海马区域小胶质细胞激活,进而降低海马区域炎性反应,改善认知功能。     

神经病理性痛大鼠海马CA1区锥体神经元树突及树突棘的变化

目的:观察神经病理性痛条件下大鼠海马CA1区锥体神经元树突形态和树突棘密度的变化。方法:建立大鼠腰5脊神经结扎(L5 spinal nerve ligation,SNL)神经病理性痛模型,对照组为假手术组即只暴露腰5脊神经,不结扎。利用Von Frey纤维丝检测其50%机械性缩足阈值(paw withdrawal threshold,PWT),判断SNL模型是否成功。通过高尔基(Golgi)染色的方法观察SNL模型后14 d海马CA1区锥体神经元树突形态和树突棘密度的变化。结果:SNL模型组大鼠CA1区锥体神经元树突棘密度升高,与对照组相比有统计学差异(P0.05),而锥体神经元树突分支数无明显差异。结论:神经病理性痛会导致海马CA1区锥体神经元树突棘密度升高。     

c-fos反义寡核苷酸探针对大鼠炎性痛和神经病理性痛的影响

研究c-fos反义寡核苷酸探针(antisense oligodeoxynucleotides,ASO)对大鼠炎性痛和神经病理性痛的作用效果,为临床慢性疼痛的治疗提供理论依据。以大鼠足底注射福尔马林(formalin)致炎性痛模型和腰5脊神经结扎(spinal nerve ligation,SNL)诱导神经病理性痛模型为基础,分别观察鞘内给予c-fosASO对两种模型大鼠的行为学及腰5脊髓背角Fos阳性神经元表达的影响。行为学检测结果表明:和正义探针(SO)组以及生理盐水(NS)对照组相比,鞘内预先给予c-fosASO后,大鼠足底注射福尔马林引起自发缩足反射次数和SNL诱导的机械性缩足反射阈值(mechanical withdrawal threshold,MWT)和热缩足反射潜伏期(thermal withdrawal latency,TWL)均有明显缓解(P<0.05),而c-fosASO不能逆转SNL诱导的神经病理性痛。免疫荧光组织化学染色结果发现:和对照组相比,鞘内给予c-fosASO能显著抑制福尔马林和SNL诱导的大鼠腰髓背角Fos阳性神经元的增加。上述结果提示,脊髓背角c-fos的活化对于炎性痛和神经病理性痛的发展都有显著意义,而抑制其活化在预防阶段比治疗阶段对慢性疼痛的缓解作用更为显著。     

亮蓝G减轻急性CO中毒大鼠海马的损伤

 目的：观察亮蓝G（BBG）对急性一氧化碳中毒（ACMP）引起的大鼠海马组织损伤的影响,探讨配体门控离子通道嘌呤能P2X₇受体（P2X₇R）在一氧化碳中毒脑损伤中的作用。方法：80只8~10周龄SD大鼠分为对照（control）组、ACMP组、ACMP+BBG组和BBG组。采用腹腔注射CO（100 mL/kg）的方法制备大鼠ACMP模型。检测静脉血中的碳氧血红蛋白（HbCO）浓度来判断CO中毒程度。RT-qPCR检测大鼠海马组织P2X₇R的mRNA表达。干湿称重法检测海马组织水肿程度。ELISA法检测海马组织促炎因子白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）和IL-6的含量。Morris水迷宫实验评价学习记忆能力。HE染色观察海马CA1区细胞形态学改变。结果：与control组（100%）相比,染毒6 h后ACMP组大鼠的存活率降低至55%;海马组织P2X₇R的mRNA表达及促炎因子IL-1β、TNF-α和IL-6的含量均明显增加（P<0.05）;含水量升高到80.72%±0.93%;大鼠逃避潜伏期明显延长,在目标象限探索时间明显缩短。值得注意的是,BBG预处理可以明显改善ACMP诱导的这些海马损伤。另外,大鼠染毒后3 d,海马CA1区的神经元排列紊乱稀疏,细胞形态不完整,细胞核深染、固缩。而BBG预处理能够显著改善CA1区的细胞形态。结论：P2X₇R与ACMP诱导的海马损伤相关。BBG能够增加ACMP大鼠的存活率,减少促炎因子的释放,减轻海马水肿,改善学习记忆能力,减轻ACMP引起的CA1区神经元损伤。     

