P2X7受体在全脑缺血复灌损伤中的作用及其机制研究
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摘要
研究背景：
缺血性脑血管病危害人类健康,目前已经成为我国居民的第一大死亡原因。随着人口老龄化加剧,缺血性脑血管病的发病率逐年增高。在心脏骤停、溺水以及手术失血过多导致的严重低血压等急诊事件中,全脑缺血是最常见并且最严重的并发症。经常遗留严重的神经功能缺损。目前,治疗全脑缺血最有效的方法是尽快的恢复大脑的血供,临床上常采用药物或者介入手段改善脑部循环,但在大脑灌注恢复过程中钙超载、氧自由基过饱和等产生的兴奋性毒性作用反而可能会进一步加重脑组织的损伤,称为脑缺血再灌注损伤(Cerebral ischemia/reperfusion injury, cerebral I/R injury)。所以目前对于治疗脑缺血的研究不应只局限在如何恢复和保留脑部血流,也应该针对缺血再灌注损伤的机制采取措施。
脑缺血后病变以神经元损伤和脑组织炎症为主要特点,其中脑组织炎症又是以星形胶质细胞、小胶质细胞的激活和细胞毒性物质(如细胞因子、基质金属蛋白酶、一氧化氮和活性氧物质等)的释放为特点,这些毒性物质可以导致血脑屏障破坏和神经元死亡,从而产生更多的细胞毒性物质激活更多的免疫细胞,形成恶性循环,加重炎症反应和脑损伤。因此针对脑缺血再灌注损伤后炎症机制的关键环节加以干预可能成为治疗脑缺血再灌注损伤的有效途径。
P2X7受体(P2X7receptor, P2X7R)是一种嘌呤受体,首先在巨噬细胞中被发现。在中枢神经系统(central nervous system,CNS)中主要表达在小胶质细胞中,P2X7R可以被较高浓度的ATP激活继而导致小胶质细胞的活化、活性氧物质释放以及一些前炎症因子如白介素-1β (Interleukin-1β, IL-1β),肿瘤坏死因子-α (Tumor necrosis factor-α, TNF-α)和白介素-6(Interleukin-6, IL-6)等的释放。近年来,研究提示P2X7R参与了多种中枢神经系统疾病的发生和发展如阿尔兹海默病(Alzheimer's disease, AD)、癫痫、脊髓损伤以及多发性硬化等。应用P2X7R阻断剂可以减轻这些疾病模型中的炎症反应。但P2X7R是否参与脑缺血再灌注损伤,是否调节脑缺血再灌注损伤后的炎症反应以及其机制,有关的研究较少,并且存在争议,值得深入探索。
研究目的：
本文拟观察P2X7R对脑缺血再灌注损伤诱导的神经元死亡及炎症反应的影响,做以下3个方面的研究：
(1)制备大鼠全脑缺血再灌注损伤模型,观察全脑缺血再灌注损伤敏感部位海马CA1区P2X7R的表达分布变化。
(2)应用P2X7R阻断剂干预,观察P2X7R在全脑缺血再灌注损伤模型中的作用及其对炎症水平的调节。
(3)探索P2X7R与NADPH氧化酶(NADPH Oxidiase, NOX)之间的相互作用以及其对炎症水平的调节。
第一部分P2X7R在大鼠全脑缺血再灌注损伤模型中的表达变化
研究方法：取体重280g-360g的成年SD雄性大鼠,应用四血管法(four-vessel occlusion,4-VO)建立全脑缺血再灌注损伤模型,缺血时间20分钟,使用激光多普勒脑血流仪检测缺血前10分钟到复灌后90分钟内大鼠区域脑血流(regional cerebral blood flow, rCBF)的变化情况,再灌注时间选取6小时,1天,3天,7天,14天。在上述时间点进行HE染色观察海马CA1区神经元死亡；免疫组织化学法观察星形胶质细胞(GFAP+)、小胶质细胞(Iba-1+)在海马CA1区的分布变化,免疫组织化学法和、Western blot法观察P2X7R在在海马CA1区的表达、分布变化。
结果：四血管法全脑缺血可以有效降低大鼠区域脑血流至20%以下,20min缺血复灌损伤7天后海马CA1区神经元90%发生死亡,随着复灌时间的延长星形胶质细胞及小胶质细胞增生活化明显增多。缺血复灌损伤后P2X7R在海马CA1区表达明显增加,主要表达在小胶质细胞样细胞中。
结论：P2X7R参与全脑缺血再灌注损伤,并与损伤后炎症有关。
