脑缺血再灌注后酪氨酸蛋白激酶和蛋白磷酸酶的活性变化及其机制(英文)

目的:研究脑缺血再灌注后沙土鼠海马突触体蛋白质酪氨酸激酶(PTK)和蛋白质酪氨酸磷酸酶(PTP)活性的变化及其引起变化的机制。方法:双侧颈总动脉结扎(15min)形成全脑缺血模型;放射性同位素γ-~(32)P掺入法和比色法分别测定了总PTK和PTP的活性,免疫沉淀和γ-~(32)P放射性同位素测定Src和PYK_2活性,免疫印渍测定Src和PYK_2蛋白表达量。结果:①脑缺血再灌注引起PTK活性升高,而PTP活性不变。②在假手术对照组中,Src比PYK_2活性高,脑缺血再灌引起Src活性明显升高,而PYK_2活性无显著变化。③脑缺血前腹腔分别给予氯胺酮和硝苯地平,都能拮抗脑缺血再灌注引起的PTK和Src活性的升高,但对PTP活性无影响。结论:脑缺血再灌注能诱导沙土鼠海马突触体总的PTK活性升高,而对PTP活性无影响,PTK活性的升高主要是Src活性的增加而与PYK_2无关;脑缺血再灌注诱导PTK和Src活性升高是通过NR和L-型电压门控钙通道介导的,即与这两种钙通道的激活有关,而与其蛋白表达量无关。     

脑缺血后IκB激酶的自身磷酸化及活性调节(英文)

目的:观察脑缺血后IκB激酶的自身磷酸化及活性变化的调节机制。方法:采用IKKα/B抗体进行免疫印迹和免疫沉淀 分析,测定IKK蛋白表达,体外激酶活性分析测定IKK的自身磷酸化及活性。结果:IKK蛋白的表达在脑缺血不同时间以及脑缺血复灌不同时间都没有明显的变化;IKK的活性在缺血30分钟时达到最高;NMDA受体拮抗剂ketamine、非NMDA受体拮抗剂DNQX和NF-κB抑制剂PDTC均能抑制IKK的自身磷酸化及活性。结论:NMDA受体和非NMDA受体介导了脑缺血后IKK的自身磷酸化及活性调控,从而介导其下游因子NF-κB的蛋白表达及活性变化,参与脑缺血的损伤调控。     

NVP-AAM077和Ro25-6981对全脑缺血小鼠海马神经元损伤及BDNF表达的影响

目的探讨N-甲基-D-天冬氨酸受体(NMDA)亚基NR2A和NR2B特异性拮抗剂对脑缺血/再灌注后海马CA1区神经元损伤的不同影响及其可能机制。方法制作三动脉阻断(3-VO)小鼠全脑缺血模型,小鼠随机分为假手术组、脑缺血/再灌注(I/R)对照组、NVP-AAM077(NVP)干预组和Ro25-6981(Ro)干预组;应用Fluoro-JadeB(F-JB)和Nissl染色检测海马神经元变性死亡和存活情况,Western blot对脑源性神经生长因子(BDNF)蛋白表达水平进行定量分析。结果①小鼠全脑缺血12min/再灌注3d后,海马CA1区出现选择性迟发性神经元死亡,NVP干预组增加了缺血所致的海马神经元死亡(P<0.05),而Ro干预组CA1区神经元存活数量明显多于缺血/再灌注组(P<0.01);②NVP干预能明显下调缺血/再灌注所致的海马组织BDNF蛋白表达升高(P<0.01),而Ro干预能明显上调BDNF蛋白的表达(P<0.05)。结论 NMDA受体亚基NR2A和NR2B在小鼠脑缺血/再灌注损伤中具有不同的作用,其机制可能与调节BDNF表达改变有关。     