Effects of probenecid and brilliant blue G on rat enteric glial cells following intestinal ischemia and reperfusion

The P2X7 receptor participates in several intracellular events and acts with the pannexin-1 channel. This study examined the effects of probenecid (PB) and brilliant blue G (BBG), which are antagonists of the pannexin-1 channel and P2X7 receptor, respectively, on rat ileum enteric glial cells after on ischemia and reperfusion. The ileal vessels were occluded for 45 min with nontraumatic vascular tweezers, and reperfusion was performed for periods of 24 h and 14 and 28 days. After ischemia (IR groups), the animals were treated with BBG (BG group) or PB (PB group). The double-labeling results demonstrated the following: the P2X7 receptor was present in enteric glial cells (S100β) and enteric neurons positive for HuC/D; enteric glial cells exhibited different phenotypes; some enteric glial cells were immunoreactive to only S100β or GFAP; and the pannexin-1 channel was present in enteric glial cells (GFAP). Density (in cells/cm²) analyses showed that the IR group exhibited a decrease in the number of cells immunoreactive for the P2X7 receptor, pannexin-1, and HuC/D and that treatment with BBG or PB resulted in the recovery of the numbers of these cells. The number of glial cells (S100β and GFAP) was higher in the IR group, and the treatments decreased the number of these cells to the normal value. However, the PB group did not exhibit recovery of S100β-positive glia. The cell profile area (μm²) of S100β-positive enteric glial cells decreased to the normal value after BBG treatment, whereas no recovery was observed in the PB group. The ileum contractile activity was decreased in the IR group and returned to baseline in the BG and PB groups. BBG and PB can effectively induce the recovery of neurons and glia cells and are thus potential therapeutic agents in the treatment of gastrointestinal tract diseases.     

1,8-桉叶素对背根神经节内P2X3受体介导神经病理痛的作用

目的:探讨1,8-桉叶素对大鼠背根神经节(DRG)神经元P2X3受体介导神经病理痛的作用。方法:建立大鼠坐骨神经慢性压迫性损伤模型(CCI)。SD大鼠随机分为假手术(Sham)组,坐骨神经慢性压迫性损伤(模型组,CCI)组、低剂量1,8-桉叶素治疗组、高剂量1,8-桉叶素治疗组、二甲亚砜对照组。检测大鼠术后7、14 d机械缩足反射(MWT)及热缩足反射潜伏期(TWL),观察大鼠行为学变化。免疫组织化学和原位杂交观察神经病理痛大鼠第4~5腰(L_(4-5))DRG神经元P2X3受体表达变化。结果:术后第7和14天,模型组大鼠MWT和TWL明显低于假手术组,低、高剂量治疗组较模型组明显升高,二甲亚砜组与模型组比较无差别;L_(4-5)DRG内P2X3受体表达模型组明显高于假手术组,低、高剂量治疗组较模型组均明显降低,二甲亚砜组与模型组比较无明显区别。结论:1,8-桉叶素抑制CCI大鼠L_(4-5)DRG神经元P2X3受体过表达,从而缓解神经病理性疼痛症状。     

Pregabalin Alters Nociceptive Behavior and Expression Level of P2X3 Receptor in the Spinal Dorsal Horn in a Rat Model Induced by Chronic Compression of the Dorsal Root Ganglion

