大鼠局灶性脑缺血后继发性脑损伤机制及替米沙坦脑保护作用的研究
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摘要
脑血管病是造成中国居民死亡的第一原因和成年年人残疾的第一原因,以缺血性脑血管病占80%。研究表明,脑缺血触发一系列复杂的病理级联反应,包括能量代谢异常、炎症反应、氧化应激、自由基生成、兴奋性氨基酸的毒性作用等,直接或者间接导致神经细胞凋亡／死亡,破坏血脑屏障形成脑水肿,并最终导致神经功能缺失。在脑缺血后复杂的病理损伤机制中,炎症反应是继发性脑损伤的最重要原因之一。尽管目前对缺血性脑损伤机制有了比较深入的了解,但仍缺乏有效的治疗手段。除了极少数脑缺血患者在超早期经过溶栓治疗取得满意效果外,绝大多数患者将面临残疾甚至死亡的厄运。探讨有效阻止缺血性脑损伤机制,保护神经细胞的方法,是目前神经科学研究的热点和难点。
尽管已经有多种药物经过实验研究,证明具有抗炎、抗氧化、抗凋亡等作用,但多数未能在临床得到广泛应用,使理论和实践大大脱节。替米沙坦是一种临床广泛应用的血管紧张素受体阻断剂类(angiotensin type1receptor blocker,ARB)降血压药物。近年来,动物和临床实验发现,替米沙坦不仅能够有效治疗高血压、保护靶器官和减轻心室重构,还能够对脑缺血具有良好的抗炎作用,能够显著减轻脑梗死体积、脑水肿和继发性脑损伤,其机制多与激活过氧化物酶体增殖物激活受体γ(peroxisome prolixferator-activated receptors, PPARy)有关。国内外多项研究证明,PPARγ是一个关键性核转录因子,与其激动剂或者配体结合后活化,具有抗炎、抗氧化、抗凋亡、神经保护等多重作用。替米沙坦是唯一一种具有活化PPARγ功能的ARB类药物。结合基础研究和临床应用的实际需要,替米沙坦可能是治疗缺血性脑血管病的主要药物之一,但替米沙坦的作用机制仍需深入研究。
应用线栓法建立大鼠永久性局灶脑缺血模型,分别对炎症反应代表性因子PPARγ、 NF-κB、Egr-1和MMP-9,神经保护因子PEDF,和以Claudin-5为代表的血脑屏损伤程度等方面进行动态观察,并应用PPARy激动剂替米沙坦和PPARy特异性拮抗剂GW9662进行干预,观察上述各因子的变化,以及评价神经功能缺失、脑水肿和梗死体积等。以PPARy为切入点,深入探讨脑缺血后脑损伤机制及替米沙坦的脑保护作用机制。
本研究将分三部分对上述内容进行探讨,各部分内容概述如下。
第一部分大鼠脑缺血后炎症反应及替米沙坦抗炎作用机制研究
目的：动态观察PPARγ、NF-κB、Egr-1和MMP-9等在大鼠脑缺血后的表达变化,探讨替米沙坦的抗炎作用机制。
方法：应用Longa等的线栓法建立MCAO大鼠模型,采用成年健康雄性Sprague-Dawley大鼠,体重230g-250g。实验1：将大鼠随机分为正常组(Normal)和手术组(MCAO),手术组按照MCAO术后不同时间点分为3h、6h、24h、48h、72h5个亚组。实验2：将大鼠随机分为假手术组(Sham)、溶剂对照组(Vehicle)、替米沙坦组(Telmisartan)及替米沙坦+GW9662组(Telmisartan+GW9662)。替米沙坦30mg/kg于MCAO手术前1h以灌胃方式给予,以防止大鼠麻醉时呕吐；假手术组和对照组给予等体积的生理盐水；替米沙坦+GW9662组于MCAO手术前1h以灌胃方式给予30mg/kg替米沙坦,术后立即腹腔注射4mg/kgGW9662。将各组大鼠分别于相应时间点进行神经功能评分后断头处死,采用免疫组化、western blot和qR-T PCR来观察脑缺血后PPARγ、NF-κB、Egr-1和MMP-9等：mRNA和蛋白水平的动态变化,TTC染色评价脑梗死体积,干湿重法评价患侧脑水肿。
结果：MCAO组大鼠脑缺血后自24h开始,出现明显的神经功能缺失,持续并进行性加重至所观察到的72h,与正常组比较差异有统计学意义(P<0.05)。同时,自脑缺血24h开始,PPARy表达呈明显减少趋势,而NF-κB.Egr-1、MMP-9等呈明显增加趋势,且持续至本研究所观察到的72h,与正常组比较,差异有统计学意义(P<0.05)。术后24h,替米沙坦干预组与溶剂对照组比较,神经功能缺失有明显改善,脑水肿明显减轻、梗死体积明显减小,保护性因子PPARγ等表达明显增加,而NF-κB、Egr-1、MMP-9等明显减少,差异有统计学意义(P<0.05)。替米沙坦+GW9662组与替米沙坦组比较,神经功能缺失有明显加重、脑水肿和梗死体积增加,并且PPARy表达明显减少,而NF-κB、Egr-1、MMP-9等明显增加,差异有统计学意义(P<0.05)。溶剂对照组和替米沙坦+GW9662组比较无差别。
结论：大鼠脑缺血早期炎症反应迅速出现,脑缺血后24h开始明显出现,并持续至所观察的72h,炎症反应程度与神经功能缺损程度具有相关性。替米沙坦单独和联合GW9662干预表明,PPARγ是炎症反应的关键性调控基因。替米沙坦通过激活PPARγ,下调NF-кB、Egr-1和MMP-9,显著抑制了炎症反应,减轻脑缺血体积和脑水肿,改善了神经功能缺失。
第二部分大鼠脑缺血后PEDF表达变化及替米沙坦调节机制的研究
目的：动态观察大鼠脑缺血后PEDF的表达变化,探讨替米沙坦的脑保护作用机制。方法：应用Longa等的线栓法建立MCAO大鼠模型,采用成年健康雄性Sprague-Dawley大鼠,体重230g～250g。实验1：将大鼠随机分为正常组(Normal)和手术组(MCAO),手术组按照MCAO术后不同时间点分为3h、6h、24h、48h、72h5个亚组。实验2：将大鼠随机分为假手术组(Sham)、溶剂对照组(Vehicle)、替米沙坦组(Telmisartan)和替米沙坦+GW9662组(Telmisartan+GW9662)。替米沙坦30mg/kg于MCAO手术前1h以灌胃方式给予,以防止大鼠麻醉时呕吐；假手术组和对照组给予等体积的生理盐水；替米沙坦+GW9662组于MCAO手术前1h以灌胃方式给予30mg/kg替米沙坦,术后立即腹腔注射4mg/kgGW9662。将各组大鼠分别于相应时间点进行神经功能评分后断头处死,采用免疫组化、western blot和qR-T PCR来观察脑缺血后PEDF的mRNA和蛋白水平动态变化,TTC染色评价脑梗死体积,干湿重法评价患侧脑水肿。
结果：MCAO组大鼠脑缺血后自24h开始,出现明显的神经功能缺失,持续并进行性加重至所观察到的72h,与正常组比较差异有统计学意义(P<0.05)。脑缺血自24h开始,PEDF表达呈明显减少趋势,持续至本研究所观察到的72h,与正常组比较,差异有统计学意义(P<0.05)。术后24h,替米沙坦干预组与对照组比较,神经功能缺失有明显改善,脑水肿明显减轻、梗死体积明显减小,神经保护因子PEDF表达明显增加,差异有统计学意义(P<0.05)。替米沙坦+GW9662组与替米沙坦组比较,神经功能缺失有明显加重、脑水肿和梗死体积增加,并且PEDF表达明显减少,差异有统计学意义(P<0.05)。溶剂对照组和替米沙坦+GW9662组比较无差别。