转导血红素氧合酶-1蛋白减轻脑缺血/再灌注沙土鼠海马神经元的损伤

目的评价血红素氧合酶-1(HO-1)蛋白转导对脑缺血/再灌注损伤沙土鼠海马神经元的影响。方法构建11R(11个精氨酸残基)-HO-1蛋白,50只沙土鼠随机分为5组(n=10):脑缺血/再灌注组(C组),沙土鼠行脑缺血/再灌注损伤。脑缺血/再灌注+生理盐水组(S组)、脑缺血/再灌注+11R组(R组)、脑缺血/再灌注+5 mg·kg-111R-HO-1组(H1组)和脑缺血/再灌注+25 mg·kg-111R-HO-1组(H2组),各组沙土鼠腹腔分别注射5 mg·kg-1生理盐水、5mg·kg-111R蛋白、5 mg·kg-111R-HO-1蛋白或25 mg·kg-111R-HO-1蛋白,3 h后行脑缺血/再灌注损伤。24 h后取沙土鼠海马,电镜下观察海马组织线粒体的变化,检测海马神经元凋亡、Caspase-3和HO-1蛋白表达、cAMP水平。结果C组、S组和R组3组间海马神经元线粒体变性率、神经元凋亡率、Caspase-3和HO-1蛋白表达、cAMP水平变化无差异(P>0.05);H1组海马神经元线粒体变性率降低、神经元凋亡率降低、Caspase-3蛋白表达下调、HO-1蛋白表达上调、cAMP水平升高(vs C组、S组和R组,P<0.01);H2组海马神经元线粒体变性率降低、神经元凋亡率降低、Caspase-3蛋白表达下调、HO-1蛋白表达上调、cAMP水平升高(vs H1组,P<0.01)。结论 HO-1蛋白转导减轻了脑缺血/再灌注沙土鼠海马神经元的损伤。     

NMDA受体NR2A,NR2B亚基对大鼠脑缺血再灌注海马CA1区神经细胞损伤的作用

目的 观察N-甲基-D-天冬氨酸受体(NMDA)NR2A,NR2B亚基对脑缺血-再灌注后海马CA1区神经细胞存活的不同影响.方法 采用Pulsinelli-Brierley四动脉阻塞(4-VO)大鼠全脑缺血模型,缺血前连续3天脑室注射NR2A,NR2B反义寡核苷酸(AS ODN)后缺血15 min,复灌5 d,石蜡切片,以焦油紫染色,图像分析测定单位面积内焦油紫染色细胞面积总和,与缺血组及错义寡核仟酸组(MS ODN)进行形态学分析.结果 NR2A、NR2B反义寡核苷酸对脑缺血再灌注后海马 CA1神经细胞均有明显保护作用,以NR2A AS ODN保护作用更明显,与缺血组比较约有50%细胞仃活(P＜0.05).结论 NR2A,NR2B亚基的含量降低对脑缺血再灌注后海马神经细胞有明显保护作用.     

二苯乙烯苷对脑缺血啮齿动物脑NMDA受体及细胞内钙离子的影响

目的　研究二苯乙烯苷对脑缺血再灌注啮齿动物脑组织N 甲基 D 天 (门 )冬氨酸 (NMDA)受体结合力的影响 ,同时观察二苯乙烯苷对神经细胞内钙离子浓度的影响 ,初步探讨二苯乙烯苷对缺血再灌注动物的脑保护作用的机制。方法　麻醉后夹闭沙土鼠和小鼠双侧颈总动脉 10min后 ,小心撤去动脉夹制作缺血再灌注动物模型 :(1)沙土鼠再灌注 7d后处死 ,取前脑进行NMDA受体结合力实验 ;(2 )小鼠再灌注 15min后迅速断头取脑 ,切成脑片 ,加入Fluo 3/AM负载后 ,在激光扫描共聚焦显微镜上进行光切 ,观察不同层面皮层和海马区细胞内游离钙的荧光强度。结果　模型组的NMDA受体结合力比假手术组明显升高 ,而二苯乙烯苷治疗组可以降低模型动物的NMDA受体结合力 ;脑片的光切结果显示 ,脑缺血再灌注模型组神经细胞内游离钙离子浓度比假手术组明显升高 ;与模型组相比 ,二苯乙烯苷治疗组的细胞内游离钙离子浓度降低。结论　二苯乙烯苷能够抑制啮齿动物脑缺血再灌注所导致的脑组织NMDA受体结合力及神经细胞内钙离子浓度的升高 ,减轻钙超载导致的脑组织损伤 ,可能具有脑保护作用     