第二部分P2X7R抑制剂通过抑制炎症反应减轻全脑全脑缺血再灌注损伤
研究方法：在全脑缺血再灌注损伤前分别给予大鼠生理盐水(Saline); P2X7R阻断剂BBG (Brilliant blue G)、OxATP (adenosine5'-triphosphate-2',3'-dialdehyde)以及A-438079侧脑室注射,统计7天动物死亡率,HE染色观察海马复灌7天时CA1区神经元死亡,TUNEL染色观察复灌3天时CA1区神经元DNA断裂,Morris水迷宫检测大鼠学习记忆能力,免疫组织化学法检测复灌3天时胶质细胞增生,使用RT-PCR方法检测三种细胞因子IL-1B,TNF-a和IL-6复灌3天时在海马中的表达变化,使用丙二醛(Malondialdehyde, MDA)试剂盒检测复灌3天时海马内脂质过氧化水平。
结果：三种P2X7R阻断剂均有剂量依赖的脑缺血再灌注损伤保护作用,与生理盐水组相比,阻断剂组大鼠生存率增加；Morris水迷宫显示BBG和A-438079组大鼠训练第二天后逃避潜伏期较生理盐水组明显缩短,OxATP组大鼠训练4天后逃避潜伏期较生理盐水组明显缩短,空间探索实验中生理盐水组在平台象限逗留时间较三种阻断剂组均缩短；HE染色提示阻断剂组大鼠海马CA1区神经元死亡率较生理盐水组减少,以BBG和A-438079两种抑制剂效果更为明显；三种阻断剂组大鼠CA1区神经元DNA断裂均减少；星形胶质细胞和小胶质细胞的激活以及细胞因子表达也较生理盐水组下降,A-438079组大鼠海马中MDA含量较生理盐水组下降。
结论：阻断P2X7R通过抑脑内炎症反应减轻全脑缺血再灌注损伤。
第三部分P2X7R-NOX参与调节全脑缺血再灌注损伤后炎症反应
研究方法：将大鼠分为假手术(Shaml, Sham2)组、全脑缺血复灌(Saline, Vehicle)组、侧脑室注射A-438079组以及腹腔注射NOX抑制剂Apocynin组。HE染色观察海马CA1区神经元存活；western bolt方法观察NOX的两种亚基gp91phox以及p47phox蛋白表达变化；Lucigenin化学发光法检测海马内NOX活性；免疫组织化学法检测复灌3天时胶质细胞增生。
结果：全脑缺血再灌注损伤后脑内NOX被激活,表现在NOX酶活性增强和gp91phox, p47phox蛋白表达升高以及p47phox蛋白的向膜转位。A-438079和apocynin预处理均可以抑制全脑缺血再灌注损伤诱导的NOX激活。Apocynin可以减少全脑缺血再灌注损伤诱导的海马CA1区神经元死亡和星形胶质细胞、小胶质细胞的增生活化。
结论：P2X7R-NOX参与全脑缺血再灌注损伤以及损伤后炎症调节。
Background
Transient global cerebral ischemia is one of the major complications of clinical emergencies such as cardiac arrest, drowning or severe systemic hypotension during a surgical procedure. Currently, the most adequate treatment for these patients is re-establishing perfusion of the brain as soon as possible. However, reperfusion may
paradoxically exacerbate brain injury, which is called cerebral ischemia/reperfusion (I/R) injury. Therefore, efforts need to be made that not only to preserve cerebral blood flow, but also to prevent the actual mechanisms that trigger brain damage after I/R injury.