结论：首次证明局灶性脑缺血早期,神经保护因子PEDF开始显著减少,可能是神经功能缺失、脑水肿等缺血性脑损伤的原因之一。首次通过大鼠脑缺血模型证明,核转录因子PPARy是PEDF的上游调控基因,上调PEDF是是替米沙坦脑保护机制之一。替米沙坦干预通过激活PPARγ上调PEDF的表达,明显改善了脑缺血的神经功能缺失、脑水肿,减小了梗死体积。
第三部分大鼠脑缺血后血脑屏障通透性变化及替米沙坦血脑屏障保护作用的研究
目的：动态观察大鼠脑缺血后血脑屏障通透性变化,评价替米沙坦对血脑屏障的保护作用及机制。
方法：应用Longa等的线栓法建立MCAO大鼠模型,采用成年健康雄性Sprague-Dawley大鼠,体重230g-250g。实验1：将大鼠随机分为正常组(Normal)和手术组(MCAO),手术组按照MCAO术后不同时间点分为3h、6h、24h、48h、72h5个亚组。实验2：将大鼠随机分为假手术组(Sham)、溶剂对照组(Vehicle)、替米沙坦组(Telmisartan)和替米沙坦+GW9662组(Telmisartan+GW9662).替米沙坦30mg/kg于MCAO手术前1h以灌胃方式给予,以防止大鼠麻醉时呕吐；假手术组和对照组给予等体积的生理盐水；替米沙坦+GW9662组于MCAO手术前1h以灌胃方式给予30mg/kg替米沙坦,术后立即腹腔注射4mg/kgGW9662。血脑屏障通透性通过检测血管内皮细胞紧密连接蛋白成分Claudin-5进行评价。将各组大鼠分别于相应时间点进行神经功能评分后断头处死,采用免疫组化、western blot和qR-T PCR来观察脑缺血后Claudin-5以及MMP-9的mRNA和蛋白水平动态变化,TTC染色评价脑梗死体积,干湿重法评价患侧脑水肿。
结果：MCAO组大鼠脑缺血后自24h开始,出现明显的神经功能缺失,持续并进行性加重至所观察到的72h,与正常组比较差异有统计学意义(P<0.05)。脑缺血自24h开始,Cludin-5表达呈明显减少趋势,持续至本研究所观察到的72h,与正常组比较,差异有统计学意义；此时,MMP-9表达显著升高(P<0.05)。术后24h,替米沙坦干预组与对照组比较,神经功能缺失有明显改善,脑水肿明显减轻、梗死体积明显减小,Cludin-5表达明显增加,MMP-9表达减少,差异有统计学意义(P<0.05)。替米沙坦+GW9662组与替米沙坦组比较,神经功能缺失有明显加重、脑水肿和梗死体积增加,Cludin-5表达明显减少,差异有统计学意义(P<0.05)。溶剂对照组和替米沙坦+GW9662组比较无差别。
结论：局灶性脑缺血早期,血脑屏障通透性显著增加是导致明显的神经功能缺失、脑水肿形成的直接原因。替米沙坦干预通过激活PPARγ、抑制MMP-9、上调Cludin-5的表达,保护了血脑屏障的完整性,因此改善了脑缺血的神经功能缺失、脑水肿,减小了梗死体积。
Stroke is the second commonest cause of death and the leading cause of adult disability worldwide and ischemic stroke accounts for about80%.
Studies showed that cerebral ischemia triggered massive physiopathologic processes including energy metabolism, inflammatory responses, oxidative stress, increased release of excitatory amion acids, etc., which direct/indirect resulting in blood-brain barrier disruption and brain edema formation, as well as neurological deficit. Among those inflammatory responses is one of the most important mechanisms. Nowadays, beyond only a restricted number of hospitalized patients profiting from thrombolytic therapy, the vast majority of patients suffering from stroke resulted in disability even death owing to the limited therapies. Delaying the development of brain damage and protecting neuron are remained.
There a large number of agents have been proved to display anti-inflammation, anti-oxidation and anti-apoptosis properties but never employed in treating cerebral ischemia. Telmisartan, an angiotensin type1receptor blocker (ARB), is a classical medicine controlling hypertention and protecting target organs. It's well established that telmisartan alleviated the infarct volume owing to anti-inflammation property by activating peroxisome prolixferator-activated receptors (PPARy). PPARy, activated by ligands, is a key transcription factor possessing multiple protective roles. So on the basis. telmisartan, the unique ARB as agonist of PPARy, attracted more attention for cerebral ischemia treatment.
Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (pMCAO). The time course expression of PEDF, PPARy, NF-κB, Egr-1, MMP-9, PEDF and Claudin-5were evaluated. Telmisartan (PPARγ agonist) and GW9662(PPARy antagonist) were systemic administrated to explore the effect on PPARγ, PEDF, NF-κB, Egr-1, MMP-9, PEDF and Claudin-5expression at24h after cerebral ischemia by immunohistochemistry, western blot, qRT-PCR. The neurological deficits, brain water content and infarct volume were measured.
The study was divided into three part list as below.
Part I The inflammatory responses after focal cerebral ischemia in rats and the anti-inflammatory role of telmisartan
Object:This study is to evaluate the time course expression of PPARy, NF-κB, Egr-1and MMP-9, then explore the regulation mechanisms of telmisartan on them.
Methods:Male Sprague-Dawley rats,230g～250g weight, were subjected to permanent middle cerebral artery occlusion (pMCAO). Experiment1:SD rats were randomly divided into normal,3h,6h,24h,48h and72h groups after operation. Experiment2:SD rats were randomly divided into4groups:sham-operated group, Vehicle group, Telmisartan group, and Telmisartan+GW9662group. Telmisartan group received telmisartan (Boehringer Ingelheim, Ingelheim, Germany) by feeding tubes at30mg/kg dissolved in PBS1hour before MCAO for avoiding emesis and then once daily thereafter. Vehicle groups received equal volume PBS in the same manner. Telmisartan+GW9662group received telmisartan before operation and GW9662(Sigma-Aldrich, USA) after MCAO immediately injected intraperitoneally at4mg/kg dissolved in3%DMSO diluted in PBS. Neurological deficit scores were evaluated by an examiner blinded to the experimental groups at respective time point. After that, rats were anesthetized with10%chloral hydrate and the brains were quickly removed. The time course expression of PPARy, NF-κB, Egr-1and MMP-9were evaluated by western blot and quantitative real-time PCR. The underling regulation mechanisms at24h were explored by administration of telmisartan and GW9662by immunohistochemistry, western blot and quantitative real-time PCR. Infarct volume was determined by2,3,5-triphenyltetrazolium chloride (TTC) and brain water content was measured using the standard wet-dry method at24h in the4groups.
Results:Compared with normal group, the neurological deficits were obviously exacerbated from24h to72h after MCAO in our study (P<0.05). At the same time, PPARy expression was obviously decreased, but NF-κB, Egr-1and MMP-9expression were increased, relative to those in normal group(P<0.05). Compared with the vehicle group, the neurological deficits, brain water content and infarct volume were dramatically alleviated by telmisartan at24h after MCAO (P<0.05), and the decrease of PPARy was significantly up-regulated and the increase of Egr-1, NF-κB and MMP-9was down-regulated by telmisartan at24h (P<0.05). Telmisartan's profits were all reversed by GW9662co-administration(P<0.05). No significant differences were observed between Vehicle group and Telmisartan+GW9662group animals (P>0.05).
Conclusions:At the early stage of focal cerebral ischemia, the inflammatory responses were triggered and responsible for the neurological deficits. PPARy was the key regular controlling Egr-1, NF-kB and MMP-9expression. Telmisartan down-regulated Egr-1, NF-κB and MMP-9by PPARy signaling, alleviated the neurological deficits, brain water content and infarct volume.
Part Ⅱ The intrinsic PEDF expression after focal cerebral ischemia in rats and the regulation of telmisartan
Object:This study is to evaluate the time course expression of PEDF and explore the underling regulation mechanisms of telmisartan.
Methods:Male Sprague-Dawley rats,230g^250g weight, were subjected to permanent middle cerebral artery occlusion (pMCAO). Experiment1:SD rats were randomly divided into normal,3h,6h,24h,48h and72h groups after operation. Experiment2:SD rats were randomly divided into4groups:sham-operated group, Vehicle group, Telmisartan group, and Telmisartan+GW9662group. Telmisartan group received telmisartan (Boehringer Ingelheim, Ingelheim, Germany) by feeding tubes at30mg/kg dissolved in PBS1hour before MCAO for avoiding emesis and then once daily thereafter. Vehicle groups received equal volume PBS in the same manner. Telmisartan+GW9662group received telmisartan before operation and GW9662(Sigma-Aldrich, USA) after MCAO immediately injected intraperitoneally at4mg/kg dissolved in3%DMSO diluted in PBS. Neurological deficit scores were evaluated by an examiner blinded to the experimental groups at respective time point. After that, rats were anesthetized with10%chloral hydrate and the brains were quickly removed. The time course expression of PEDF was evaluated by western blot and quantitative real-time PCR. The underling regulation mechanisms at24h were explored by administration of telmisartan and GW9662by immunohistochemistry, western blot and quantitative real-time PCR. Infarct volume was determined by2,3,5-triphenyltetrazolium chloride (TTC) and brain water content was measured using the standard wet-dry method at24h in the4groups.
Results:Compared with normal group, the neurological deficits were obviously exacerbated from24h to72h after MCAO in our study (P0.05). At the same time, the intrinsic PEDF expression was obviously decreased, relative to that in normal group (P<0.05). Compared with the vehicle group, the neurological deficits, brain water content and infarct volume were dramatically alleviated by telmisartan at24h after MCAO (P<0.05), and the decrease of the intrinsic PEDF was significantly up-regulated by telmisartan at24h (P<0.05). Telmisartan's profits were all reversed by GW9662co-administration (P<0.05). No significant differences were observed between Vehicle group and Telmisartan+GW9662group animals (P>0.05).
Conclusions:It's the first report that the intrinsic PEDF were reduced at the early stage of focal cerebral ischemia, which was probably responsible for the neurological deficits and brain edema. PPARγ was the key regular controlling the intrinsic PEDF expression. Telmisartan normalizing the intrinsic PEDF by PPARy signal could contribute to the alleviated infarct volume, neurological deficits and brain edema in the study.