小檗碱及其衍生物对2型糖尿病大鼠骨骼肌PTP1B蛋白表达的影响

目的:观察小檗碱及其衍生物对2型糖尿病大鼠骨骼肌蛋白酪氨酸磷酸酯酶1B(PTP1B)蛋白表达的影响。方法:采用尾静脉注射小剂量链脲佐菌素(STZ)和高脂高热卡饮食喂养的方法建立大鼠2型糖尿病模型。随机分为模型组、小檗碱组、8-羟基二氢小檗碱(Hdber)高、中、低剂量组和二甲双胍组,另设正常对照组。干预8周后,检测各组大鼠血糖、血脂、空腹胰岛素(FINS)水平及骨骼肌PTP1B、胰岛素受体β(InsRβ)亚基蛋白及其酪氨酸磷酸化水平的差异。结果:与正常组比较,模型组大鼠血糖升高,血脂代谢紊乱,FINS及骨骼肌PTP1B蛋白表达升高(P<0.05,P<0.01),而InsRβ亚基蛋白表达及其酪氨酸磷酸化水平降低(P<0.01);与模型组比较,各治疗组代谢紊乱均得到明显改善、FINS降低(P<0.05,P<0.01),但骨骼肌组织PTP1B蛋白表达水平无明显差异;与小檗碱组比较,Hdber中高剂量组对各指标的改善程度相接近。结论:小檗碱及Hdber均可改善2型糖尿病大鼠糖脂代谢紊乱,其机制可能与增加骨骼肌组织InsRβ亚基蛋白表达及其酪氨酸磷酸化水平有关,而未观察到其对PTP1B蛋白表达水平的显著影响。     

羟丁酸钠对沙土鼠脑缺血再灌注损伤的作用研究

目的　研究缺血前后羟丁酸钠 (sodiumgamma hy droxybutyrate,γ OH)对沙土鼠脑缺血再灌注损伤的保护作用。方法　采用沙土鼠双侧颈总动脉结扎法制作全脑缺血再灌注损伤模型 ,观察γ OH对脑缺血再灌注沙土鼠大脑皮层、海马和纹状体ATP酶活性、超氧化物歧化酶 (SOD)活性及丙二醛 (MDA)含量的影响。结果　缺血前给γ OH能保护脑缺血再灌注沙土鼠脑组织ATP酶和SOD的活性 ,降低MDA含量 ,缺血后给药仍有一定疗效。结论　γ OH对脑缺血再灌注损伤有保护作用 ,其机制与保护脑组织ATP酶和SOD活性 ,清除氧自由基 ,减少脂质过氧化有关。     

亚低温对沙土鼠前脑缺血再灌注海马神经元凋亡及p38活性的影响

目的通过动态观察亚低温(33℃,4h)对沙土鼠前脑缺血再灌注后不同时间点海马神经元凋亡细胞及磷酸化p38表达的影响,探讨亚低温脑保护的可能机制。方法采用沙土鼠双侧颈总动脉阻断5min前脑缺血再灌注损伤模型,随机分为假手术组,常温再灌注组,低温假手术组,低温再灌注组。每组根据再灌注的不同时间点(2h、4h、d1、3、d5)又分为5个对应的亚组(n=6)。在预定时间点行开阔法迷宫检查,TUNEL法检测海马CA1/3区的凋亡细胞,免疫组化检测pp38在海马各区的动态变化。结果4h亚低温可显著减少缺血沙土鼠d1、d3、d5的探索活动及CA1/区的凋亡细胞,明显抑制脑缺血后海马CA1区pp38早期的表达(2h、4h)。结论4h亚低温治疗对沙土鼠5min前脑缺血有明确的保护作用,抑制海马CA1区缺血再灌注早期pp38的激活可能是其减少海马细胞凋亡、产生脑保护作用的机制之一。     

