【作者】 徐艳炜；

【导师】 程焱； 孙莉；

【作者基本信息】 天津医科大学 ， 神经病学， 2010， 博士

【摘要】 研究目的：1.观察脑缺血再灌注损伤时过氧化物酶体增殖物激活受体(peroxisome proliferators-activated receptors, PPARs)各亚型蛋白(PPARα、PPARβ/δ、PPARγ)表达的变化以及PPARs激动剂对PPARs表达的影响,并探讨PPARs的改变在脑缺血再灌注损伤中的意义；2.以PPARs三亚型中的PPARRγ为研究对象,观察脑缺血再灌注损伤时其核移位的变化,以及PPARγ激动剂和拮抗剂对核移位的影响,并初步探讨PPARγ核移位在脑缺血再灌注损伤中的意义；3.观察脑缺血再灌注损伤时12/15-脂氧酶(12/15-lipoxygenase,12/15-LOX)表达及其产物15-HETE含量的改变,以及12/15-LOX抑制剂和产物对PPARγ表达的影响,初步探讨12/15-LOX途径在PPARγ表达调控中的意义。研究方法：1.将SD大鼠随机分为假手术组(Sham组)、缺血再灌注模型组(I/R组)、治疗组。制作大鼠MCAO/R模型,缺血60min,再灌注24h。治疗组于MCAO/R前30min给予PPAR全激动剂苯扎贝特(Bezafibrate, Beza)。使用神经功能缺损评分、2%氯化三苯四唑(2,3,5-triphenyltetrazolium, TTC)染色对苯扎贝特在脑缺血再灌注损伤中的作用进行评价,免疫组化、Western blot检测PPARs各亚型蛋白表达情况；2.将大鼠随机分为假手术组(Sham组)、缺血再灌注模型组(I/R组)、PPARγ激动剂干预组(Ros组)、PPARγ抑制剂干预组(GW9662组)。其中I/R组,缺血均为60min,又根据再灌注时间分为2h、4h、8h、24h组。Ros组、GW9662组均于MCAO/R前30min给予相应的药物。使用神经功能缺损评分、TTC染色对PPARγ激动剂、抑制剂在脑缺血再灌注损伤中的作用进行评价,免疫组化、Western blot检测PPARγ表达水平,免疫荧光检测PPARγ在神经细胞内的定位；3.将大鼠随机分为Sham组、I/R组(60min/24h)、Baicalein干预组、15-羟基二十碳四烯酸(15-hydroxyeicosatetraenoic,15-HETE)干预组。Baicalein干预组、15-HETE干预组均于MCAO/R前30min给予相应的药物。使用免疫荧光双标检测和Western blot检测12/15-LOX、PPARγ表达,酶联免疫法检测15-HETE含量。研究结果：1.与假手术组相比,缺血60min再灌注24h(1)引起平均梗塞体积为44.30%,同时神经功能缺损评分增加,P<0.01,差异有统计学意义；(2)使脑组织PPARs各亚型表达均增加,分别增加了1.47倍、3.52倍和2.25倍,差别有统计学意义(免疫组化检测t值分别为8.63、9.29和13.62,Western blot检测t值分别为8.16、9.24和6.43,均P=0.000)；同时PPARs各亚型免疫阳性细胞增加主要表现在缺血侧半暗带区域,而非缺血侧(对侧)表达没有明显改变。与I/R组相比,PPAR全激动剂Beza(1)显著降低脑梗死体积,平均减少54.36%,差异有统计学意义(t=7.69,P=0.000),同时mNSS降低,P<0.01,差异有统计学意义；(2)进一步增加PPARs各亚型表达,分别增加了95.45%、183.47%、224.61%,差异有统计学意义(免疫组化检测t值分别为7.36、5.64和10.50,Western blotting检测t值分别为13.02、17.52和13.64,均P=0.000)；同时不但使缺血侧PPARs免疫阳性细胞增加,而且使非缺血侧(对侧)也增加。2. (1)I/R组与Sham组相比,①PPARγ总蛋白表达：Western blot检测显示,再灌注2h组未见明显改变(t=-0.701,P>0.05,差异无统计学意义),再灌注4h组、8h组、24h组PPARγ总蛋白表达水平均增高,且呈再灌注时间依赖性增强(t=-15.749,-16.715,-22.839,均P<0.05,差异有统计学意义)；免疫组化检测与Western blot结果一致；②PPARγ成分蛋白表达：Western blot检测显示,Sham组胞浆、胞核内均有PPARγ表达；I/R后,再灌注2h、4h、8h、24h组PPARγ在胞核内的表达增加(与Sham组相比,t=-11.343、-27.191、-24.861、-25.75,均P<0.05),同时在胞浆内的表达减少(与Sham组相比,t=7.197、9.031、10.777、13.78,均P<0.05),且PPARγ的核移位改变呈再灌注时间依赖性；免疫荧光检测与Western blot结果一致；(2)与I/R(60min/24h)组相比,①Ros组梗死体积减小了48.27%,而GW9662组梗死体积增加了39.52%；神经功能缺损评分也呈现一致的改变(均为P<0.05,差异均有统计学意义)；②PPARγ总蛋白和成分蛋白表达：Western blot检测显示,与I/R组(60min/24h)相比,PPARγ激动剂组(Ros组)PPARγ的总蛋白表达水平显著增高(t=-23.465,P<0.05),核蛋白表达水平亦显著增高(t=-33.788,P<0.05),浆蛋白表达水平则降低(t=6.428,P<0.05),差异均有统计学意义；而PPARγ抑制剂组(GW9662组)PPARγ的总蛋白表达水平显著降低(t=29.403,P<0.05),核蛋白表达水平亦显著降低(t=22.414,P<0.05),浆蛋白表达水平则增高(t=-6.826,P<0.05),差异均有统计学意义；免疫荧光检测与Western blot结果一致。3. (1)与Sham组相比,Western blot检测显示,I/R(60min/24h)组12/15-LOX总蛋白表达增高(t=-8.195,P<0.05,差异有统计学意义)；免疫荧光检测显示,12/15-LOX的表达主要位于缺血半暗带,且其表达位置与PPARγ具有一致性；同时酶联免疫检测显示,I/R组12/15-LOX产物15-HETE的含量明显增加(t=-12.787,P<0.05,差异有统计学意义)。(2)与I/R(60min/24h)组相比,同时给予不同剂量Baicalein (150mg/kg、200mg/kg)使PPARγ总蛋白(t=7.253,8.61,P<0.05)、核蛋白(t=15.361,26.922,P<0.05)表达均降低,浆蛋白(t=-17.579，-25.719,P<0.05)表达增高,差异均有统计学意义；Baicalein 200mg/kg处理不同时间(缺血60min再灌注4h和24h)均引起PPARγ总蛋白(t=2.773,11.650,P<0.05)、核蛋白(t=4.313,37.468,P<0.05)表达水平均降低,浆蛋白(t=-2.553,-8.246,P<0.05)表达水平增高,差异均有统计学意义；(3)与I/R(60min/24h)组相比,12/15-LOX产物15-HETE干预组PPARγ总蛋白(t=-23.884,P<0.05)、核蛋白(t=-15.36,P<0.05)表达水平均增高,浆蛋白(t=5.86,P<0.05)表达水平降低,差异均有统计学意义。