Part Ⅲ The permeability of blood-brain barrier after focal cerebral ischemia in rats and the protection of telmisartan
Object:This study is to evaluate the time course expression of Claudin-5and MMP-9and explore the underling regulation mechanisms of telmisartan.
Methods:Male Sprague-Dawley rats,230g-250g weight, were subjected to permanent middle cerebral artery occlusion (pMCAO). Experiment1:SD rats were randomly divided into normal,3h,6h,24h,48h and72h groups after operation. Experiment2:SD rats were randomly divided into4groups:sham-operated group, Vehicle group, Telmisartan group, and Telmisartan+GW9662group. Telmisartan group received telmisartan (Boehringer Ingelheim, Ingelheim, Germany) by feeding tubes at30mg/kg dissolved in PBS1hour before MCAO for avoiding emesis and then once daily thereafter. Vehicle groups received equal volume PBS in the same manner. Telmisartan+GW9662group received telmisartan before operation and GW9662(Sigma-Aldrich, USA) after MCAO immediately injected intraperitoneally at4mg/kg dissolved in3%DMSO diluted in PBS. Neurological deficit scores were evaluated by an examiner blinded to the experimental groups at respective time point. After that, rats were anesthetized with10%chloral hydrate and the brains were quickly removed. The time course expression of Claudin-5and MMP-9were evaluated by western blot and quantitative real-time PCR. The underling regulation mechanisms at24h were explored by administration of telmisartan and GW9662by immunohistochemistry, western blot and quantitative real-time PCR. Infarct volume was determined by2,3,5-triphenyltetrazolium chloride (TTC) and brain water content was measured using the standard wet-dry method at24h in the4groups.
Results:Compared with normal group, the neurological deficits were obviously exacerbated from24h to72h after MCAO in our study(P<0.05). At the same time, the Claudin-5and MMP-9expression was obviously decreased, relative to that in normal group (P<0.05). Compared with the vehicle group, the neurological deficits, brain water content and infarct volume were dramatically alleviated by telmisartan at24h after MCAO (P<0.05), and the decrease of the intrinsic PEDF was significantly up-regulated by telmisartan at24h(P<0.05). Telmisartan's profits were all reversed by GW9662co-administration (P<0.05). No significant differences were observed between Vehicle group and Telmisartan+GW9662group animals (P>0.05).
Conclusions:The blood-brain barrier was dramatically disrupted at the early stage of focal cerebral ischemia, which was responsible for the brain edema. PPARy was the key factor regulating Claudin-5and protecting BBB integrity. Telmisartan normalizing Claudin-5by inhibiting MMP-9through PPARγ signal could contribute to the alleviated infarct volume, neurological deficits and brain edema in the study.
尽管已经有多种药物经过实验研究,证明具有抗炎、抗氧化、抗凋亡等作用,但多数未能在临床得到广泛应用,使理论和实践大大脱节。替米沙坦是一种临床广泛应用的血管紧张素受体阻断剂类(angiotensin type1receptor blocker,ARB)降血压药物。近年来,动物和临床实验发现,替米沙坦不仅能够有效治疗高血压、保护靶器官和减轻心室重构,还能够对脑缺血具有良好的抗炎作用,能够显著减轻脑梗死体积、脑水肿和继发性脑损伤,其机制多与激活过氧化物酶体增殖物激活受体γ(peroxisome prolixferator-activated receptors, PPARy)有关。国内外多项研究证明,PPARγ是一个关键性核转录因子,与其激动剂或者配体结合后活化,具有抗炎、抗氧化、抗凋亡、神经保护等多重作用。替米沙坦是唯一一种具有活化PPARγ功能的ARB类药物。结合基础研究和临床应用的实际需要,替米沙坦可能是治疗缺血性脑血管病的主要药物之一,但替米沙坦的作用机制仍需深入研究。
应用线栓法建立大鼠永久性局灶脑缺血模型,分别对炎症反应代表性因子PPARγ、 NF-κB、Egr-1和MMP-9,神经保护因子PEDF,和以Claudin-5为代表的血脑屏损伤程度等方面进行动态观察,并应用PPARy激动剂替米沙坦和PPARy特异性拮抗剂GW9662进行干预,观察上述各因子的变化,以及评价神经功能缺失、脑水肿和梗死体积等。以PPARy为切入点,深入探讨脑缺血后脑损伤机制及替米沙坦的脑保护作用机制。