二十二碳六烯酸乙酯对脑缺血再灌注氧化损伤及脑水肿的影响

目的 研究二十二碳六烯酸乙酯(ethyl docosahexaenoate，E-DHA)对沙土鼠脑缺血再灌注引起的氧化损伤和脑水肿的保护作用。方法 采用沙土鼠双侧颈总动脉阻断法制作全脑缺血再灌注损伤模型。检测E-DHA对缺血再灌注后脑中丙二醛(MDA)和谷胱甘肽(GSH)含量；谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、ATP酶活性；水及Na^ 、Ca^2 含量的影响．并进行了病理检查。结果 缺血前E-DHA预防给药10周有效阻止了脑缺血再灌注引起的MDA升高，GsH降低，GsH-Px、CAT活性降低．ATP酶活性降低，水及Ca^2 、Na^ 含量升高。结论 E-DHA对沙土鼠脑缺血再灌注具有保护作用，其机理与清除自由基和减轻脑水肿有关。     

Changes and mechanisms of protein-tyrosine kinase and protein-tyrosine phosphatase activities after brain ischemia/reperfusion

AIM: To study the changes and mechanisms of protein-tyrosine kinase (PTK) and protein-tyrosine phosphatase (PTP) activities in the hippocamal synaptosome following cerebral ischemia/reperfusion (I/R) in gerbil. METHODS: Transient (15 min) global ischemia was produced by bilateral carotid artery occlusion. Total PTK and PTP activities were measured by [r-32P] incorporation and colorimetric analysis, respectively. Src and proline-rich tyrosine kinase2 (PYK2) activities were measured by immunoprecipitation and [r-32P] incorporation. RESULTS: Total PTK activity increased significantly after I/R, but the PTP activity did not change. The Src activity was much higher than PYK2 activity in sham-operated controls. I/R mainly caused a pronounced increase in Src activity, but not PYK2 activity. The increase in Src activity had no relation to the expression of Src protein. Administration of ketamine (KT) or nifedipine (ND) 20 min before ischemia caused a decrease in total PTK and Src activities, and no change in the PYK2 and PTP activities. CONCLUSION: The increase in PTK activity caused by I/R may be mainly due to the increase in Src activity. This increase in Src activity has no relation to the expression of Src protein. But it is related to the activation of NMDA (N-methyl-D-aspartate) receptor (NR) and L-type voltage-gated calcium channel (L-type VGCC). In other words, the increase in total PTK and Src activities induced by I/R may be mediated via NR and L-type VGCC. The PTP activity did not change during I/R.     

Dehydroepiandrosterone sulfate prevents ischemia-induced impairment of long-term potentiation in rat hippocampal CA1 by up-regulating tyrosine phosphorylation of NMDA receptor

