脊髓p38丝裂原活化蛋白激酶对神经痛大鼠热痛觉过敏影响的研究

目的:观察脊髓p38丝裂原活化蛋白激酶(p38 MAPK)的激活对神经病理性疼痛大鼠热痛觉过敏的影响,探讨神经痛的机制。方法:40只雄性SD大鼠,建立坐骨神经结扎性损伤(CCI)疼痛模型,随机分成五组,对照组、SB 0.1μg组、SB0.5μg组、SB2.5μg组和SB5μg组,(n=8),CCI后7 d,鞘内注射5%二甲基亚砜或不同剂量的p38 MAPK抑制剂SB203580(0.1μg、0.5μg、2.5μg、5μg)10μL,在给药前和给药后0.5 h、3 h、6 h、12 h、24 h,用热辐射刺激仪测定大鼠术侧后爪热缩足反射潜伏期(TWL)。另24只大鼠随机分为假手术组、CCI组、溶媒对照组和SB203580组(n=6),分别于假手术后7d、CCI后7 d、CCI后7 d鞘内注射5%二甲基亚砜后第6 h或SB203580 5μg后第6 h取材脊髓,用免疫组织化学方法观察脊髓背角磷酸化p38 MAPK表达的变化。结果:鞘内注射SB203580剂量依赖性地延长了TWL(P<0.05或P<0.01),SB203580同时抑制脊髓背角磷酸化p38 MAPK的表达。结论:脊髓p38 MAPK的激活参与了CCI...     

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目的：探讨鞘内注射p38丝裂原蛋白激酶(p38MAPK)特异性抑制剂SB203580对大鼠慢性压迫性损伤(CCI)术后脊髓背角环氧化酶-2(COX-2)蛋白表达的影响。方法：鞘内置管成功的SD雄性大鼠24只,随机分为4组：Sham组、NS组、DMSO组和SB组,其中Sham组做假手术,后3组大鼠右侧坐骨神经建立CCI模型。模型成功后,术后第6天开始分别鞘内注射生理盐水、2%二甲亚砜、SB203580,每天2次,连续5 d。术前1 d、术后第1,3,5,7,9,11天测定机械性痛阈。术后第11天测定机械性痛阈后处死大鼠,取脊髓腰膨大进行免疫组织化学分析测定COX-2蛋白表达。结果：与术前相比,Sham组术后机械性痛阈无明显改变(P>0.05),NS组和DMSO组在术后第1至11天机械性痛阈降低(P<0.05),SB组在术后第1至5天机械性痛阈降低(P<0.05),与NS组和DMSO组比较,SB组在术后第7至11天,机械性痛阈增加(P<0.05);免疫组织化学分析,NS组和DMSO组脊髓背角COX-2蛋白阳性细胞数和阳性评分均高于Sham组(P<0.05),SB组COX-2蛋白阳性细胞数和阳性评分低于NS组和DMSO组(P<0.05)。结论：鞘内注射SB203580可使神经病理性疼痛大鼠产生明显镇痛效应;可减少神经病理性疼痛大鼠脊髓背角COX-2蛋白的表达,p38MAPK参与COX-2蛋白表达的调节。     

右美托咪啶对神经病理性疼痛大鼠脊髓p38丝裂原活化蛋白激酶和脑源性神经营养因子表达的影响

目的 观察右美托咪啶对坐骨神经慢性压迫性损伤(CCI)所致神经病理性疼痛大鼠的镇痛作用以及对脊髓p38丝裂原活化蛋白激酶(p38MAPK)和脑源性神经营养因子(BDNF)mRNA表达的影响.方法 雄性SD大鼠72只,随机分为3组,每组24只,分别为假手术组(S组)、CCI组(C组)和右美托咪啶组(D组),S组大鼠仅分离坐骨神经不结扎,C组和D组建立CCI模型,D组于CCI术后即刻开始至取材时点每天腹腔注射右美托咪啶50 μg/kg,S组和C组注射相同体积的生理盐水.各组大鼠分别于术前l d,术后1、3、7、14 d测定机械痛阈和热痛阈,在术后第1、3、7、14 d测定痛阈后每组随机取6只大鼠取L4-6脊髓组织,逆转录PCR(RT-PCR)法检测p38MAPK mRNA和BDNF mRNA的表达.结果 与S组比较,C组术后1、3、7、14 d机械痛阈和热痛阈明显降低,p38MAPK mRNA和BDNF mRNA的表达明显上调(P<0.05);与C组比较,D组术后3、7、14 d机械痛阈和热痛阈显著升高,p38MAPK mRNA和BDNF mRNA表达明显下调(P<0.05);与术前比较,C组、D组机械痛阈和热痛阈术后1、3、7、14 d显著降低,第7天时降到最低(P<0.05);与术后1 d比较,C组、D组p38MAPK mRNA和BDNF mRNA于术后3、7、14 d明显升高,第7天达到最高(P<0.05);对p38MAPK和BDNF mRNA的表达进行相关性分析,Pearson相关系数为0.901,表明p38MAPK mRNA和BDNF mRNA表达呈正相关(P<0.05).结论 右美托咪啶对大鼠坐骨神经慢性压迫性损伤疼痛具有明显的镇痛效应,其机制可能与抑制p38MAPK的活化,从而下调BDNF的表达有关.     

