【作者】 孙宝柱；

【导师】 张岫美；

【作者基本信息】 山东大学 ， 医学基础药理学， 2011， 博士

【摘要】 研究背景与目的脑缺血再灌注损伤是临床常见的病理生理表现,在重症创伤、心脏停博复苏、心脏手术、缺血性脑血管疾病的溶栓治疗、器官移植手术及低血容量休克的复苏治疗等病理条件下均可发生脑缺血再灌注损伤(cerebral ischemia-reperfusion injury, CIRI)。近年来大量研究表明,脑缺血再灌注后脑组织局部过度的炎症反应是造成再灌注损伤的主要原因之一,也是脑缺血继发性损伤的重要病理生理机制。缺血和再灌注早期产生的细胞因子(IL-1、IL-6. TNF-α、TGF-β等)和黏附分子(ICAM-1、VCAM-1、P-selectin、E-selectin等)构成了缺血性损伤向炎症性损伤转变的基础。在阻断大鼠大脑中动脉复制出的脑缺血再灌注模型(middle cerebral artery occlusion, MCAO)中早期便可检测到中性粒细胞在微血管、缺血脑实质及海马组织中的聚集和浸润,并且海马是大鼠脑组织中对缺血再灌注损伤最为敏感的部位之一。NF-κB (nuclear factor-κB)是脑缺血再灌注损伤后所激活的一类重要的转录因子,NF-κB活化后可促进黏附分子、细胞表面受体和细胞因子的基因表达,进一步加重脑缺血过程中的炎症反应。因此,及时有效的抗炎措施将有助于缓解缺血再灌注造成的脑损伤程度。脑缺血再灌注损伤是多因素、多环节的级联过程,而凋亡是导致缺血再灌注损伤后神经元死亡的主要机制之一。在大鼠脑缺血2h再灌注24h后,可在脑皮层中检测到大量TUNEL染色阳性细胞及Bcl-2、Bax蛋白的变化。抑凋亡因子Bcl-2和促凋亡因子Bax以及两者之间的平衡关系在细胞凋亡中发挥着重要作用。另外,信号通路Akt/GSK-3β对细胞凋亡和Bcl-2与Bax蛋白之间的平衡也起到重要调控作用。抑制神经细胞发生以凋亡为主的迟发性死亡,可以诱导脑缺血耐受,有助于减轻脑缺血损伤。COX-2活性的增加将通过氧化应激或合成具有神经毒性的前列腺素(PGA1和PGE1)等机制造成中枢神经系统的损伤。因此,有些研究表明非甾体抗炎药在脑缺血条件下具有脑保护作用。在脑中风的动物模型研究中发现,布洛芬能减轻全脑缺血时的神经元损伤并改善中枢血流量和神经功能损害评分情况；还能减少局灶性脑缺血造成的脑梗死容积。氟比洛芬酯是一种新剂型的非甾体类抗炎镇痛药,它以脂微球作为药物载体,可以选择性地蓄积在炎症组织及血管损伤部位,从而更好地发挥抗炎作用。氟比洛芬以脂微球为载体,可以靶向性地在炎症部位聚集,还可使氟比洛芬快速穿透细胞膜从而利于其吸收而缩短起效时间。氟比洛芬的抗炎机制主要是抑制环氧合酶,阻断花生四烯酸代谢,抑制前列腺素生物合成。本研究将着重从炎症和凋亡两个角度探讨氟比洛芬酯对大鼠局灶性脑缺血再灌注损伤的保护作用及作用机制。研究方法健康清洁级雄性Wistar大鼠随机分为假手术组(S组)、缺血再灌组(I/R组)、脂微球溶剂组(V组)、氟比洛芬酯5mg/kg组(FA-L组)和氟比洛芬酯lOmg/kg组(FA-H组)。各组分别于再灌注即刻由尾静脉注射给药,给药容积均为1mL/kg,SH组、IR组和V组分别给予等容量生理盐水和脂微球溶剂。改良线栓法阻断大鼠大脑中动脉供血120min,再灌注24h制备大鼠MCAO模型。本实验分为四部分：1.大鼠局灶性脑缺血再灌注损伤模型的建立,并观察氟比洛芬酯对大鼠脑缺血再灌注损伤后神经病学、病理学变和脑梗死容积的影响。采用Zea Longa的5级4分制评分法进行神经功能缺陷评分,常规苏木精-伊红(HE)染色观察大鼠缺血海马组织的病理形态学变化,TTC染色法测定大鼠脑梗死容积。2.检测大鼠脑缺血再灌注损伤后海马组织中细胞因子(IL-1β、IL-6、TNF-α)表达和髓过氧化物酶(myeloperoxidase, MPO)活性的改变,并观察氟比洛芬酯对上述指标的影响。RT-PCR检测肿瘤坏死因子α(tumor necrosis factor-alpha, TNF-a),白介素1β(interlukin-1β, IL-1β)及白介素6(interlukin-6, IL-6)的表达,分光光度法测定MPO活性的改变。3.检测大鼠脑缺血再灌注损伤后海马组织中转录因子NF-κB活性的改变和P38 MAPK蛋白表达情况,并观察氟比洛芬酯对两者的影响。采用免疫组化和Western Blot法检测磷酸化NF-κB (p65)蛋白水平,Western Blot法检测磷酸化P38 MAPK蛋白水平。4.观察氟比洛芬酯对大鼠局灶性脑缺血再灌注后脑皮层神经元凋亡的影响,并探讨其作用机制。TUNEL法检测缺血侧脑皮层神经细胞的凋亡,Western Blot法检测缺血侧脑皮层中Bcl-2与Bax的蛋白表达及磷酸化Akt和GSK-3p蛋白水平。结果1.在再灌注24h时,缺血再灌注组和脂微球溶剂组神经功能缺陷评分明显高于氟比洛芬酯组和假手术组,并且氟比洛芬酯10 mg/kg组的神经功能缺陷评分低于氟比洛芬酯5 mg/kg组(P<0.05)。缺血再灌注组与脂微球溶剂组神经功能缺陷评分无统计学差异(P>0.05)。与假手术组相比,缺血再灌注组和脂微球溶剂组的脑梗死容积显著增大(P<0.05),而两组之间的脑梗死容积无显著性差异(P>0.05)。氟比洛芬酯组与缺血再灌注组和脂微球溶剂组相比,脑梗死容积显著降低,并且氟比洛芬酯高剂量组的脑梗死容积显著低于低剂量组(P<0.05)。缺血再灌注组和脂微球溶剂组的海马组织HE染色显示脑损伤改变明显,而氟比洛芬酯用药组HE染色结果显示脑损伤程度减轻。2.与假手术组相比,缺血再灌注组和脂微球溶剂组RT-PCR结果显示大鼠脑缺血再灌注损伤后海马组织中细胞因子IL-1β、IL-6、TNF-α基因表达增强；所测定的MPO活性也显著增强(P<0.05)。而氟比洛芬酯能够明显降低上述指标的表达,并且高低两个剂量组之间的差异有统计学意义(P<0.05)。3.免疫组化结果提示缺血再灌注组和脂微球溶剂组的海马组织中NF-κB(p65)亚基发生核转位,大部分位于细胞核内,而氟比洛芬酯能明显抑制NF-κB(p65)亚基由细胞质到细胞核的转位,并且结合细胞核提取物的Western Blot检测,缺血再灌注组和脂微球溶剂组的磷酸化NF-κB (p65)蛋白水平显著高于氟比洛芬酯组和假手术组(P<0.05)。各组Western Blot检测结果显示磷酸化P38MAPK蛋白水平与磷酸化NF-κB (p65)蛋白水平具有相似的变化趋势。4.再灌注24h时,与氟比洛芬酯组和假手术组相比,缺血再灌注组和脂微球溶剂组的脑皮层区TUNEL染色阳性细胞数明显增多,Bcl-2蛋白表达下调,Bax蛋白表达上调,磷酸化Akt蛋白水平降低,而磷酸化GSK-3p蛋白水平增加(P<0.05)。与缺血再灌注组和脂微球溶剂组比较,氟比洛芬酯两剂量组TUNEL染色阳性细胞数显著降低,Bcl-2蛋白表达上调,Bax蛋白表达下调,磷酸化Akt蛋白水平增加,而磷酸化GSK-3β蛋白水平降低(P<0.05)。结论1.本实验采用改良线栓法复制的MCAO模型成功有效,且稳定性良好。氟比洛芬酯能显著改善大鼠脑缺血再灌注后的神经功能缺陷评分、脑梗死容积和海马组织的病理学改变。2.氟比洛芬酯能抑制大鼠脑缺血再灌注损伤后海马组织中的细胞因子表达和髓过氧化物酶的活性改变。3.氟比洛芬酯能抑制海马组织中转录因子NF-κB (p65)亚基的核转位,并降低磷酸化NF-κB (p65)蛋白和磷酸化P38 MAPK蛋白水平。4.氟比洛芬酯通过Akt/GSK-3β信号途径调节Bcl-2和Bax蛋白表达抑制脑皮层区的神经细胞凋亡。更多还原