We have reported that dehydroepiandrosterone sulfate (DHEAS) reduces the threshold for long-term potentiation (LTP) in Shaffer collateral-CA1 synapses through the amplification of Src-dependent NMDA receptor signaling. The present study is a follow-up of the above reports, aiming at evaluating the effects of DHEAS on the impaired LTP in reversible forebrain ischemic rats. Transient (20 min) incomplete forebrain ischemia led to an impaired LTP in the hippocampal CA1 region without damages to the basal synaptic transmission between the Shaffer collaterals and pyramidal neurons. Repetitive administrations of DHEAS (20 mg/kg for 3 days) from the first 3 h of reperfusion, but not acute DHEAS application (50 microM), prevent the impairment of LTP produced by ischemia. Co-administration of the specific sigma(1) receptor antagonist NE100 with DHEAS completely prevented the protective effect of DHEAS. In contrast, progesterone (PRGO) not only had no protective effect against the ischemic LTP impairment, but also attenuated the protective effect of DHEAS on the impaired LTP. Tyrosine phosphorylation of NMDA receptor subunit 2B (NR2B) significantly decreased after ischemia, whereas that of NR1 had no obvious change. Furthermore, the repetitive administration of DHEAS improved the reduction in tyrosine phosphorylation of NR2B. These findings suggest that the repetitive activation of sigma(1) receptor induced by DHEAS might prevent the ischemic LTP impairment through regulating the tyrosine phosphorylation of NR2B.     

NR2A反义寡核苷酸对短暂性脑缺血/再灌注大鼠海马神经元损伤的保护作用

目的探讨NMDA受体亚单位2A(NR2A)反义寡核苷酸在短暂性脑缺血/再灌注大鼠海马神经元损伤中的保护作用,为研制和开发针对NR2A的特异性新药提供理论基础和形态学依据。方法健康♂SD大鼠随机分为正常对照组、假手术对照组和缺血/再灌注组。经生理盐水、错义寡核苷酸和反义寡核苷酸预处理后,以四血管阻断法建立短暂性全脑缺血(15min)/再灌注(1、2、3和5d)动物模型。在确定的时间点进行灌注固定、取材、石蜡包埋和组织切片(片厚8μm),然后行TUNEL反应、焦油紫染色、原位杂交染色以及免疫组织化学染色。结果短暂性脑缺血/再灌注(I/R)3d,大鼠海马CA1区出现大量的凋亡阳性细胞;I/R5d大鼠海马CA1区细胞严重受损,与对照组相比二组差异具有显著性(P<0.05)。经NR2A反义寡核苷酸预处理后,I/R3d和I/R5d海马CA1区的细胞凋亡和细胞损伤明显减轻,与对照组相比二组差异具有显著性(P<0.05)。NR2A反义寡核苷酸能抑制I/R1d NR2A mRNA表达和I/R2d蛋白质表达,与对照组相比差异具有显著性(P<0.05)。结论短暂性全脑缺血后,NR2A反义寡核苷酸能明显地减轻缺血诱导的大鼠海马CA1区细胞凋亡和细胞损伤,且这种作用与NR2A反义寡核苷酸特异性抑制NR2A及其mRNA的表达密切相关。     

Competitive binding of postsynaptic density 95 and Ca^2＋-calmodulin dependent protein kinase Ⅱ to N-methyl-D-aspartate receptor subunit 2B in rat brain

AIM: To investigate the interactions among postsynaptic density 95 (PSD-95), Ca^2 -calmodulin dependent protein kinase Ⅱα (CaMKⅡα), and N-methyl-D-aspartate receptor subunit 2B (NR2B) during ischemia and reperfusion in hippocampus of rats. METHODS: Brain ischemia was induced by four-vessel occlusion procedure in rats. Immunoprecipitation and immunoblotting were performed to study the interactions and phosphorylation of proteins. The association-dissociation of PSD-95 and CaMKⅡα to and from N-methyl-D-aspartate (NMDA) receptor induced by ischemia and reperfusion and the effects of 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62, a selective inhibitor of CaMKⅡ) on these protein interactions were investigated. Coimmunoprecipitation and immunoblotting were performed for the studies of interactions among proteins. RESULTS: The alternations of the binding level of PSD-95 and CaMKⅡα to NR2B during ischemia and reperfusion demonstrated the negative correlation to each other. Pre-administration of KN62 through both cerebral ventricles inhibited the 10min ischemia-induced increase of the binding of PSD-95 to NR2B and, on the contrary, promoted the binding of CaMKⅡα to NR2B. CONCLUSION: PSD-95 competes with CaMKⅡ to bind to NR2B during ischemia and reperfusion in rat hippocampus.     