身痛逐瘀汤对大鼠神经病理性疼痛及脊髓p38MAPK蛋白表达的影响

目的:观察中药复方身痛逐瘀汤对CCI大鼠神经病理性疼痛及脊髓p38丝裂原活化蛋白激酶(p38 MAPK)蛋白的影响。方法:SD雄性大鼠随机分为假手术组、模型组、身痛逐瘀汤单倍剂量组(6.5g/kg)、身痛逐瘀汤双倍剂量组(13 g/kg)。假手术组切开暴露坐骨神经而不结扎,模型组结扎坐骨神经诱导神经痛模型,术后未给予干预治疗。给药组在造模后第3天开始灌胃给药,在造模后第3,5,7,14d观察大鼠热痛阈、机械痛阈、运动评分值的变化,免疫荧光检测CCI大鼠小胶质细胞活化情况,Western-Blot检测大鼠脊髓p38蛋白表达变化。结果:在造模后第7d和第14d,与假手术组相比,模型组大鼠热痛阈值、机械痛阈值出现显著降低,右侧后肢运动功能评分显著升高(P<0.01),小胶质细胞标记物OX-42免疫阳性物质表达显著增多;与模型组相比,身痛逐瘀汤给药组均可显著提高热痛、机械痛阈值(P<0.05;P<0.01),并降低右侧后肢运动评分值(P<0.01)。与假手术组相比,模型组大鼠在造模后第7d和第14d脊髓磷酸化p38蛋白(p-p38)表达显著上调(P<0.01);与模型组相比,身痛逐瘀汤给药组在造模后第14d可显著下调脊髓p-p38蛋白的表达(P<0.01)。结论:中药复方身痛逐瘀汤可抑制CCI大鼠的痛敏反应,改善患侧后肢运动功能,其机制可能与下调脊髓磷酸化p38蛋白表达,降低脊髓神经炎症反应有关。     

鞘内注射补体调节蛋白衰变加速因子对大鼠神经病理性疼痛的影响

目的 观察鞘内注射补体调节蛋白衰变加速因子（decay accelerating factor,DAF）对大鼠神经病理性疼痛（neuropathic pain,NPP）的影响,探索其在NPP发病机制中的作用.方法 健康雄性SD大鼠,体质量200～250g,选取鞘内置管成功的大鼠30只,采用随机数字表法,将大鼠随机分为3组（n=10）,即假手术组、CCI组和DAF组.后2组采用慢性坐骨神经损伤法制作动物模型,假手术组分离大鼠坐骨神经干后逐层缝合.DAF组于术前1d开始鞘内注射DAF溶液20μL,1次/d,连续7d,假手术组和CCI组在相同时间点鞘内注射等容量生理盐水.于术前,术后1、3、7d,用热刺激和机械刺激法测定大鼠的痛阈值变化.术后7d处死大鼠,取L4～6段脊髓背角,用Western-Blot测定其DAF蛋白含量.结果 与假手术组相比,CCI组和DAF组大鼠术后1、3、7d热痛阈值和机械痛阈值降低（P＜0.05）;与CCI组相比,DAF组大鼠术后3、7d热痛阈值和机械痛阈值升高（P＜0.05）.Western-Blot 检测显示,CCI组大鼠脊髓背角DAF表达显著低于假手术组（P＜0.05）;与CCI组相比,DAF组大鼠脊髓背角DAF表达上调（P ＜0.05）.结论 鞘内注射DAF可抑制大鼠NPP的痛觉过敏,脊髓背角DAF的表达变化与NPP形成有关.     