Neuroinflammation, which is characterized by microglial and astroglial activation, as well as the release of cytotoxic agents (cytokines, matrix metalloproteinases, nitric oxide and reactive oxygen species) can be triggered by cerebral I/R injury, which can contribute to blood-brain barrier disruption and delayed neuronal death. Subsequently, these damaged cells release more toxic mediators, which in turn activate more immune cells. Thus, prolonged inflammation caused by this vicious circle exacerbates brain damage. Taken together, anti-inflammation therapy may become a promising therapeutic strategy for the treatment of cerebral I/R injury.
The P2X7receptor(P2X7R), a purinergic receptor, was first discovered in macrophages. In the central nervous system (CNS), the P2X7R is predominantly expressed in microglia which are the resident macrophages of the brain. The P2X7R can be activated by high concentrations of ATP. Stimulating the P2X7R leads to microglial activation, reactive oxygen species production and increased secretion of pro-inflammatory cytokines such as IL-1β, TNF-α and IL-6. Recently, the P2X7R has been reported to be involved in neuroinflammation in many CNS diseases
including A lzheimer's disease (AD), epilepsy, spinal cord injury and multiple sclerosis, and treatment with P2X7R antagonists reduces experimentally induced neuroinflammation in animal models of such diseases.
The P2X7receptor(P2X7R), a purinergic receptor, was first discovered in macrophages. In the central nervous system (CNS), the P2X7R is predominantly expressed in microglia which are the resident macrophages of the brain. The P2X7R can be activated by high concentrations of ATP. Stimulating the P2X7R leads to microglial activation, reactive oxygen species production and increased secretion of pro-inflammatory cytokines such as Interleukin-1β(IL-1β), Tumor necrosis factor-α (TNF-a) and Interleukin-6(IL-6). Recently, the P2X7R has been reported to be involved in neuroinflammation in many CNS diseases including Alzheimer's disease (AD), epilepsy, spinal cord injury and multiple sclerosis, and treatment with P2X7R antagonists reduces experimentally induced neuroinflammation in animal models of such diseases. Only a few study have reported the effect of P2X7R in cerebral ischemic injury, and the results are inconsistent. So it is still worth to be discussed.
Aim
To clarify the role of P2X7R in global cerebral I/R-induced injury and neruoinflammation, in the present study we performed the following investigations.
(1) We examined the P2X7R expression of P2X7R after global cerebral I/R-induced injury in rats.
(2) Using P2X7R antagonist, we investigated the role of P2X7R in global cerebral I/R injury. We also explored the association between the P2X7R and neuroinflammation after transient global cerebral I/R injury.
(3) We also investigated the interaction between P2X7R and NADPH Oxidiase(NOX), and thier effect in the regulation on neuroinflammation.
Part Ⅰ Expression properties of P2X7R in the hippocampal CA1region after global cerebral I/R injury.
Methods:Twenty mins global cerebral I/R injury was conducted on male Sprague-Dawley rats weighing260-320g using four-vessel occlusion (4-VO) method. Regional cerebral blood flow (rCBF) from10mins before ischemia to90mins after reperfusion was measured using a laser Doppler flowmetry; HE staining was performing to observe the neuronal death6hours,1day,3days,7days and14days after reperfusion; activated microglia and astrocytes were detected by immunohistochemical staining; P2X7R expression was examined by western bolts and immunobistochemical staining.
Results:During the ischemic period, the rCBF was lower than20%; more than90%CA1neurons dead after3days-reperfusion. glial activation, gliosis and P2X7R expression were increased in a time-dependent manner after reperfusion. P2X7R mainly expressed on microglia-like cells.
Conclusion:P2X7R is involved in global cerebral I/R injury and is associated with I/R-induced neuroinflammation.
Part Ⅱ Inhibition of P2X7R ameliorates transient global cerebral I/R injury via modulating inflammatory responses in the rat hippocampus
Methods:Immediately after infusion with the P2X7R antagonists Brilliant blue G (BBG), adenosine5'-triphosphate-2',3'-dialdehyde (OxATP) or A-438079, tweenty minutes of transient global cerebral I/R was induced using the4-VO method in rats. Survival rate was calculated, neuronal death in the hippocampal CA1region was observed using HE staining, and DNA cleavage was observed by deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL). In addition, behavioral deficits were measured using the Morris water maze, and RT-PCR and immunohistochemical staining were performed to measure the expression of IL-1β,TNF-α and IL-6, and to identify activated microglia and astrocytes.