【Abstract】 Background and objective Ischemic cerebrovascular disease is a neurological disorder caused by insufficient blood supply to a particular brain area and has become a common clinically pathological sypmptom. Ischemia and reperfusion (I/R) occurs in a wide range of clinical settings, including trauma, cardiac arrest, cardiac surgery, thrombolysis treatment, organ transplantation and hypovolemic shock with resuscitation. Experimental data obtained from animal models of middle cerebral artery occlusion indicates that inflammation plays a vital role in the pathogenesis of cerebral ischemia and secondary damage. Inflammatory cytokines such as tumor necrosis factorα(TNF-a) and interleukin-1β(IL-1β) as well as adhesion molecules which are produced at the early stage of ischemia promote both the recruitment of neutrophil, their adherence to brain endothelial cells and the resultant activation of inflammatory processes. In various animal models of MCAO, increased neutrophil accumulation has been detected in the microvessels and ischemic cerebral parenchyma,and the hippocampus is one of the most sensitive to ischemia/reperfusion injury in rats. Nuclear factor-KB (NF-κB) is one of the most important transcription factors activated after cerebral ischemia. NF-κB is involved in inflammatory responses that potentiate ischemic injury activating many genes involved in the pathogenesis of cerebral ischemia, such as iNOS, IL-1β, TNF-α, ICAM-1, COX-2, and IL-6. Consequently anti-inflammatory therapies may be of benefit to ischemic cerebrovascular disease outcome.Apoptosis is one of the major mechanisms that lead to cell death after cerebral ischemia and reperfusion. Ischemia followed by restoration of blood flow exacerbates neuronal apoptosis in the cortex and hippocampus. Cerebral ischemia reperfusion injury could induce apoptosis. A large number of TUNEL-positive cells and the alteration of Bax/Bcl-2 protein ratios were observed 24h after reperfusion. It has been suggested that the balance between the protein levels of anti-apoptotic Bcl-2 and pro-apoptotic Bax played an important role in regulating apoptotic cell death. Moreover, Akt activation promotes cell survival by phosphorylation and subsequent inactivation of apoptosis-inducing factors, including the Bcl-2 family member and glycogen synthase kinase GSK-3β.The increased COX-2 activity may contribute to neuro-degeneration by either oxidative stress, or the neurotoxic effect of prostaglandins such as PGA1 and PGE1. Therefore, some studies suggest that NSAIDS might be neural protective in cerebral ischemic conditions. In animal models of stroke, ibuprofen reduces neuronal injury and improves cerebral blood flow and neurological outcome in global ischemia and decreases infarct size in focal ischemia. The COX inhibitor flurbiprofen, which utilizes a lipid microsphere drug delivery system, may facilitate effective target therapy. In addition, the lipid microsphere easily permeates