SB203580对神经病理性疼痛早期脊髓γ-氨基丁酸受体亚单位1表达的影响

目的 观察鞘内注射特异性小胶质细胞p38有丝分裂原激活蛋白激酶(MAPK)抑制剂SB203580对神经病理性痛大鼠脊髓背角γ-氨基丁酸受体1[GABAB(1)]表达的影响,探讨脊髓小胶质细胞参与神经病理性疼痛发生的机制.方法 成年雄性Sprague-Dawley大鼠32只,全部行鞘内置管,建立L5神经根结扎模型或假手术,按模型和给药方式分为4组,每组各8只.Ⅰ组:神经根结扎+鞘内注射0.1%SB203580[溶于2%二甲亚砜(DMSO)]10μL;Ⅱ组:神经根结扎+鞘内注射溶剂(2%DMSO)10μL;Ⅲ组:假手术+鞘内注射0.1%SB203580(溶于2%DMSO)10μL;Ⅳ组:假手术+鞘内注射溶剂(2%DMSO)10μL.每组于术前1 h及术后连续6 d经鞘内注射相应药物.分别于术前1 h和术后1、3、7 d给药前1 h测定大鼠的50%机械缩爪阈值(50%PWT),术后第7天完成50%PWT测定后各组取6只大鼠,采用Western印迹法测定脊髓背角GABAB(1)的表达.结果 与Ⅱ组相比,Ⅰ组在术后1、3、7 d的50%PWT显著升高(P值均＜0.01),脊髓背角GABAB(1a)表达显著上调(P值均＜0.01);与Ⅳ组相比,Ⅱ组在术后1、3、7 d术后50%PWT显著降低(P值均＜0.01),脊髓背角GABAB(1a)表达显著下调(P值均＜0.01).各组间GABAB(1b)表达的差异均无统计学意义(P值均＞0.05).结论 小胶质细胞可能通过抑制突触前GABAB(1a)表达参与神经病理性疼痛的发生,小胶质细胞抑制剂与GABAB激动剂的联合用药可能成为治疗神经病理性疼痛更有效的方法.     

大鼠SNI神经痛模型不同时相脊髓背角 p-p38MAPK和p-ATF2表达的变化

目的观察坐骨神经分支选择性损伤(SNI)模型大鼠不同时间点术侧腰段L4~L6脊髓背角神经元磷酸化p38丝裂原活化蛋白激酶(p-p38MAPK)和磷酸化活化转录因子2(p-ATF2)的表达情况,探讨脊髓背角p-p38MAPK和p-ATF2在神经病理性痛模型不同阶段中的作用。方法健康雄性SD大鼠36只,完全随机分为空白对照组、假手术组和手术组,各12只。通过结扎腓总神经及切断胫神经,保留腓肠神经的方法建立SNI大鼠模型。观察造模前、造模后3天和14天术侧足跖缩足阈值(PWT);免疫荧光法检测造模后3天和14天术侧腰段脊髓背角p-p38MAPK和p-ATF2阳性细胞表达情况。结果选模后3天和14天,手术组大鼠术侧足跖PWT较假手术组与空白对照组明显降低(P0.01),假手术组大鼠与空白对照组大鼠比较差异无统计学意义(P0.05)。造模后3天和14天,手术组大鼠术侧腰段脊髓背角p-p38MAPK和p-ATF2阳性细胞表达率较假手术组和空白对照组均明显升高(P0.01),假手术组和空白对照组SNI模型大鼠造模后各时间点,术侧腰段脊髓背角p-p38MAPK和p-ATF2阳性细胞表达率差异均无统计学意义(P0.05)。结论 SNI模型神经病理痛的产生和维持可能与术侧腰段脊髓背角p-p38MAPK和p-ATF2表达上调有关。     