Results:The P2X7R antagonists protected against transient global cerebral I/R injury in a dosage-dependent manner. A high dosage of BBG (10μg) and A-438079(3μg). and a low dosage of OxATP (1μg) significantly increased survival rates, reduced I/R-induced learning memory deficit, and reduced I/R-induced neuronal death, DNA cleavage, glial activation and inflammatory cytokine overexpression in the hippocampus.
Conclusions:Our study indicates that inhibiting P2X7R.S protects against transient global cerebral I/R injury by reducing the I/R-induced inflammatory response, which suggests inhibition of P2X7Rs may be a promising therapeutic strategy for clinical treatment of transient global cerebral I/R injury.
Part Ⅲ P2X7R-NOX participates in transient global cerebral I/R-induced neuroinflammation
Methods:Rats were divided into six group:Sham1, Sham2, Saline, Vehicle, A-438079(3μg i.c.v.), Apocynin (10mg/kg i.p. twice). NOX activity was measured with Lucigenin-enhanced chemiluminescence; expression of gp91phox and p47phox, p47phox translocation were detected by western bloting; neuronal death in the hippocampal CA1region was observed using HE staining and immunohistochemical staining were performed to identify activated microglia and astrocytes.
Results:NOX activity was elevated at the third day after global cerebral ischemia reflected by elevated relative light units (RLU) values, and increased gp91phox, p47phox expression and less p47phox translocation. The P2X7R antagonists A-438079suppressed the I/R-induced NOX activity elevation. NOX antagonist apocynin protected against transient global cerebral I/R injury and reduced I/R-induced glial activation.
Conclusions:P2X7R-NOX participated in the I/R-induced brain injury and neurolinflammation after I/R injury.
缺血性脑血管病危害人类健康,目前已经成为我国居民的第一大死亡原因。随着人口老龄化加剧,缺血性脑血管病的发病率逐年增高。在心脏骤停、溺水以及手术失血过多导致的严重低血压等急诊事件中,全脑缺血是最常见并且最严重的并发症。经常遗留严重的神经功能缺损。目前,治疗全脑缺血最有效的方法是尽快的恢复大脑的血供,临床上常采用药物或者介入手段改善脑部循环,但在大脑灌注恢复过程中钙超载、氧自由基过饱和等产生的兴奋性毒性作用反而可能会进一步加重脑组织的损伤,称为脑缺血再灌注损伤(Cerebral ischemia/reperfusion injury, cerebral I/R injury)。所以目前对于治疗脑缺血的研究不应只局限在如何恢复和保留脑部血流,也应该针对缺血再灌注损伤的机制采取措施。
脑缺血后病变以神经元损伤和脑组织炎症为主要特点,其中脑组织炎症又是以星形胶质细胞、小胶质细胞的激活和细胞毒性物质(如细胞因子、基质金属蛋白酶、一氧化氮和活性氧物质等)的释放为特点,这些毒性物质可以导致血脑屏障破坏和神经元死亡,从而产生更多的细胞毒性物质激活更多的免疫细胞,形成恶性循环,加重炎症反应和脑损伤。因此针对脑缺血再灌注损伤后炎症机制的关键环节加以干预可能成为治疗脑缺血再灌注损伤的有效途径。