本研究将分三部分对上述内容进行探讨,各部分内容概述如下。
第一部分大鼠脑缺血后炎症反应及替米沙坦抗炎作用机制研究
目的：动态观察PPARγ、NF-κB、Egr-1和MMP-9等在大鼠脑缺血后的表达变化,探讨替米沙坦的抗炎作用机制。
方法：应用Longa等的线栓法建立MCAO大鼠模型,采用成年健康雄性Sprague-Dawley大鼠,体重230g-250g。实验1：将大鼠随机分为正常组(Normal)和手术组(MCAO),手术组按照MCAO术后不同时间点分为3h、6h、24h、48h、72h5个亚组。实验2：将大鼠随机分为假手术组(Sham)、溶剂对照组(Vehicle)、替米沙坦组(Telmisartan)及替米沙坦+GW9662组(Telmisartan+GW9662)。替米沙坦30mg/kg于MCAO手术前1h以灌胃方式给予,以防止大鼠麻醉时呕吐；假手术组和对照组给予等体积的生理盐水；替米沙坦+GW9662组于MCAO手术前1h以灌胃方式给予30mg/kg替米沙坦,术后立即腹腔注射4mg/kgGW9662。将各组大鼠分别于相应时间点进行神经功能评分后断头处死,采用免疫组化、western blot和qR-T PCR来观察脑缺血后PPARγ、NF-κB、Egr-1和MMP-9等：mRNA和蛋白水平的动态变化,TTC染色评价脑梗死体积,干湿重法评价患侧脑水肿。
结果：MCAO组大鼠脑缺血后自24h开始,出现明显的神经功能缺失,持续并进行性加重至所观察到的72h,与正常组比较差异有统计学意义(P<0.05)。同时,自脑缺血24h开始,PPARy表达呈明显减少趋势,而NF-κB.Egr-1、MMP-9等呈明显增加趋势,且持续至本研究所观察到的72h,与正常组比较,差异有统计学意义(P<0.05)。术后24h,替米沙坦干预组与溶剂对照组比较,神经功能缺失有明显改善,脑水肿明显减轻、梗死体积明显减小,保护性因子PPARγ等表达明显增加,而NF-κB、Egr-1、MMP-9等明显减少,差异有统计学意义(P<0.05)。替米沙坦+GW9662组与替米沙坦组比较,神经功能缺失有明显加重、脑水肿和梗死体积增加,并且PPARy表达明显减少,而NF-κB、Egr-1、MMP-9等明显增加,差异有统计学意义(P<0.05)。溶剂对照组和替米沙坦+GW9662组比较无差别。
结论：大鼠脑缺血早期炎症反应迅速出现,脑缺血后24h开始明显出现,并持续至所观察的72h,炎症反应程度与神经功能缺损程度具有相关性。替米沙坦单独和联合GW9662干预表明,PPARγ是炎症反应的关键性调控基因。替米沙坦通过激活PPARγ,下调NF-кB、Egr-1和MMP-9,显著抑制了炎症反应,减轻脑缺血体积和脑水肿,改善了神经功能缺失。
第二部分大鼠脑缺血后PEDF表达变化及替米沙坦调节机制的研究
目的：动态观察大鼠脑缺血后PEDF的表达变化,探讨替米沙坦的脑保护作用机制。方法：应用Longa等的线栓法建立MCAO大鼠模型,采用成年健康雄性Sprague-Dawley大鼠,体重230g～250g。实验1：将大鼠随机分为正常组(Normal)和手术组(MCAO),手术组按照MCAO术后不同时间点分为3h、6h、24h、48h、72h5个亚组。实验2：将大鼠随机分为假手术组(Sham)、溶剂对照组(Vehicle)、替米沙坦组(Telmisartan)和替米沙坦+GW9662组(Telmisartan+GW9662)。替米沙坦30mg/kg于MCAO手术前1h以灌胃方式给予,以防止大鼠麻醉时呕吐；假手术组和对照组给予等体积的生理盐水；替米沙坦+GW9662组于MCAO手术前1h以灌胃方式给予30mg/kg替米沙坦,术后立即腹腔注射4mg/kgGW9662。将各组大鼠分别于相应时间点进行神经功能评分后断头处死,采用免疫组化、western blot和qR-T PCR来观察脑缺血后PEDF的mRNA和蛋白水平动态变化,TTC染色评价脑梗死体积,干湿重法评价患侧脑水肿。
结果：MCAO组大鼠脑缺血后自24h开始,出现明显的神经功能缺失,持续并进行性加重至所观察到的72h,与正常组比较差异有统计学意义(P<0.05)。脑缺血自24h开始,PEDF表达呈明显减少趋势,持续至本研究所观察到的72h,与正常组比较,差异有统计学意义(P<0.05)。术后24h,替米沙坦干预组与对照组比较,神经功能缺失有明显改善,脑水肿明显减轻、梗死体积明显减小,神经保护因子PEDF表达明显增加,差异有统计学意义(P<0.05)。替米沙坦+GW9662组与替米沙坦组比较,神经功能缺失有明显加重、脑水肿和梗死体积增加,并且PEDF表达明显减少,差异有统计学意义(P<0.05)。溶剂对照组和替米沙坦+GW9662组比较无差别。
结论：首次证明局灶性脑缺血早期,神经保护因子PEDF开始显著减少,可能是神经功能缺失、脑水肿等缺血性脑损伤的原因之一。首次通过大鼠脑缺血模型证明,核转录因子PPARy是PEDF的上游调控基因,上调PEDF是是替米沙坦脑保护机制之一。替米沙坦干预通过激活PPARγ上调PEDF的表达,明显改善了脑缺血的神经功能缺失、脑水肿,减小了梗死体积。
第三部分大鼠脑缺血后血脑屏障通透性变化及替米沙坦血脑屏障保护作用的研究
目的：动态观察大鼠脑缺血后血脑屏障通透性变化,评价替米沙坦对血脑屏障的保护作用及机制。
方法：应用Longa等的线栓法建立MCAO大鼠模型,采用成年健康雄性Sprague-Dawley大鼠,体重230g-250g。实验1：将大鼠随机分为正常组(Normal)和手术组(MCAO),手术组按照MCAO术后不同时间点分为3h、6h、24h、48h、72h5个亚组。实验2：将大鼠随机分为假手术组(Sham)、溶剂对照组(Vehicle)、替米沙坦组(Telmisartan)和替米沙坦+GW9662组(Telmisartan+GW9662).替米沙坦30mg/kg于MCAO手术前1h以灌胃方式给予,以防止大鼠麻醉时呕吐；假手术组和对照组给予等体积的生理盐水；替米沙坦+GW9662组于MCAO手术前1h以灌胃方式给予30mg/kg替米沙坦,术后立即腹腔注射4mg/kgGW9662。血脑屏障通透性通过检测血管内皮细胞紧密连接蛋白成分Claudin-5进行评价。将各组大鼠分别于相应时间点进行神经功能评分后断头处死,采用免疫组化、western blot和qR-T PCR来观察脑缺血后Claudin-5以及MMP-9的mRNA和蛋白水平动态变化,TTC染色评价脑梗死体积,干湿重法评价患侧脑水肿。
结果：MCAO组大鼠脑缺血后自24h开始,出现明显的神经功能缺失,持续并进行性加重至所观察到的72h,与正常组比较差异有统计学意义(P<0.05)。脑缺血自24h开始,Cludin-5表达呈明显减少趋势,持续至本研究所观察到的72h,与正常组比较,差异有统计学意义；此时,MMP-9表达显著升高(P<0.05)。术后24h,替米沙坦干预组与对照组比较,神经功能缺失有明显改善,脑水肿明显减轻、梗死体积明显减小,Cludin-5表达明显增加,MMP-9表达减少,差异有统计学意义(P<0.05)。替米沙坦+GW9662组与替米沙坦组比较,神经功能缺失有明显加重、脑水肿和梗死体积增加,Cludin-5表达明显减少,差异有统计学意义(P<0.05)。溶剂对照组和替米沙坦+GW9662组比较无差别。
结论：局灶性脑缺血早期,血脑屏障通透性显著增加是导致明显的神经功能缺失、脑水肿形成的直接原因。替米沙坦干预通过激活PPARγ、抑制MMP-9、上调Cludin-5的表达,保护了血脑屏障的完整性,因此改善了脑缺血的神经功能缺失、脑水肿,减小了梗死体积。
Stroke is the second commonest cause of death and the leading cause of adult disability worldwide and ischemic stroke accounts for about80%.