研究结论：1.局灶性脑缺血再灌注损伤大鼠脑组织PPARs各亚型(PPARα、PPARβ/δ和PPARγ)表达增加,该改变可能是脑组织的一种代偿性神经保护反应；2.(1)脑缺血再灌注损伤可诱导PPARγ表达上调及核移位,最终引起细胞核内PPARγ表达水平增高,即PPARγ核活性增加；(2)I/R时PPARγ核移位的发生早于表达水平上调,相对于增加蛋白水平,可能是机体调节该转录因子的更为直接、快捷的方式；(3)PPARγ激动剂增强、PPARγ抑制剂抑制I/R诱导的PPARγ表达及核移位改变,表明I/R诱导的PPARγ表达及核移位改变可能与内源性PPARγ配体的释放有关；3.I/R时12/15-LOX表达和活性增加；12/15-LOX抑制剂和产物可分别抑制和促进缺血再灌注诱导的PPARγ改变,提示脑缺血再灌注时PPARγ的改变可能(至少部分)与12/15-LOX途径有关。更多还原

【Abstract】 Objectives:1. To examine PPAR isotypes (PPARa, PPARβ/δ, and PPARγ) expression, in rats exposed to cerebral I/R and I/R in combination with pan-PPAR co-agonist, as well as explore the significance of altered PPARs expression in the context of brain injury.2. To evaluate the alteration in protein expression and nuclear translocation of endogenous PPARγafter focal cerebral I/R in rats, and the influence of PPARγagonist and antagonist to nuclear translocation, and to investigate the meaning of PPARγnuclear translocation in brain ischemia reperfusion injury.3. To examine 12/15-LOX and its metabolite 15-HETE expression in rats exposed to cerebral I/R, and the influence of 12/15-LOX antagonist and metabolite to expression, and to investigate the meaning of 12/15-LOX pathway in PPARγexpression.Methods:1. Adult male SD rats underwent 60min middle cerebral artery occlusion followed by a 24-hour reperfusion (MCAO/R), receiving either vehicle (I/R-group) or bezafibrate (6mg/kg) treatment (Beza-group) 30min before operation. mNSS.was used to evaluate the neurological function. TTC staining was adopted to determine the volume of cerebral infarction. The expressions of PPAR isotypes were characterized by immunohistochemical staining (IHC) and Western blot, respectively. Furthermore, the spatial localizations of PPAR isotypes were detected with respect to ipsilateral ischemic (core and penumbra) and contralateral non-ischemic hemisphere. 2. Adult male SD rats underwent 60min middle cerebral artery occlusion followed by 2,4,8 or 24 hour reperfusion (MCAO/R), receiving either vehicle (I/R-group) or rosiglitazone (6mg/kg) treatment (Ros-group) or GW9662 (4mg/kg) treatment (GW9662-group) 30min before operation. mNSS was used to evaluate the neurological severity. TTC staining was adopted to determine the volume of cerebral infarction. The expressions of PPARγwas characterized by immunohistochemical staining (IHC) and Western blot, respectively. The nuclear translocation of PPARγwas characterized by immunoflorescene staining.3. Adult male SD rats underwent 60min middle cerebral artery occlusion followed by a 24-hour reperfusion (MCAO/R), receiving either vehicle (I/R-group) or Baicalein treatment (Baicalein-group) or 15-hydroxyeicosatetraenoic (15-HETE) treatment (15-HETE-group) 30min before operation. Immunoflorescene staining and Western blot were used to evaluate expression