P2X7受体(P2X7receptor, P2X7R)是一种嘌呤受体,首先在巨噬细胞中被发现。在中枢神经系统(central nervous system,CNS)中主要表达在小胶质细胞中,P2X7R可以被较高浓度的ATP激活继而导致小胶质细胞的活化、活性氧物质释放以及一些前炎症因子如白介素-1β (Interleukin-1β, IL-1β),肿瘤坏死因子-α (Tumor necrosis factor-α, TNF-α)和白介素-6(Interleukin-6, IL-6)等的释放。近年来,研究提示P2X7R参与了多种中枢神经系统疾病的发生和发展如阿尔兹海默病(Alzheimer's disease, AD)、癫痫、脊髓损伤以及多发性硬化等。应用P2X7R阻断剂可以减轻这些疾病模型中的炎症反应。但P2X7R是否参与脑缺血再灌注损伤,是否调节脑缺血再灌注损伤后的炎症反应以及其机制,有关的研究较少,并且存在争议,值得深入探索。
研究目的：
本文拟观察P2X7R对脑缺血再灌注损伤诱导的神经元死亡及炎症反应的影响,做以下3个方面的研究：
(1)制备大鼠全脑缺血再灌注损伤模型,观察全脑缺血再灌注损伤敏感部位海马CA1区P2X7R的表达分布变化。
(2)应用P2X7R阻断剂干预,观察P2X7R在全脑缺血再灌注损伤模型中的作用及其对炎症水平的调节。
(3)探索P2X7R与NADPH氧化酶(NADPH Oxidiase, NOX)之间的相互作用以及其对炎症水平的调节。
第一部分P2X7R在大鼠全脑缺血再灌注损伤模型中的表达变化
研究方法：取体重280g-360g的成年SD雄性大鼠,应用四血管法(four-vessel occlusion,4-VO)建立全脑缺血再灌注损伤模型,缺血时间20分钟,使用激光多普勒脑血流仪检测缺血前10分钟到复灌后90分钟内大鼠区域脑血流(regional cerebral blood flow, rCBF)的变化情况,再灌注时间选取6小时,1天,3天,7天,14天。在上述时间点进行HE染色观察海马CA1区神经元死亡；免疫组织化学法观察星形胶质细胞(GFAP+)、小胶质细胞(Iba-1+)在海马CA1区的分布变化,免疫组织化学法和、Western blot法观察P2X7R在在海马CA1区的表达、分布变化。
结果：四血管法全脑缺血可以有效降低大鼠区域脑血流至20%以下,20min缺血复灌损伤7天后海马CA1区神经元90%发生死亡,随着复灌时间的延长星形胶质细胞及小胶质细胞增生活化明显增多。缺血复灌损伤后P2X7R在海马CA1区表达明显增加,主要表达在小胶质细胞样细胞中。
结论：P2X7R参与全脑缺血再灌注损伤,并与损伤后炎症有关。
第二部分P2X7R抑制剂通过抑制炎症反应减轻全脑全脑缺血再灌注损伤
研究方法：在全脑缺血再灌注损伤前分别给予大鼠生理盐水(Saline); P2X7R阻断剂BBG (Brilliant blue G)、OxATP (adenosine5'-triphosphate-2',3'-dialdehyde)以及A-438079侧脑室注射,统计7天动物死亡率,HE染色观察海马复灌7天时CA1区神经元死亡,TUNEL染色观察复灌3天时CA1区神经元DNA断裂,Morris水迷宫检测大鼠学习记忆能力,免疫组织化学法检测复灌3天时胶质细胞增生,使用RT-PCR方法检测三种细胞因子IL-1B,TNF-a和IL-6复灌3天时在海马中的表达变化,使用丙二醛(Malondialdehyde, MDA)试剂盒检测复灌3天时海马内脂质过氧化水平。
结果：三种P2X7R阻断剂均有剂量依赖的脑缺血再灌注损伤保护作用,与生理盐水组相比,阻断剂组大鼠生存率增加；Morris水迷宫显示BBG和A-438079组大鼠训练第二天后逃避潜伏期较生理盐水组明显缩短,OxATP组大鼠训练4天后逃避潜伏期较生理盐水组明显缩短,空间探索实验中生理盐水组在平台象限逗留时间较三种阻断剂组均缩短；HE染色提示阻断剂组大鼠海马CA1区神经元死亡率较生理盐水组减少,以BBG和A-438079两种抑制剂效果更为明显；三种阻断剂组大鼠CA1区神经元DNA断裂均减少；星形胶质细胞和小胶质细胞的激活以及细胞因子表达也较生理盐水组下降,A-438079组大鼠海马中MDA含量较生理盐水组下降。
结论：阻断P2X7R通过抑脑内炎症反应减轻全脑缺血再灌注损伤。
第三部分P2X7R-NOX参与调节全脑缺血再灌注损伤后炎症反应
研究方法：将大鼠分为假手术(Shaml, Sham2)组、全脑缺血复灌(Saline, Vehicle)组、侧脑室注射A-438079组以及腹腔注射NOX抑制剂Apocynin组。HE染色观察海马CA1区神经元存活；western bolt方法观察NOX的两种亚基gp91phox以及p47phox蛋白表达变化；Lucigenin化学发光法检测海马内NOX活性；免疫组织化学法检测复灌3天时胶质细胞增生。
结果：全脑缺血再灌注损伤后脑内NOX被激活,表现在NOX酶活性增强和gp91phox, p47phox蛋白表达升高以及p47phox蛋白的向膜转位。A-438079和apocynin预处理均可以抑制全脑缺血再灌注损伤诱导的NOX激活。Apocynin可以减少全脑缺血再灌注损伤诱导的海马CA1区神经元死亡和星形胶质细胞、小胶质细胞的增生活化。
结论：P2X7R-NOX参与全脑缺血再灌注损伤以及损伤后炎症调节。
Background
Transient global cerebral ischemia is one of the major complications of clinical emergencies such as cardiac arrest, drowning or severe systemic hypotension during a surgical procedure. Currently, the most adequate treatment for these patients is re-establishing perfusion of the brain as soon as possible. However, reperfusion may