cell membrane and greatly promotes the absorption of the drug and results in shortened onset time. Their primary mechanism of action is through the inhibition of COX activity in the arachidonic acid cascade which in turn inhibits the biosynthesis of prostaglandin (PG), which is involved in causing inflammation of cerebral ischemia/reperfusion injury. The aim of this study was to investigate the protective effects and mechanisims of flurbiprofen axetil on the injury of cerebral ischemia-reperfusion in rats from apoptosis and inflammation.Methods Healthy clean male Wistar rats weighing 260-320 g were randomLy divided into five groups:sham group (group S), ischemia/reperfusion (I/R) group (group I/R),5 mg/kg,10 mg/kg dose of flurbiprofen axetil groups (group FA-L and group FA-H) and 1 mL/kg lipo-microballoons group (group V). All drags were administered via tail-vein injection at the onset of reperfusion. Focal cerebral ischemia/reperfusion model was conducted by occluding left middle cerebral artery for 2 hours followed by 24 hours of reperfusion. There are four parts in this study:1. The suture method was used to block the middle cerebral artery for 120 mins, and then a reperfusion for 24 hours was performed in rats. We evaluated the influence of flurbiprofen on cerebral infarction volume, neurological outcome and pathological issue change following focal cerebral ischemia-reperfusion (I/R) injury in rats. The hematoxylin-eosin (HE) staining was used to observe pathological changes of ischemic hippocampus. Zea-Longa method was used to assessment the neurological disfunction scores. Triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction size.2. To investigate the effects of flurbiprofen on myeloperoxidase (MPO) activity and cytokines (IL-1β, TNF-α, IL-6) gene expression in ischemic hippocampus following focal cerebral ischemia-reperfusion (I/R) injury in rats.10% hippocampus homogenate was prepared to analyze MPO activity by spectrophotometry. Pro inflammation cytokine TNF-α, IL-1βand IL-6 were analyzed by RT-PCR.3. To investigate the effects of flurbiprofen on NF-κB activity and the level of phospho-P38 in ischemic hippocampus following focal cerebral ischemia-reperfusion (I/R) injury in rats. The expression of NF-κB (p65) in ischemic hippocampus was detected by immunohistochemistry assay and western blot assay. Western blot was used to determine the phospho-P38.4. The present study aimed to show that administration of flurbiprofen may provide neuroprotection in focal cerebral ischemia-reperfusion injury and this neuroprotection is associated with inhibition of apoptosis mediated by activation of the Akt/GSK-3b pathway. The number of apoptotic neurons in the ischemic penumbra region was counted using TUNEL. Western blot was used to test the level of Bcl-2, Bax, phospho-Akt and phospho-GSK3βin the ischemic penumbra.Results1. The neurologic deficit scores were significantly higher in both group I/R and group V than in group S and group FA 24 hours after reperfusion, and significant differences were found in neurological deficits between the 5 mg·kg-1 