Studies showed that cerebral ischemia triggered massive physiopathologic processes including energy metabolism, inflammatory responses, oxidative stress, increased release of excitatory amion acids, etc., which direct/indirect resulting in blood-brain barrier disruption and brain edema formation, as well as neurological deficit. Among those inflammatory responses is one of the most important mechanisms. Nowadays, beyond only a restricted number of hospitalized patients profiting from thrombolytic therapy, the vast majority of patients suffering from stroke resulted in disability even death owing to the limited therapies. Delaying the development of brain damage and protecting neuron are remained.
There a large number of agents have been proved to display anti-inflammation, anti-oxidation and anti-apoptosis properties but never employed in treating cerebral ischemia. Telmisartan, an angiotensin type1receptor blocker (ARB), is a classical medicine controlling hypertention and protecting target organs. It's well established that telmisartan alleviated the infarct volume owing to anti-inflammation property by activating peroxisome prolixferator-activated receptors (PPARy). PPARy, activated by ligands, is a key transcription factor possessing multiple protective roles. So on the basis. telmisartan, the unique ARB as agonist of PPARy, attracted more attention for cerebral ischemia treatment.
Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (pMCAO). The time course expression of PEDF, PPARy, NF-κB, Egr-1, MMP-9, PEDF and Claudin-5were evaluated. Telmisartan (PPARγ agonist) and GW9662(PPARy antagonist) were systemic administrated to explore the effect on PPARγ, PEDF, NF-κB, Egr-1, MMP-9, PEDF and Claudin-5expression at24h after cerebral ischemia by immunohistochemistry, western blot, qRT-PCR. The neurological deficits, brain water content and infarct volume were measured.
The study was divided into three part list as below.
Part I The inflammatory responses after focal cerebral ischemia in rats and the anti-inflammatory role of telmisartan
Object:This study is to evaluate the time course expression of PPARy, NF-κB, Egr-1and MMP-9, then explore the regulation mechanisms of telmisartan on them.
Methods:Male Sprague-Dawley rats,230g～250g weight, were subjected to permanent middle cerebral artery occlusion (pMCAO). Experiment1:SD rats were randomly divided into normal,3h,6h,24h,48h and72h groups after operation. Experiment2:SD rats were randomly divided into4groups:sham-operated group, Vehicle group, Telmisartan group, and Telmisartan+GW9662group. Telmisartan group received telmisartan (Boehringer Ingelheim, Ingelheim, Germany) by feeding tubes at30mg/kg dissolved in PBS1hour before MCAO for avoiding emesis and then once daily thereafter. Vehicle groups received equal volume PBS in the same manner. Telmisartan+GW9662group received telmisartan before operation and GW9662(Sigma-Aldrich, USA) after MCAO immediately injected intraperitoneally at4mg/kg dissolved in3%DMSO diluted in PBS. Neurological deficit scores were evaluated by an examiner blinded to the experimental groups at respective time point. After that, rats were anesthetized with10%chloral hydrate and the brains were quickly removed. The time course expression of PPARy, NF-κB, Egr-1and MMP-9were evaluated by western blot and quantitative real-time PCR. The underling regulation mechanisms at24h were explored by administration of telmisartan and GW9662by immunohistochemistry, western blot and quantitative real-time PCR. Infarct volume was determined by2,3,5-triphenyltetrazolium chloride (TTC) and brain water content was measured using the standard wet-dry method at24h in the4groups.
Results:Compared with normal group, the neurological deficits were obviously exacerbated from24h to72h after MCAO in our study (P<0.05). At the same time, PPARy expression was obviously decreased, but NF-κB, Egr-1and MMP-9expression were increased, relative to those in normal group(P<0.05). Compared with the vehicle group, the neurological deficits, brain water content and infarct volume were dramatically alleviated by telmisartan at24h after MCAO (P<0.05), and the decrease of PPARy was significantly up-regulated and the increase of Egr-1, NF-κB and MMP-9was down-regulated by telmisartan at24h (P<0.05). Telmisartan's profits were all reversed by GW9662co-administration(P<0.05). No significant differences were observed between Vehicle group and Telmisartan+GW9662group animals (P>0.05).
Conclusions:At the early stage of focal cerebral ischemia, the inflammatory responses were triggered and responsible for the neurological deficits. PPARy was the key regular controlling Egr-1, NF-kB and MMP-9expression. Telmisartan down-regulated Egr-1, NF-κB and MMP-9by PPARy signaling, alleviated the neurological deficits, brain water content and infarct volume.
Part Ⅱ The intrinsic PEDF expression after focal cerebral ischemia in rats and the regulation of telmisartan
Object:This study is to evaluate the time course expression of PEDF and explore the underling regulation mechanisms of telmisartan.