paradoxically exacerbate brain injury, which is called cerebral ischemia/reperfusion (I/R) injury. Therefore, efforts need to be made that not only to preserve cerebral blood flow, but also to prevent the actual mechanisms that trigger brain damage after I/R injury.
Neuroinflammation, which is characterized by microglial and astroglial activation, as well as the release of cytotoxic agents (cytokines, matrix metalloproteinases, nitric oxide and reactive oxygen species) can be triggered by cerebral I/R injury, which can contribute to blood-brain barrier disruption and delayed neuronal death. Subsequently, these damaged cells release more toxic mediators, which in turn activate more immune cells. Thus, prolonged inflammation caused by this vicious circle exacerbates brain damage. Taken together, anti-inflammation therapy may become a promising therapeutic strategy for the treatment of cerebral I/R injury.
The P2X7receptor(P2X7R), a purinergic receptor, was first discovered in macrophages. In the central nervous system (CNS), the P2X7R is predominantly expressed in microglia which are the resident macrophages of the brain. The P2X7R can be activated by high concentrations of ATP. Stimulating the P2X7R leads to microglial activation, reactive oxygen species production and increased secretion of pro-inflammatory cytokines such as IL-1β, TNF-α and IL-6. Recently, the P2X7R has been reported to be involved in neuroinflammation in many CNS diseases
including A lzheimer's disease (AD), epilepsy, spinal cord injury and multiple sclerosis, and treatment with P2X7R antagonists reduces experimentally induced neuroinflammation in animal models of such diseases.
The P2X7receptor(P2X7R), a purinergic receptor, was first discovered in macrophages. In the central nervous system (CNS), the P2X7R is predominantly expressed in microglia which are the resident macrophages of the brain. The P2X7R can be activated by high concentrations of ATP. Stimulating the P2X7R leads to microglial activation, reactive oxygen species production and increased secretion of pro-inflammatory cytokines such as Interleukin-1β(IL-1β), Tumor necrosis factor-α (TNF-a) and Interleukin-6(IL-6). Recently, the P2X7R has been reported to be involved in neuroinflammation in many CNS diseases including Alzheimer's disease (AD), epilepsy, spinal cord injury and multiple sclerosis, and treatment with P2X7R antagonists reduces experimentally induced neuroinflammation in animal models of such diseases. Only a few study have reported the effect of P2X7R in cerebral ischemic injury, and the results are inconsistent. So it is still worth to be discussed.