慢性压迫损伤性神经病理性疼痛大鼠模型中脊髓背角ERK、CREB、BDNF表达的变化

目的通过建立慢性神经结扎性损伤（CCI）大鼠模型,揭示在神经病理性疼痛发生、发展过程中细胞外调节蛋白激酶（ERK）、环磷腺苷效应元件结合蛋白（CREB）、脑源性神经营养因子（BDNF）表达的变化。方法将21只大鼠随机双盲分为模型组、假手术组、U0126组,每组各7只,模型组及U0126组建立CCI模型,假手术组暴露坐骨神经不做结扎,3组均进行鞘内置管,U0126组鞘内注射阻断剂U012610滋g/次,连续3d;模型组造模成功后、假手术组术后予0.9%氯化钠溶液0.12ml/kg灌胃至术后14d。对各组大鼠分别于术前、术后1、7、14d进行热痛觉过敏行为测试和机械刺激测试,采用Westernblot法检测模型大鼠脊髓背角ERK、CREB、BDNF的表达水平。结果与假手术组相比,模型组大鼠术后热板及机械缩足反射阈值明显降低,差异均有统计学意义（均P＜0.05）。CCI导致大鼠脊髓背角内胞浆与胞核内ERK、CREB、BDNF水平均增加,差异均有统计学意义（均P＜0.01）。ERK阻滞剂U0126组能明显升高大鼠术后热板及机械痛敏阈值（P＜0.05）,U0126组导致大鼠脊髓背角内胞质与胞核内CREB、BDNF水平均明显降低,差异均有统计学意义（均P＜0.05）。结论CCI模型导致痛觉过敏,U0126可以减轻CCI大鼠的疼痛。ERK-CREB磷酸化的通路参与BDNF对于背根神经节的神经元的保护和修复过程。     

姜黄素对神经病理性疼痛大鼠脊髓11β羟基固醇脱氢酶1表达的影响

目的 探讨皮质醇和11β羟基固醇脱氢酶1(11βHSD1)在神经病理性疼痛大鼠痛觉过敏中的作用,并揭示姜黄素缓解大鼠神经病理性疼痛的作用机制.方法 雄性SD大鼠随机分成4组:假手术(Sham)组、坐骨神经慢性压榨损伤模型(CCI)组、CCI+溶剂(SC)组、CCI+姜黄素100 mg/kg(Cur100)组.于术前2 d和术后1、3、5、7、10、14 d测定鼠爪热痛阈(PTWL)和机械痛阈(PMWT),术后3、7和14 d取血,同时取大鼠术侧L4、L5脊髓背角和背根节,用ELISA法测血清皮质醇浓度,用免疫组化法和Western印迹法分析脊髓背角和背根节11βHSD1表达的动态变化.结果 与Sham组相比,CCI组大鼠术后1~14 d PTWL和PMWT明显降低(P<0.05),血清皮质醇浓度明显升高(P<0.05),术侧脊髓背角和背根节内11βHSD1阳性神经元的表达显著增多(P<0.05).Cur100大鼠PMWT和PTWL显著提高(P<0.05),同时组大鼠血清皮质醇浓度显著降低(P<0.05),脊髓背角和背根节11βHSD1阳性神经元细胞的表达也明显降低(P<0.05).结论 神经病理性疼痛所致应激引起血清皮质醇浓度升高、脊髓背角和背根节11βHSD1阳性神经元细胞表达增加,姜黄素能减轻CCI大鼠机械性痛觉过敏和热痛觉过敏,降低血清皮质醇浓度及脊髓背角和背根节11βHSD1阳性神经元细胞表达.皮质醇及脊髓背角和背根节的11βHSD1阳性神经元细胞可能与神经病理性疼痛的发病和维持有关.     

姜黄素对神经病理性疼痛大鼠脊髓背角和背根神经节GR和NMDAR-NR1表达的影响

目的探讨姜黄素对神经病理性疼痛大鼠脊髓背角和背根神经节皮质激素受体(GR)和NMDAR-NR1表达的影响。方法将雄性SD大鼠72只(体质量220～250g),随机分成4组:假手术组(Sham组)、慢性坐骨神经结扎损伤组(CCI组)、CCI+溶剂组(SC组)、CCI+姜黄素100mg/kg组(Cur100组)。SC组和Cur100组大鼠在坐骨神经结扎后,分别腹腔注射溶剂或100mg.kg-1.d-1姜黄素,至术后14d。于术前2d和术后1、3、5、7、10、14d测定机械缩足反射阈值(MWT)和热缩足反射潜伏期(TWL);术后3、7、14d取大鼠术侧L4、L5脊髓背角和背根神经节,用免疫组化法检测脊髓背角和背根神经节GR和NMDAR-NR1表达情况。结果与Sham组相比,CCI组术后TWL、MWT明显降低(P<0.01),术侧脊髓背角和背根神经节内GR和NMDAR-NR1表达明显增多(P<0.01)。与CCI组相比,Cur100组大鼠TWL和MWT明显提高(P<0.05),脊髓背角和背根神经节GR和NMDAR-NR1表达明显减少(P<0.05)。结论姜黄素可通过抑制脊髓背角和背神经根节GR和NMDAR-NR1的表达而减轻CCI大鼠神经病理性疼痛。     