Silica induces nuclear factor-kappa B activation through tyrosine phosphorylation of I kappa B-alpha in RAW264.7 macrophages

It was previously reported that protein tyrosine kinase (PTK) but not protein kinase C or A plays an important role in silica-induced activation of NF-kappa B in macrophages. The question is raised whether PTK stimulation and NF-kappa B activation in silica-stimulated macrophages are directly connected through tyrosine phosphorylation of I kappa B-alpha. Results indicate that stimulation of macrophages with silica led to NF-kappaB activation through tyrosine phosphorylation without serine phosphorylation. Specific inhibitors of protein tyrosine kinase, such as genistein and tyrophostin AG126, prevented tyrosine phosphorylation of I kappa B-alpha in response to silica. I kappa B-alpha protein levels remained relatively unchanged for up to 60 min after silica stimulation. Moreover, inhibition of proteasome proteolytic activity did not affect NF-kappa B activation by silica. Antioxidants, such as superoxide dismutase (SOD), N-acetylcysteine (NAC), and pyrrolidine dithiocarbamate (PDTC), blocked tyrosine phosphorylation of I kappa B-alpha induced by silica, suggesting reactive oxygen species (ROS) may be important regulatory molecules in NF-kappa B activation through tyrosine phosphorylation of I kappa B-alpha. The results suggest that tyrosine phosphorylation of I kappa B-alpha represents a proteasome proteolytic activity-independent mechanism for NF-kappa B activation that directly couples NF-kappa B to cellular tyrosine kinase in silica-stimulated macrophages. This proposed mechanism of NF-kappa B activation induced by silica could be used as a target for development of antiinflammatory and antifibrosis drugs.     

金雀异黄素对脑缺血／再灌注诱导大鼠海马组织STAT3激活的抑制作用

目的：观察脑缺血／再灌注(I／R)后海马组织信号转导与转录激活子-3(STAT3)的激活及金雀异黄素对其的影响．方法：免疫印迹测定STAT3蛋白表达及磷酸化水平,电泳迁移率改变分析STAT3的DNA结合活性．结果：I／R3h后胞浆STAT3持续磷酸化激活,再灌注3h和72h达两高峰(1．7和2．5倍);而STAT3蛋白表达I／R24h开始升高,72h达高峰(1.7倍);核内STAT3的磷酸化水平及DNA结合活性缺血后即显著增高,再灌注3h和72h达两高峰(分别为2.6、3.1及3.6、4.4倍）。金雀异黄素呈剂量依赖方式明显抑制I/R 72h诱导STAT3的磷酸化及DNA结合活性的升高,但不影响STAT3蛋白表达。结论：脑缺血/再灌注导致STAT3的磷酸化激活及DNA结合活性的增加,蛋白酪氨酸激酶参与STAT3的激活调控。     