and 10 mg·kg-1 groups (P<0.05). No great differences were found in neurological deficits between group I/R and group V (P>0.05). The infarct volumes in group FA were significantly smaller than those of group I/R and group V (P<0.05). Furthermore, there was a significant difference in infarct volume between 5 mg·kg-1 and 10 mg·kg-1 groups (P<0.05).The results of HE staining showed that the ischemic damage of the group I/R and group V was great and the group S and group FA had a light pathological damage.2. The mRNA levels of the inflammatory cytokines (TNF-α, IL-6,IL-1β) and MPO activity were greatly increased in group I/R and group V compared with the group S (P<0.05). However, treatment with flurbiprofen in doses of 5 mg·kg-1 and 10 mg·kg-1 significantly relieved the increase of these variables induced by MCAO. Furthermore, significant differences were found in these variables between group FA-L and group FA-H (P< 0.05).3. The expression of NF-κB (p65) in ischemic hippocampus was detected by immunohistochemistry assay and western blot assay. NF-κB (p65) was predominantly located in the nucleus in group I/R and group V and flurbiprofen can significantly inhibit its translocation from cytoplasm into the nucleus (P<0.05). Furthermore, through western blot assay of nuclear extract, the level of p-NF-KB (p65) in group I/R and group V was markedly higher than that in the sham and flurbiprofen groups (P<0.05).4. TUNEL positive cells and expression of Bax were significantly higher and expression of Bcl-2 were significantly lower in the ischemic penumbra in group I/R and group V than in group S at 24 h after reperfusion (P<0.05).However, TUNEL positive cells and expression of Bax were significantly lower and expression of Bcl-2 in the ischemic penumbra were significantly higher in group FA than in group I/R and group V (P<0.05). The present study showed that phospho-Akt level in ischemic penumbra was increased significantly after administration of flurbiprofen, whereas the level of phospho-GSK3βsignificantly decreased compared with compared with group I/R and group V(P<0.05). No significant differences in the levels of phospho-Akt and phospho-GSK3P were found between flurbiprofen 5 and 10 mg/kg groups (P>0.05). Conclosion1. The modified suture is reliable in making MCAO model in rats. Flurbiprofen could not only reduce infarct volume and pathological damage but also improve the neurological outcome in the rat model after cerebral ischemia.2. Flurbiprofen can inhibit the inflammatory response in the hippocampus of rats exposed to cerebral ischemia/reperfusion by limiting the increase of MPO activity and cytokines gene expression.3. Flurbiprofen can significantly inhibit NF-κB (p65) translocation from cytoplasm into the nucleus and reduce the levelof p-NF-KB (p65) and p-P38 protein in the hippocampus of rats.4. Flurbiprofen protected against cerebral ischemia/reperfusion injury by reducing apoptosis via up-regulation of Bcl-2 expression and down-regulation of Bax expression in rats. These effects may be partly due to the activation of Akt/GSK-3βsignaling pathway.更多还原