P2X3 receptors are present in the spinal dorsal horn (SDH) and play an essential role in the regulation of nociception and pain. Pregabalin (PGB) has been used as a new antiepileptic drug in the treatment of neuropathic pain. However, it is unclear whether PGB‐induced analgesia was associated with the P2X3 receptor in SDH. Here, rats were randomly divided into four groups (n = 12 per group), including 2 sham operation groups, which was treated by normal saline (Sham + NS group) or PGB (Sham + PGB group), other 2 groups with chronic compression of the dorsal root ganglion, a normal saline‐treated CCD group (CCD+NS group), and a PGB‐treated CCD group (CCD + PGB group). A rat model of neuropathic pain was used by compressing the right L4 and L5 dorsal root ganglia. Each group was evaluated using the mechanical withdrawal threshold (MWT). The mRNA and protein levels of the P2X3 receptor in the ipsilateral SDH were measured by RT‐PCR, western blot, and immunofluorescence on 14 day after CCD operation. CCD rats showed the highest mechanical hyperalgesia and the lowest pain threshold in the four groups. Simultaneously, CCD rats showed higher P2X3 mRNA and protein expression in ipsilateral side of the SDH than the sham operation rats. However, the MWT was increased and expression of P2X3 mRNA and protein in the ipsilateral SDH in CCD rats was decreased 3 days after PGB treatment. Thus, PGB may partially reverse mechanical hyperalgesia in CCD rats by inhibiting P2X3 receptor expression in the ipsilateral SDH. Anat Rec, 296:1907–1912, 2013. © 2013 Wiley Periodicals, Inc.     

白藜芦醇预处理介导Wnt/β-catenin信号通路保护脑缺血再灌注大鼠海马区神经元损伤

目的:研究白藜芦醇(resveratrol,Res)预处理对脑缺血再灌注(ischemia reperfusion,I/R)大鼠海马区Wnt/β-catenin信号通路相关蛋白的表达量、星形胶质细胞及神经元形态的影响,探讨白藜芦醇对脑缺血再灌注大鼠海马区神经元损伤的保护作用机制。方法:将SD大鼠60只随机分为四组,每组15只:正常对照组(Control)、假手术组(Sham)、缺血再灌注组(I/R)和白藜芦醇治疗组(Res)。Res组大鼠连续7 d腹腔注射白藜芦醇(每日30 mg/kg,由2%DMSO溶解),Sham组和I/R组注射同等量2%DMSO;7 d后Res组和I/R组采用改良线栓法制备大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MACO)模型,缺血90 min,再灌注24 h。Sham组仅分离暴露一侧颈内动脉,不插入线栓;24 h后对各组大鼠进行神经功能学评分;脑组织TTC染色;尼氏染色;免疫组化检测海马区GSK-3β和星型胶质细胞(GFAP)数量及形态变化;Western Blot分别检测Wnt信号通路相关因子GSK-3β、p-GSK-3β、β-catenin蛋白水平表达量。结果:再灌注24 h后,尼氏染色显示与I/R组相比Res组海马区仅有少量的神经元破碎和消失,形态尚完整(P0.05),同时行为学评分和梗死面积也低于I/R组(P0.05)。免疫组化显示,与I/R组相比Res组GSK-3β的表达量明显降低,伴随星形胶质细胞活化状态和数量减少(P0.05)。Western Blot显示,与免疫组化表达趋势结果一致Res组GSK-3β的的表达量明显低于I/R组,而p-GSK-3β、β-catenin的表达量均增高(P0.05)。结论:白藜芦醇可能是通过调节Wnt/β-catenin信号通路相关蛋白的表达进而有效的降低大鼠脑缺血再灌注后神经元的损伤。     