Aim
To clarify the role of P2X7R in global cerebral I/R-induced injury and neruoinflammation, in the present study we performed the following investigations.
(1) We examined the P2X7R expression of P2X7R after global cerebral I/R-induced injury in rats.
(2) Using P2X7R antagonist, we investigated the role of P2X7R in global cerebral I/R injury. We also explored the association between the P2X7R and neuroinflammation after transient global cerebral I/R injury.
(3) We also investigated the interaction between P2X7R and NADPH Oxidiase(NOX), and thier effect in the regulation on neuroinflammation.
Part Ⅰ Expression properties of P2X7R in the hippocampal CA1region after global cerebral I/R injury.
Methods:Twenty mins global cerebral I/R injury was conducted on male Sprague-Dawley rats weighing260-320g using four-vessel occlusion (4-VO) method. Regional cerebral blood flow (rCBF) from10mins before ischemia to90mins after reperfusion was measured using a laser Doppler flowmetry; HE staining was performing to observe the neuronal death6hours,1day,3days,7days and14days after reperfusion; activated microglia and astrocytes were detected by immunohistochemical staining; P2X7R expression was examined by western bolts and immunobistochemical staining.
Results:During the ischemic period, the rCBF was lower than20%; more than90%CA1neurons dead after3days-reperfusion. glial activation, gliosis and P2X7R expression were increased in a time-dependent manner after reperfusion. P2X7R mainly expressed on microglia-like cells.
Conclusion:P2X7R is involved in global cerebral I/R injury and is associated with I/R-induced neuroinflammation.
Part Ⅱ Inhibition of P2X7R ameliorates transient global cerebral I/R injury via modulating inflammatory responses in the rat hippocampus
Methods:Immediately after infusion with the P2X7R antagonists Brilliant blue G (BBG), adenosine5'-triphosphate-2',3'-dialdehyde (OxATP) or A-438079, tweenty minutes of transient global cerebral I/R was induced using the4-VO method in rats. Survival rate was calculated, neuronal death in the hippocampal CA1region was observed using HE staining, and DNA cleavage was observed by deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL). In addition, behavioral deficits were measured using the Morris water maze, and RT-PCR and immunohistochemical staining were performed to measure the expression of IL-1β,TNF-α and IL-6, and to identify activated microglia and astrocytes.
Results:The P2X7R antagonists protected against transient global cerebral I/R injury in a dosage-dependent manner. A high dosage of BBG (10μg) and A-438079(3μg). and a low dosage of OxATP (1μg) significantly increased survival rates, reduced I/R-induced learning memory deficit, and reduced I/R-induced neuronal death, DNA cleavage, glial activation and inflammatory cytokine overexpression in the hippocampus.
Conclusions:Our study indicates that inhibiting P2X7R.S protects against transient global cerebral I/R injury by reducing the I/R-induced inflammatory response, which suggests inhibition of P2X7Rs may be a promising therapeutic strategy for clinical treatment of transient global cerebral I/R injury.
Part Ⅲ P2X7R-NOX participates in transient global cerebral I/R-induced neuroinflammation
Methods:Rats were divided into six group:Sham1, Sham2, Saline, Vehicle, A-438079(3μg i.c.v.), Apocynin (10mg/kg i.p. twice). NOX activity was measured with Lucigenin-enhanced chemiluminescence; expression of gp91phox and p47phox, p47phox translocation were detected by western bloting; neuronal death in the hippocampal CA1region was observed using HE staining and immunohistochemical staining were performed to identify activated microglia and astrocytes.
Results:NOX activity was elevated at the third day after global cerebral ischemia reflected by elevated relative light units (RLU) values, and increased gp91phox, p47phox expression and less p47phox translocation. The P2X7R antagonists A-438079suppressed the I/R-induced NOX activity elevation. NOX antagonist apocynin protected against transient global cerebral I/R injury and reduced I/R-induced glial activation.
Conclusions:P2X7R-NOX participated in the I/R-induced brain injury and neurolinflammation after I/R injury.
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