P2X4 receptor and brain-derived neurotrophic factor in neuropathic pain

Objective： To investigate whether the activation of p38MAPK is involved in the neuropathic pain induced by P2X4 receptor, and the effects of activated P2X4 receptor and p38MAPK on expression of brain-derived neurotrophic factor （BDNF） in the chronic neuropathic pain. Methods： Lumbar intrathecal catheters were chronically implanted in male Sprague-Dawley rats. The right sciatic nerve was loosely ligated proximal to the sciatica＇s trifurcation at approximately 1.0 mm intervals with 4-0 silk sutures. The microglia inhibitor minocycline, P2X4 antagonist （TNP-ATP） and p38MAPK inhibitor （SB203580） were intrathecally administered every 12 h, 3 d postchronic constriction injury （CCI）. Mechanical nociceptive thresholds were assessed with the paw withdrawal threshold （PWT） to von Frey filaments. The expression of P2X4 and BDNF were assessed by both immunohistochemical analysis and RT-PCR. Results： Intrathecal injection of minocycline or TNP-ATP or SB203580 significantly attenuated CCI-induced mechanical allodynia. The time courses of P2X4 receptor and BDNF expression were increased at all points after CCI and reached a peak level on postoperative d 7. Intrathecal injection of minocycline or TNP-ATP or SB203580 markedly suppressed the increase of CCI-induced P2X4 receptor and BDNF expression in the spinal cord. Conclusion： The activation of P2X4 receptor BDNF pathways contributes to neuropathic pain in CCI rats, and the activation of p38MAPK is involved in the neuropathic pain induced by P2X4 receptor.     

p38MAPK磷酸化水平及Caspase-3在NMDA诱导的体外培养神经元中的激活及SB203580的保护作用

目的探讨p38丝裂原活化蛋白激酶磷酸化(P-MAPK)水平及半胱氨酸蛋白酶-3(Caspase-3)在N-甲基-D-天冬氨酸(NMDA)诱导的体外培养神经元中的激活及p38MAPK抑制剂SB203580的保护作用。方法随机将原代培养7 d的大鼠皮质神经元分为对照组,NMDA组,SB203580低、中、高剂量组。对照组仅用DMEM/F12完全培养基,NMDA组去除正常神经元培养液,加入50μmol/L NMDA,处理时间10 min;SB203580低、中、高剂量组浓度分别为5、10、20μmol/L,继续培养24 h,加入50μmol/L NMDA。采用MTT法评价细胞生存力,丫啶橙(AO)染色检测凋亡细胞数量及乳酸脱氢酶(LDH)含量;免疫细胞化学染色法(IHC)及免疫印记法(WB)检测皮质神经元内P-MAPK及Caspase-3的表达水平。结果与对照组比较,NMDA组的吸光度值明显降低、神经元凋亡明显增加、P-MAPK及Caspase-3表达增加(P均<0.01),SB203580低、中、高剂量组P-MAPK及Caspase-3随着浓度的增加表达减少,以高剂量组为明显(P<0.05,P<0.01);与NMDA组比较,SB203580低、中、高剂量组吸光度值均升高,以高剂量组为明显,细胞凋亡数量明显减少(P均<0.05)。结论 P-MAPK及Caspase-3在NMDA诱导的皮质神经元中表达增强,SB203580对NMDA诱导的神经元损伤具有保护作用,其作用机制可能与p38MAPK的活化,进而直接或间接激活Caspase-3的表达有关。     