Synaptic terminals from mice midbrain exhibit functional P2X7 receptor

P2X(7) receptor has been recently localized in mice cerebellar granule neuron fibers. Here, the expression of this subunit has been detected in wild type mice midbrain, by quantitative real time-polymerase chain reaction, immunocytochemistry and Western blot assays. The functionality of this P2X(7) subunit has been confirmed using microfluorimetric experiments in isolated synaptic terminals from mice midbrain. 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP) was 30-fold more potent than ATP and EC(50) values were 20 microM and 630 microM respectively. Brilliant Blue G (BBG) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) produced an inhibition in the responses induced by BzATP, with IC(50) values of 0.027 nM and 2.23 nM, respectively. In addition, P2X(7) inhibitors as ZnSO(4), BBG and suramin abolished partially or totally the responses induced by the physiological agonist ATP. According to immunochemical and PCR assays the presence of a "P2X(7)-like" protein in synaptosomes from validated P2X(7) knockout (KO) model have been detected. In KO animals, BzATP was sixfold more potent than ATP and the EC(50) values were 87 microM and 590 microM respectively. BBG and KN-62 also produced an inhibition in the responses induced by BzATP, with IC(50) value of 0.61 nM and 118 nM respectively, both of them higher than in wild type mice. Moreover, the calcium mobilization ability of native P2X(7) receptors was higher in control compared with KO mice. These biochemical and pharmacological experiments are consistent with the presence of a functional P2X(7) receptor in wild type mice midbrain, and the existence of a less efficient "P2X(7)-like" receptor in the KO model.     

氯胺酮鞘内给药对神经病理性痛大鼠脊髓背角小胶质细胞活化、TNF-α和IL-1β产生的影响

目的:探究鞘内注射氯胺酮(ketamine,KTM)对坐骨神经结扎(CCI)神经病理性疼痛大鼠模型行为、脊髓背角肿瘤坏死因子-α(TNF-α)和白介素1β(IL-1β)以及小胶质细胞标记蛋白OX42表达的影响。方法:(1)建立CCI模型大鼠,鞘内注射KTM或MI(米诺四环素)进行干预,测定大鼠机械缩足阈值(MWT)和热缩足潜伏期(TWL)。(2)运用ELISA法检测TNF-α和IL-1β表达水平。(3)使用Western Blot法检测OX42的表达情况。结果:(1)KTM组和MI组大鼠MWT和TWL值较CCI组显著升高;KTM组大鼠MWT和TWL值高于CCI组(P0.05)。(2)KTM组和MI组TNF-α和IL-1β表达水平低于CCI组(P0.05);其中KTM组TNF-α和IL-1β表达水平显著高于MI组(P0.05)。(3)KTM组和MI组OX42表达水平显著低于CCI组;KTM组OX42表达水平高于MI组(P0.05)。结论:鞘内KTM可以抑制脊髓背角小胶质细胞激活,减少TNF-α和IL-1β表达,显著改善坐骨神经结扎引起的神经病理性疼痛。     

异氟烷预处理增强大鼠海马内M2型小胶质细胞活化减轻电磁脉冲所致神经损伤

目的:观察异氟烷预处理是否通过增强大鼠海马内M2型小胶质细胞活化,减轻炎症反应改善电磁脉冲所致神经元损伤。方法:24只SD大鼠随机分为4组,分别为对照组(CON组)、异氟烷预处理组(IP组)和电磁脉冲照射组(EMP组)和异氟烷预处理加电磁脉冲照射组(IP+EMP组)。各组于照射后24 h进行尼氏染色;采用Western Blot和免疫荧光组织化学染色方法检测M2型小胶质细胞标志物Arg-1的表达;并采用ELISA方法测定海马组织中TNF-α和IL-1β的含量。结果:与CON组相比,EMP组海马齿状回颗粒细胞的尼氏体明显减少,而与EMP组相比,IP+EMP组海马齿状回颗粒细胞的尼氏体明显增多增大。Western Blot结果显示:与CON组相比,EMP组海马内Arg-1蛋白的表达明显减少(P0.05),与EMP组相比,IP+EMP组Arg-1蛋白的表达明显增加(P0.05)。免疫荧光染色结果显示:与CON组相比,EMP组海马内Arg-1的表达减弱;而与EMP组相比,IP+EMP组Arg-1的表达明显增强。EMP组海马组织中炎症因子TNF-α和IL-1β的含量较CON组增加(P0.05),IP+EMP组脑组织中炎症因子TNF-α和IL-1β的含量较EMP组减少(P0.05)。结论:异氟烷预处理可通过增加M2型小胶质细胞,减少脑组织中炎症因子含量,减轻EMP所致神经元损伤。