Methods:Male Sprague-Dawley rats,230g^250g weight, were subjected to permanent middle cerebral artery occlusion (pMCAO). Experiment1:SD rats were randomly divided into normal,3h,6h,24h,48h and72h groups after operation. Experiment2:SD rats were randomly divided into4groups:sham-operated group, Vehicle group, Telmisartan group, and Telmisartan+GW9662group. Telmisartan group received telmisartan (Boehringer Ingelheim, Ingelheim, Germany) by feeding tubes at30mg/kg dissolved in PBS1hour before MCAO for avoiding emesis and then once daily thereafter. Vehicle groups received equal volume PBS in the same manner. Telmisartan+GW9662group received telmisartan before operation and GW9662(Sigma-Aldrich, USA) after MCAO immediately injected intraperitoneally at4mg/kg dissolved in3%DMSO diluted in PBS. Neurological deficit scores were evaluated by an examiner blinded to the experimental groups at respective time point. After that, rats were anesthetized with10%chloral hydrate and the brains were quickly removed. The time course expression of PEDF was evaluated by western blot and quantitative real-time PCR. The underling regulation mechanisms at24h were explored by administration of telmisartan and GW9662by immunohistochemistry, western blot and quantitative real-time PCR. Infarct volume was determined by2,3,5-triphenyltetrazolium chloride (TTC) and brain water content was measured using the standard wet-dry method at24h in the4groups.
Results:Compared with normal group, the neurological deficits were obviously exacerbated from24h to72h after MCAO in our study (P0.05). At the same time, the intrinsic PEDF expression was obviously decreased, relative to that in normal group (P<0.05). Compared with the vehicle group, the neurological deficits, brain water content and infarct volume were dramatically alleviated by telmisartan at24h after MCAO (P<0.05), and the decrease of the intrinsic PEDF was significantly up-regulated by telmisartan at24h (P<0.05). Telmisartan's profits were all reversed by GW9662co-administration (P<0.05). No significant differences were observed between Vehicle group and Telmisartan+GW9662group animals (P>0.05).
Conclusions:It's the first report that the intrinsic PEDF were reduced at the early stage of focal cerebral ischemia, which was probably responsible for the neurological deficits and brain edema. PPARγ was the key regular controlling the intrinsic PEDF expression. Telmisartan normalizing the intrinsic PEDF by PPARy signal could contribute to the alleviated infarct volume, neurological deficits and brain edema in the study.
Part Ⅲ The permeability of blood-brain barrier after focal cerebral ischemia in rats and the protection of telmisartan
Object:This study is to evaluate the time course expression of Claudin-5and MMP-9and explore the underling regulation mechanisms of telmisartan.
Methods:Male Sprague-Dawley rats,230g-250g weight, were subjected to permanent middle cerebral artery occlusion (pMCAO). Experiment1:SD rats were randomly divided into normal,3h,6h,24h,48h and72h groups after operation. Experiment2:SD rats were randomly divided into4groups:sham-operated group, Vehicle group, Telmisartan group, and Telmisartan+GW9662group. Telmisartan group received telmisartan (Boehringer Ingelheim, Ingelheim, Germany) by feeding tubes at30mg/kg dissolved in PBS1hour before MCAO for avoiding emesis and then once daily thereafter. Vehicle groups received equal volume PBS in the same manner. Telmisartan+GW9662group received telmisartan before operation and GW9662(Sigma-Aldrich, USA) after MCAO immediately injected intraperitoneally at4mg/kg dissolved in3%DMSO diluted in PBS. Neurological deficit scores were evaluated by an examiner blinded to the experimental groups at respective time point. After that, rats were anesthetized with10%chloral hydrate and the brains were quickly removed. The time course expression of Claudin-5and MMP-9were evaluated by western blot and quantitative real-time PCR. The underling regulation mechanisms at24h were explored by administration of telmisartan and GW9662by immunohistochemistry, western blot and quantitative real-time PCR. Infarct volume was determined by2,3,5-triphenyltetrazolium chloride (TTC) and brain water content was measured using the standard wet-dry method at24h in the4groups.
Results:Compared with normal group, the neurological deficits were obviously exacerbated from24h to72h after MCAO in our study(P<0.05). At the same time, the Claudin-5and MMP-9expression was obviously decreased, relative to that in normal group (P<0.05). Compared with the vehicle group, the neurological deficits, brain water content and infarct volume were dramatically alleviated by telmisartan at24h after MCAO (P<0.05), and the decrease of the intrinsic PEDF was significantly up-regulated by telmisartan at24h(P<0.05). Telmisartan's profits were all reversed by GW9662co-administration (P<0.05). No significant differences were observed between Vehicle group and Telmisartan+GW9662group animals (P>0.05).
Conclusions:The blood-brain barrier was dramatically disrupted at the early stage of focal cerebral ischemia, which was responsible for the brain edema. PPARy was the key factor regulating Claudin-5and protecting BBB integrity. Telmisartan normalizing Claudin-5by inhibiting MMP-9through PPARγ signal could contribute to the alleviated infarct volume, neurological deficits and brain edema in the study.