p38MAPK在Aβ25-35诱导AD大鼠海马CA1区表达的研究

OBJECTIVE: To investigate the changes of p38MAPK expression in a rat model of Alzheimer disease (AD). METHODS: Seventy-two adult SD rats were randomized equally into 4 groups, and a single-dose injection of Abeta(25-35) (dementia group), normal saline (saline group), SB203580 (inhibitor group), or DMSO (inhibitor control group) was administered into the lateral cerebral ventricle. Y-maze tast was performed to evaluate the behavioral changes of the rats after the injections, and on days 4, 7 and 14 after the injection, p38MAPK expression in the hippocampal CA1 area was measured by means of immunohistochemistry. RESULTS: On days 7 and 14 following Abeta(25-35) injection, the training times, error number and total reaction time were significantly higher in dementia group than in saline group (P<0.05), but all these indices were significantly lowered in the inhibitor group as compared with the dementia group (P<0.05). Immunohistochemistry revealed obvious p38 expression in the dementia group 4 days after Abeta(25-35) injection, which increased significantly with the passage of time (P<0.01). The gray scale in the inhibitor group was significantly higher than that in the dementia group (P<0.01). CONCLUSION: p38MAPK activation in the hippocampal CA1 area is an event that persists during the entire course of Abeta(25-35)-induced AD in rats, and the inhibitor SB203580 prevents p38MAPK expression and improves the learning and memory abilities of the rats.     

p38丝裂原活化蛋白激酶在溃疡性结肠炎中的作用

目的 探讨p38丝裂原活化蛋白激酶(p38MAPK)在溃疡性结肠炎(UC)以及葡聚糖硫酸钠(DSS)诱导的小鼠结肠炎中的作用。 方法 ①7只Balb/c小鼠随机分为3组:正常对照组、DSS结肠炎组、p38MAPK选择性抑制剂SB203580干预组。结肠炎组小鼠每日饮用5% DSS溶液,干预组小鼠则在饮用5% DSS溶液72 h后,加用SB203580[1 mg/(kg·d)]进行腹腔注射。观察并记录各组小鼠的疾病活动指数(DAI);7 d后处死小鼠,观察各组小鼠肠黏膜组织中肿瘤坏死因子α(TNF-α)和白细胞介素-1β(IL-1β)的表达。②收集36例活动性UC患者的肠黏膜组织标本,并以18例结肠癌旁的正常组织标本作对照,通过体外组织培养,观察SB203580对UC患者肠黏膜活检组织中TNF-α和IL-1β表达的影响。 结果 ①DSS结肠炎小鼠的DAI评分、肠黏膜组织中TNF-α和IL-1β的水平明显升高(P均<0.01),经SB203580干预后,其DAI评分、TNF-α、IL-1β的水平均明显降低,但仍然高于正常对照组(P均<0.01)。②体外组织培养结果显示,与未用SB203580处理的UC组比较,SB203580处理后UC患者肠黏膜组织中TNF-α和IL-1β的水平明显降低(P<0.01)。 结论 SB203580能阻断p38MAPK信号转导通路,从而降低促炎性细胞因子TNF-α和IL-1β的释放。     

苦参煎与SB203580对p38MAPK信号传导通路的调控影响

目的:探讨苦参煎与SB203580对p38MAPK信号传导通路的调控影响。利用苦参煎与SB203580抑制p38 MAPK信号传导通路,来证明中药对基因转录表达具有一定的调控作用。方法:模拟血管内膜损伤术,采用ELISA方法检测188只Webster大鼠血清中P38MAPK的水平含量。将其随机平均分为正常对照组、假手术组、生理盐水组、苦参煎组和SB203580组。结果与结论:中药苦参煎有着与SB203580相同的功效,即对P38MAPK的显著抑制作用。所以对血管内皮有比较明确的保护作用,能干扰MAPK信号传导通路,既能减轻内皮损伤后的炎性细胞释放反应,也能使再生的内皮某些功能得到恢复,有利于内皮细胞结构和功能恢复,防止炎性因子对细胞的损害。     

SB203580减轻体外循环肺组织炎症介质产生的实验研究

目的 研究体外循环肺组织中P38丝裂原活化蛋白激酶(P38 MAPK)的变化对肺组织炎症反应的作用及其机制.方法 54只SD大鼠随机分为3组:全麻开胸组(S组)、体外循环组(CPB组)、体外循环+SB203580组(SB组).不同时间段处死动物,留取标本,Western blotting检测肺组织中P38 MAPK、磷酸化P38 MAPK.EMSA检测核因子(NF)-κB的DNA结合活性变化,ELIASA分别检测TNF-α和IL-1β产量.结果 CPB组磷酸化P38MAPK较S组增加.NF-κB活性水平也较S组明显增加,肺组织中TNF-α和IL-1β产量增加.SB203580减轻了肺组织中磷酸化P38MAPK活性水平,减少了肺组织中炎症闪子的产生.结论 (1)P38MAPK通过影响NF-κB的激活而参与体外循环术后肺组织炎症因子的产生;(2)SB203580通过阻断P38MAPK的激活而减轻体外循环术后肺炎症因子的产生.