of 12/15-LOX and PPARy. The content of 15-HETE was evaluated by enzyme immunoassay.Results:1. Compared with sham-group,60min ischemia and 24 h reperfusion caused (1) 44.30% infarct volume in average in ipsilateral hemisphere and mNSS increased, P<0.01; (2) marked up-regulations of all 3 PPAR isotypes expression evaluated by IHC (t=8.63,9.29,13.62, and P<0.01) and Western blot (PPARa by 1.47-fold, PPARδ/βby 3.52-fold, and PPARy by 2.25-fold;t=8.16,9.24,6.43; and P=0.000); meanwhile, dramatical increase in PPARs staining in the ischemia-affected region of the ipsilateral MCA territory, and in particular to the ischemic penumbra. No detectable increase in number and intensity was observed in the contralateral (non-ischemic) hemisphere. Compared with I/R-group, the pan-PPAR co-agonists bezafibrate (Beza) (1) dramatically decreased infarct volume by 54.36% in average (t=7.69, P=0.000), mNSS decreased too, P<0.01; (2) Bezafibrate treatment further increased the alterations of all PPARs expression, which were induced by the exposure of I/R, as determined by IHC (t=7.36,5.64,10.50, and P=0.000) and Western blot (PPARa by 95.45%, PPARβ/δby 183.47%, and PPARγby224.61%; r=13.02,17.52,13.64, and P=0.000); (3) the PPARs immunoreactive expressions were observed in not only the ipsilateral but the contralateral hemisphere of Beza-group.2. (1) Compared with Sham-group, I/R-group①PPARy whole protein expression:As shown with Western blot, the PPARy whole protein expression of 2h-reperfusion hadn’t detectable change,t=-0.701, P>0.05; however, the PPARy whole protein expression of 4,8 and 24h-reperfusion all up-regulated in a reperfusion time-dependent way (t=-15.749,-16.715,-22.839, P<0.05); we can observe the same change with immunohistochemical staining;②PPARy component protein expression:as shown with Western blot, in Sham-group, PPARy expressed in both cytosol and nuclear fractions. A increase of PPARy in the nucleus (t=-11.343,-27.191,-24.861,-25.75; P<0.05) could be noted with a simultaneous reduction in the cytosol (t=7.197,9.031,10.777,13.78; P<0.05) following I/R, a time-dependent enhancement in PPARy translocation by the analysis of cytosol and nuclear level of PPARγat 2,4,8 and 24h of reperfusion; we can observe the same change with immunoflorescene staining; (2)Compared with I/R(60min/24h)-group,①the infarct volume of Ros-group decreased by 48.27% and the infarct volume of GW9662-group increased by 39.52%, mNSS also showed the same change, P<0.05;②PPARy whole and component protein expression:as shown with Western blot, rosiglitazone dramtically increased I/R-induced PPARy total protein(t=-23.465, P<0.05) and nuclear protein level (t=-33.788, P<0.05), and decreased I/R-induced PPARy cytosol protein level(t=6.428, P<0.05); GW9662 dramtically decreased I/R-induced PPARy total protein(t=29.403, P<0.05) and nuclear protein level (t=22.414, P<0.05), and decreased I/R-induced PPARy cytosol protein level(t=-6.826, P<0.05); we can observe the same