引文
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1 Auriel E, Bornstein NM. Neuroprotection in acute ischemic stroke-current status. J Cell Mol Med,2010,14(9):2200-2202
2 Chavez JC, Hurko O, Barone FC, et al. Pharmacologic interventions for stroke:looking beyond the thrombolysis time window into the penumbra with biomarkers, not a stopwatch. Stroke,2009,40(10):e558-563
3 Toth G,Albers GW. Use of MRI to estimate the therapeutic window in acute stroke:is perfusion-weighted imaging/diffusion-weighted imaging mismatch an EPITHET for salvageable ischemic brain tissue? Stroke, 2009,40(1):333-335
4 Bilak MM, Corse AM, Bilak SR, et al. Pigment epithelium-derived factor (PEDF) protects motor neurons from chronic glutamate-mediated neurodegeneration. J Neuropathol Exp Neurol,1999,58(7):719-728
5 Sanagi T, Yabe T, Yamada H. Gene transfer of PEDF attenuates ischemic brain damage in the rat middle cerebral artery occlusion model. J Neurochem,2008,106(4):1841-1854
6 DeCoster MA, Schabelman E, Tombran-Tink J, et al. Neuroprotection by pigment epithelial-derived factor against glutamate toxicity in developing primary hippocampal neurons. J Neurosci Res,1999,56(6):604-610
7 Falk T, Zhang S, Sherman SJ. Pigment epithelium derived factor (PEDF) is neuroprotective in two in vitro models of Parkinson's disease. Neurosci Lett,2009,458(2):49-52
8 Taniwaki T, Becerra SP, Chader GJ, et al. Pigment epithelium-derived factor is a survival factor for cerebellar granule cells in culture. J Neurochem,1995,64(6):2509-2517
9 Houenou LJ, D'Costa AP, Li L, et al. Pigment epithelium-derived factor promotes the survival and differentiation of developing spinal motor neurons. J Comp Neurol,1999,412:506-514
10 Takita H, Yoneya S, Gehlbach PL, et al. Retinal neuroprotection against ischemic injury mediated by intraocular gene transfer of pigment epithelium-derived factor. Invest Ophthalmol Vis Sci,2003,44: 4497-4504
11 Drevon CA. Fatty acids and expression of adipokines. Biochim Biophys Acta,2005,1740(2):287-292
12 Sawant S, Aparicio S, Tink AR, et al. Regulation of factors controlling angiogenesis in liver development:a role for PEDF in the formation and maintenance of normal vasculature. Biochem Biophys Res Commun,2004, 325(2):408-413
13 Quan GM, Ojaimi J, Li Y, et al. Localization of pigment epithelium-derived factor in growing mouse bone. Calcif Tissue Int,2005, 76(2):146-153
14 Tombran-Tink J, Mazuruk K, Rodriguez IR, et al. Organization, evolutionary conservation, expression and unusual Alu density of the human gene for pigment epithelium-derived factor, a unique neurotrophic serpin. Mol Vis,1996,2:11
15 Ho TC, Chen SL, Yang YC, et al. PEDF induces p53-mediated apoptosis through PPAR gamma signaling in human umbilical vein endothelial cells. Cardiovasc Res,2007,76:213-223
16 Ho TC, Yang YC, Chen SL, et al. Pigment epithelium-derived factor induces THP-1 macrophage apoptosis and necrosis by the induction of the peroxisome proliferator-activated receptor gamma. Mol Immunol,2007, 45:898-909
17 Ho TC, Chen SL, Shih SC, et al. Pigment epithelium-derived factor (PEDF) promotes tumor cell death by inducing macrophage membrane tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). J Biol Chem,2011,286:35943-35954
18 Ho TC, Chen SL, Shih SC, et al. Pigment epithelium-derived factor is an intrinsic antifibrosis factor targeting hepatic stellate cells. Am J Pathol, 2010,177:1798-1811
19 Wang SH, Liang CJ, Wu JC, et al. Pigment epithelium-derived factor reduces the PDGF-induced migration and proliferation of human aortic smooth muscle cells through PPARy activation. Int J Biochem Cell Biol, 2012,44:280-289
20 Xu X, Zhang SS, Barnstable CJ, et al. Molecular phylogeny of the antiangiogenic and neurotrophic serpin, pigment epithelium derived factor in vertebrates. BMC Genomics,2006,7:248
21 Alberdi E, Aymerich MS, Becerra SP. Binding of pigment epithelium-derived factor (PEDF) to retinoblastoma cells and cerebellar granule neurons. Evidence for a PEDF receptor. J Biol Chem,1999,274: 31605-31612
22 Aymerich MS, Alberdi EM, Martinez A, et al. Evidence for pigment epithelium-derived factor receptors in the neural retina. Invest Ophthalmol Vis Sci,2001,42:3287-3293
23 Ogata N, Wang L, Jo N, et al. Pigment epithelium derived factor as a neuroprotective agent against ischemic retinal injury. Curr Eye Res,2001; 22:245-252
24 Takita H, Yoneya S, Gehlbach PL, et al. Invest Ophthalmol Vis Sci.2003 Oct;44(10):4497-504. Retinal neuroprotection against ischemic injury mediated by intraocular gene transfer of pigment epithelium-derived factor
25 Notari L, Miller A, Martinez A, et al. Pigment epithelium-derived factor is a substrate for matrix metalloproteinase type 2 and type 9:implications for downregulation in hypoxia. Invest Ophthalmol Vis Sci,2005,46(8): 2736-2747
26 Dong X, Song YN, Liu WG, et al. Mmp-9, a potential target for cerebral ischemic treatment. Curr Neuropharmacol,2009,7:269-275
27 Hu Q, Chen C, Khatibi NH, et al. Lentivirus-mediated transfer of MMP-9 shRNA provides neuroprotection following focal ischemic brain injury in rats. Brain Res,2011,1367:347-359
28 Yabe T, Wilson D, Schwartz JP. NF-κB activation is required for the neuroprotective effects of pigment epithelium-derived factor (PEDF) on cerebellar granule neurons. J Biol Chem,2001; 276:43313-43319
29 Pang IH, Zeng H, Fleenor DL, et al. Pigment epithelium-derived factor protects retinal ganglion cells. BMC Neurosci.2007,8:11
30 Yan S F, Fujita T, Lu J, et al. Egr-1, a master switch coordinating upregulation of divergent gene families underlying ischemic stress. Nature Med,2000,6:1355-1361
31 Nurmi A, Lindsberg PJ, Koistinaho M, et al. Nuclear factor-kappaB contributes to infarction after permanent focal ischemia. Stroke,2004,35: 987-991
32 Zhang HL, Xu M, Wei C, et al. Neuroprotective effects of pioglitazone in a rat model of permanent focal cerebral ischemia are associated with peroxisome proliferator-activated receptor gamma-mediated suppression of nuclear factor-KB signaling pathway. Neuroscience,2011,176:381-395
33 Lv B, Wang H, Tang Y, et al. High-mobility group box 1 protein induces tissue factor expression in vascular endothelial cells via activation of NF-kappaB and Egr-1. Thromb Haemost,2009,102:352-359
34 Blessing E, Preusch M, Kranzhofer R, et al. Anti-atherosclerotic properties of telmisartan in advanced atherosclerotic lesions in apolipoprotein E deficient mice. Atherosclerosis,2008,199:295-303
35 Okada M, Harada T, Kikuzuki R, et al. Effects of telmisartan on right ventricular remodeling induced by monocrotaline in rats. J Pharmacol Sci, 2009,111:193-200
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