Abstract：

Objective To examine the changes in P38MAPK during and after cardiopulmonary bypass (CPB) and the effect of SB203580, a specific P38MAPK inhibitor, on CPB-induced pulmonary inflammatory response. Metholds Fifty-four SD rats were randomized into 3 groups (each=18), namely sham CPB group, CPB group, and SB203580 group in which rats underwent CPB with SB203580 pretreatment. The lungs were excised immediately after the rats were sacrificed at scheduled time points and p38, nuclear factor-κB (NF-ΚB), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected. Results The activities of P38 MAPK and NF-ΚB were significantly increased in CPB group as compared with those in sham CPB group. CPB resulted in increased TNF-α and IL-1β production in the lung tissues. Administration of SB203580 prevented up-regulation of lung phosphorylated P38 MAPK, and decreased proinflammatory cytokine productions in the lung tissues. Conclusion P38 MAPK is activated in the lung tissue during and after CPB to affect the activation of NF-κB in the lung; SB203580 selectively inhibits P38 MAPK activation to reduce proinflammatory cytokine production after CPB.     

Electroacupuncture Attenuates Spinal Nerve Ligation-Induced Microglial Activation Mediated by p38 Mitogen-Activated Protein Kinase

Objective

To investigate whether analgesic effect of electroacupuncture (EA) is affected by p38 mitogen-activated protein kinase (p38 MAPK) on microglia.

Methods

There were two experiments. The experiment 1: 40 male Sprague-Dawley (SD) rats were randomly divided into the normal, surgery, EA and sham EA groups, and the L5 spinal nerve ligation (SNL) on the right side was used to establish neuropathic pain model. EA was applied to bilateral Zusanli (ST36) and Kunlun (BL60) at 24, 48 and 72 h after SNL for 30 min, once per day. The paw withdrawal thresholds (PWTs) were measured before surgery (as base) and at 24, 25, 49 and 73 h after surgery. Phospho-p38 MAPK (p-p38 MAPK), oxycocin-42 (OX-42, marker of microglia), and glial fibrillary acidic protein (GFAP, marker of astrocyte) in bilateral spinal cord dorsal horn (SCDH) were detected by immunofluorescence, respectively. The experiment 2: 40 male SD rats were cannulated for SNL-induced neuropathic pain, and then were randomly divided into the dimethyl sulfoxide (DMSO), EA plus DMSO, 4-(4-fluorophenyl)-2-(4-methylsulfonylpheny)-5-(4-pyridyl)-1H-imidazole (SB203580) and EA plus SB203580 groups. SB203580 (30 nmol/L) was administered 5 min prior to EA treatment. The PWTs and OX-42 in bilateral SCDH were measured as mentioned above.

Results

SNL-induced neuropathic pain reduced PWTs and increased the expression of p-p38 MAPK and OX-42 in bilateral lumbar SCDH of rats (P<0.01). Spinal p-p38 MAPK was only co-localized with OX-42 in our study. EA treatment significantly alleviated SNL-mediated mechanical hyperalgesia, and suppressed the expression of p-p38 MAPK and OX-42 in lumbar SCDH (P<0.05 or P<0.01). Intrathecal injection of low dose SB203580 had no influence on PWTs (P>0.05), but significantly inhibited the expression of OX-42 positive cells in bilateral SCDH (P<0.01 or P<0.05). EA plus SB203580 synergistically increased PWTs, and reduced the expression of bilateral spinal OX-42 (P<0.01 or P<0.05).

Conclusions

The central mechanism of EA-induced anti-hyperalgesia may be partially associated with the reduced expression of p-p38 MAPK, and subsequently reducing the activation of OX-42 in neuropathic pain. Therefore, EA may be a new complementary and alternative therapy for neuropathic pain.