白三烯受体拮抗剂ONO-1078对大鼠短暂性全脑缺血的神经保护作用

AIM: To determine whether ONO-1078 {pranlukast, 4-oxo-8-[p-(4-phenylbutyloxy)benzoyl-amono]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate), a potent leukotriene receptor antagonist, possesses a neuroprotective effect on global cerebral ischemia in rats, and to explore its possible mechanism of action. METHODS: Transient global cerebral ischemia was induced by four-vessel occlusion for 10 min and followed by 72-h reperfusion. ONO-1078(0.03-0.3mg/kg) and edaravone (MCI-186, 3-methyl-1-phenyl-2-pyrazolin-5-one, a neuroprotective agent) 10 mg/kg were ip injected 30 rain before ischemia and 1 h after reperfusion, and once a day afterward. Neurological outcome was evaluated before ischemia and 24, 48, 72 h after reperfusion. Neuron density, the expressions of N-methyl-D-aspartate (NMDA) receptor subunit proteins (NR1, NR2A, NA2B) and vascular cell adhesion molecule 1 (VCAM-1) in the cerebral cortex and hippocampus were measured at 72h after reperfusion. RESULTS: ONO-1078 (0.1,0.3mg/kg) and edaravone (10 mg/kg) improved ischemia-induced neurological deficiency and reduced neuron death.ONO-1078 (0.1, 0.3mg/kg) significantly inhibited the enhanced expression of NMDA receptor subunit protein NR2A in the cortex and VCAM-1 in the hippocampus of ischemic rats. CONCLUSION: ONO-1078 possesses a neuroprotective effect on global cerebral ischemia in rats, and its mechanism may be partly related to the inhibition of the upregulation of NR2A and VCAM- 1 in different regions of the brain.     

Neuroprotective effect of ONO-1078, a leukotriene receptor antagonist, on transient global cerebral ischemia in rats

AIM: To determine whether ONO-1078 {pranlukast, 4-oxo-8-[p-(4-phenylbutyloxy)benzoyl-amono]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate}, a potent leukotriene receptor antagonist, possesses a neuroprotective effect on global cerebral ischemia in rats, and to explore its possible mechanism of action. METHODS: Transient global cerebral ischemia was induced by four-vessel occlusion for 10 min and followed by 72-h reperfusion. ONO-1078 (0.03-0.3 mg/kg) and edaravone (MCI-186, 3-methyl-1-phenyl-2-pyrazolin-5-one, a neuroprotective agent) 10 mg/kg were ip injected 30 min before ischemia and 1 h after reperfusion, and once a day afterward. Neurological outcome was evaluated before ischemia and 24, 48, 72 h after reperfusion. Neuron density, the expressions of N-methyl-Daspartate (NMDA) receptor subunit proteins (NR1, NR2A, NA2B) and vascular cell adhesion molecule 1(VCAM-1) in the cerebral cortex and hippocampus were measured at 72 h after reperfusion. RESULTS: ONO-1078 (0.1, 0.3 mg/kg) and edaravone (10 mg/     

JAK-STAT信号通路在离体大鼠心肌缺血再灌注损伤激活时程的研究

目的观察JAK-STAT信号通路在离体大鼠心肌缺血再灌注损伤过程中激活的时程及意义。方法采用Langendorff离体灌流模型,将42只雄性Wistar大鼠随机分为7组:对照组:用改良的KH液持续灌流180 min;I/R组:按再灌注时间分为R0、R5、R15、R30、R60、R120 6组,用改良的KH液灌流平衡30 min后,全心停灌30 min,分别再灌注0、5、15、30、601、20 min。连续监测左室发展压(LVDP)、左室压力最大升降速度(+dp/dtmax)以评价心功能,采用改良的Gonori变色酸(GCA)亮绿特殊染色法观察心肌缺血程度;免疫印迹法检测再灌注不同时程磷酸化的STAT1、STAT3蛋白表达的变化。结果与对照组相比,再灌注不同时程LVDP及+dp/dtmax明显降低((P<0.01),GCA特殊染色显示缺血变性的心肌细胞数量明显增多(P<0.01),再灌注各时程STAT1、STAT3均处于激活状态,与对照组相比差异有统计学意义(P<0.01)。但二者表达存在时间差异,p-STAT1在缺血30 min增高不明显,再灌注过程中处于显著激活状态(P<0.01);p-STAT3在缺血30 min即明显升高(P<0.01),而再灌注期未见进一步升高(P>0.05),p-STAT1/p-STAT3与LVDP及GCA特殊染色阳性率呈显著的相关性。结论JAK-STAT信号通路参与了心肌缺血再灌注损伤的发生,磷酸化的STAT1与STAT3的比例可能决定了再灌注损伤的严重程度。