change with immunoflorescence staining; 3. (1) Compared with Sham group, as shown with Western blot, the expression of 12/15-lipoxygenase (12/15-LOX) whole protein was enhanced in I/R(60min/24h) group (t=-8.195, P<0.05); the localization of 12/15-LOX was major in ischemic penumbra as shown with immunoflorescence staining, and consistent with PPARy; and as shown with enzyme immunoassay, the content of 15-HETE in I/R group was dramatically enhanced compared with Sham group (t=-12.787, P<0.05). (2) As shown with Western blot, the majority of PPARy localized in the nucleus under the I/R condition, however, different dose of Baicalein treatment significantly suppressed I/R-induced PPARy nuclear accumulation (150mg/kg,t=15.361, P<0.05; 200mg/kg,t=26.922, P<0.05) and maintained PPARy cytoplasmic retention (150mg/kg,t=-17.579, P<0.05; 200mg/kg,t=-25.719, P<0.05), furthermore, Baicalein also inhibited the up-regulation of PPARy total protein expression (150mg/kg,t=7.253, P<0.05; 200mg/kg,t=8.61, P<0.05); Baicalein 200mg/kg treatment suppressed I/R-induced increase in PPARy total protein expression(t=2.773, P<0.05; t=11.650, P<0.05) and nuclear translocation(t=4.313, P<0.05; t=37.468, P<0.05) with vehicle-treated I/R rats at both 4h and 24h after ischemia, and maintained PPARy cytoplasmic retention (t=4.313, P<0.05; t=-8.246, P<0.05); (3) Compared with I/R(60min/24h) group, 12/15-LOX metabolite 15-HETE treatment dramatically enhanced I/R-induce increase in PPARy total protein expression (t=-23.884, P<0.05) and nuclear translocation (t=-15.36, P<0.05), and suppressed PPARγcytoplasmic retention (t=5.86, P<0.05). Conclusion:1. Cerebral I/R injury increases the expression all 3 PPAR isotypes and activation of PPARs by pan-PPAR agonist not only reduces ischemic size but further enhances the alteration of PPARs. The up-regulation of PPARs expression may represent the compensatory self-protection against brain I/R injury. 2. (1) Cerebral I/R could up-regulated PPARy expression-and promoted its nuclear’ translocation, the combined and final effect of these changes is to bring about an elevation of nuclear PPARγ, which probably associates with the enchancement of its nuclear activity; (2) The onset of PPARy nuclear translocation was earlier than that of the up-regulation of protein expression, indicating that nuclear translocation of PPARγwas a more direct and quick response to I/R; (3) The supplementation of rosiglitazone, the exogenous PPARγligand, promoted I/R-induced PPARγalterations, whereas the treatment with PPARγantagonist GW9662 inhibited the changes of PPARγ, indicate that I/R-induced changes of PPARγresulting from the activation by endogenous ligands; 3. When ischemia reperfusion, the expression and activity of 12/15-LOX increased; 12/15-LOX inhibitor reduced and metabolite 15-HETE enhanced I/R-induced PPARγchanges, it seems that the mechanism underlying I/R-induced changes of PPARγmay be related to, at least partlγ,12/15-LOX pathway.-更多还原