     

p38MAPK参与去甲肾上腺素预处理的延迟保护作用

目的 探讨p38MAPK(丝裂素活化蛋白激酶)是否参与去甲肾上腺素(NE)预处理的延迟保护作用。方法 建立大鼠心肌缺血/再灌注损伤(I/R)模型。分别应用去甲肾上腺素、p38的特异性抑制剂SB203580预处理心脏,24h后对心脏进行缺血再灌注,观察此时心肌梗塞范围、血浆乳酸脱氢酶(LDH)活性,同时用western blotting方法检测NE预处理后即刻、10 min、15 min、30 min时p~(38)磷酸化水平。结果 NE预处理明显缩小心梗范围,减轻血浆LDH活性升高程度(P<0.01 vs I/R)。p38磷酸化在NE预处理后即刻稍微增高,10min轻度升高,15min达到高峰,30min开始下降。用SB203580可明显抑制p38磷酸化,而心梗范围、血浆LDH活性同单纯缺血/再灌注组相似(P>0.05)。结论 ①去甲肾上腺素预处理对24h后缺血/再灌注心肌有保护作用。②p38参与心肌预处理后的延迟保护作用,去甲肾上腺素预处理后p38短暂而快速的激活可能是药物延迟保护作用的重要机制之一。     

The Relationship between p38MAPK and Apoptosis during Paclitaxei Resistance of Ovarian Cancer Cells

To investigate the relationship between p38 mitogen-activated protein kinase (p38MAPK) and cell apoptosis during the paclitaxel resistance of ovarian carcinoma cell lines, flow cytometry (FCM) and PI staining were employed to determine the effect of p38MAPK inhibitor SB203580 on the apoptosis of A2780/Taxol cells, a drug-resistant human ovarian carcinoma cell line. p38MAPK protein expression in SB203580-treated cells was immunochemically measured. The 50% inhibition concentration (IC(50)) of paclitaxel on A2780/Taxol cells was determined by MTT assay. MDR-1 mRNA, and expression of p38MAPK and phospho-p53 protein were detected by RT-PCR and Western blotting, respectively. The apoptosis rate of A2780/Taxol cells was (19.7+/-1.04)% 24 h after SB203580 treatment. A significant difference in apoptosis rate was found among experiment group, control group and untreated group (P<0.05). The relative reversal rate of A2780/Taxol cells to paclitaxel was (57.18+/-2.01)%. As compared with the control group and the untreated group, p38MAPK protein and MDR-1 mRNA in SB203580-treated cells was substantially decreased. The expression of p53 protein was significantly increased. It is concluded that p38MAPK pathway is related to paclitaxel resistance of ovarian carcinoma, and blockade of this pathway can promote the apoptosis of the drug-resistant cells and reverse the drug-resistance. Moreover, p38MAPK-mediated apoptosis in paclitaxel-resistant ovarian carcinoma cells depends on the activation of p53.     

P38丝裂原活化蛋白激酶抑制剂对大鼠重症急性胰腺炎的作用

目的 探讨P38丝裂原活化蛋白激酶（P38MAPK）抑制剂（SB203580）对重症急性胰腺炎的治疗作用.方法 将Wistar大鼠63只随机分为三组：假手术组（S组）,重症急性胰腺炎组（P组）,SB203580处理组（T组）,每组21只.SAP模型由5%牛磺胆酸钠大鼠胆胰管逆行注射诱发而成.模型成功后,P组不做处理 T组立即行SB203580 10 mg/kg腹腔注射 S组仅开腹,不做其他处理 术后各组于1 h、2 h、4 h 3个时间点处死大鼠,各时间点酶联免疫吸附测定（ELISA）法测血清TNF-α水平,并取胰腺组织行病理检查,免疫组化法检测p-P38MAPK表达,TUNEL法检测胰腺腺泡细胞凋亡.结果 血清TNF-α水平随SAP病情的进展而升高（P＜0.05）,其水平P组及T组各时间点均较S组高,但TNF-α水平T组各时间点显著低于P组（P＜0.05） p-P38MAPK表达随SAP病情进展而升高（P＜0.05）,1、2、4 h三个时间点P组及T组均升高（P＜0.05）,但T组活性显著低于P组（P＜0.05）：TUNEL显示假手术组胰腺组织存在极少量的凋亡细胞,T、P组细胞凋亡率均高于S组,T组凋亡率高于P组（P＜0.05） 胰腺病理损害光镜下T组明显轻于P组.结论 P38MAPK可能参与大鼠重症急性胰腺炎发病过程,SB203580可能通过抑制P38MAPK活化减少炎症递质释放,增加胰腺腺泡细胞凋亡,减轻SAP病理损害.