Cilostazol prevents focal cerebral ischemic injury by enhancing casein kinase 2 phosphorylation and suppression of phosphatase and tensin homolog deleted from chromosome 10 phosphorylation in rats

This study shows the in vivo neuroprotective effect of cilostazol against cerebral ischemic injury evoked by subjecting rats to 2-h occlusion of middle cerebral artery (MCAO) followed by 24-h reperfusion. We observed the signaling pathway by which cilostazol suppressed MCAO-induced increased phosphorylation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) and apoptosis via increased phosphorylation of casein kinase 2 (CK2). When rats received 30 mg/kg cilostazol orally two times at 5 min and 4 h after the completion of ischemia, the infarct area was significantly reduced in the cortex and striatum with improvement of neurological deterioration. Increased DNA fragmentation in the penumbral zone was significantly reduced by cilostazol. Cilostazol significantly elevated phosphorylation levels of CK2, Akt, and cyclic AMP response element-binding protein (CREB) in association with increased Bcl-2 in the ischemic area, whereas the elevated PTEN phosphorylation was significantly reduced, all of which were antagonized by iberiotoxin, a maxi-K channel blocker, administered intracisternally 30 min before ischemia. In conclusion, cilostazol ameliorates the neuronal damage by suppression of apoptotic cell death via the maxi-K channel opening-coupled up-regulation of CK2 phosphorylation and down-regulation of PTEN phosphorylation with resultant increase in the Akt and CREB phosphorylation and increased Bcl-2 protein.     

17 Beta-estradiol prevents focal cerebral ischemic damages via activation of Akt and CREB in association with reduced PTEN phosphorylation in rats

This study aimed to assess the signaling pathway of the neuroprotective action of estrogen in the cerebral ischemic injury evoked by subjecting rats to 2-h occlusion of the middle cerebral artery (MCA) followed by 24-h reperfusion. Rats received 17 beta-estradiol (1, 4 and 10 mg/kg, i.p.) 24 h before and 5 min after the completion of 2-h MCA occlusion. The cerebral infarct area was consistently observed in the cortex and striatum of the left hemisphere. Increased terminal deoxynucleotidyl transferase-mediated deoxyuridine-biotin nick-end labeling (TUNEL)-positive cells and DNA fragmentation in the penumbral zone were significantly reduced by 17 beta-estradiol. In line with these results, 17 beta-estradiol significantly increased Akt and cyclic AMP response element binding protein (CREB) with increased Bcl-2 protein in the ischemic area, whereas the elevated the phosphatase and tensin homolog deleted from chromosome10 (PTEN) phosphorylation was significantly reduced with decreased Bax protein and cytochrome c release. Inhibition of DNA fragmentation, PTEN phosphorylation, and Akt activation by 17 beta-estradiol were antagonized by iberiotoxin, a maxi-K channel blocker. Taken together, it is suggested that suppression of cerebral ischemic injury by 17 beta-estradiol may be ascribed to the maxi-K channel opening-coupled downregulation of PTEN phosphorylation and upregulation of Akt and CREB phosphorylation with resultant increase in Bcl-2 protein and decrease in Bax protein and cytochrome c release.     

Neuroprotective effect of (2S,3S,4R)-N"-cyano-N-(6-amino-3, 4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N'-benzylguanidine (KR-31378), a benzopyran analog, against focal ischemic brain damage in rats

This study shows the preventive effect of KR-31378 [(2S,3S,4R)-N"-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N'-benzylguanidine] against cerebral infarct via antioxidant and antiapoptotic actions evoked by subjecting rats to 2 h of occlusion of the left middle cerebral artery followed by 24 h of reperfusion. The brain infarct zone in the cortex and striatum of the left hemisphere was consistently identified in the cortex and striatum of the left hemisphere. The infarct area was significantly reduced after three intraperitoneal administrations of 10, 30, or 50 mg/kg KR-31378 at 5 min, 4 h, and 8 h after the completion of 2 h of ischemia. Treatment with KR-31378 (30 or 50 mg/kg) significantly reduced the increase in the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling positive cells as well as strongly suppressed the laddered feature of DNA fragmentation in the lateral cortical tissue corresponding to the penumbra. The findings of samples from penumbral zone, which showed markedly reduced Bcl-2 protein level and increased Bax protein and cytochrome c release, were wholly reversed by treatment with KR-31378. In conclusion, postischemic treatment with KR-31378 provided significant levels of cortical neuroprotection in association with inhibition of apoptotic cell death through the up-regulation of Bcl-2 expression, and the down-regulation of Bax protein and cytochrome c release.     

姜黄素对大鼠脑缺血再灌注损伤的保护作用

目的：研究姜黄素对大鼠脑缺血再灌注损伤的保护作用，并探讨其作用的机制。方法：采用线栓法制作大鼠大脑中动脉缺血再灌注模型，50只Wistar雄性大鼠被随机分为假手术组、缺血组和100mg／kg、300mg／kg姜黄素治疗组。缺血组和姜黄素治疗组分别在脑缺血再灌注后24h处死，观察大鼠神经功能缺失的评分．应用TTC染色观察梗死体积，应用TUNEL法及免疫组化染色检测脑组织凋亡细胞数及Bcl-2、Bax蛋白表达。结果：与损伤模型组比较．姜黄素治疗组改善大鼠神经功能的受损程度和减少脑梗死体积．各剂量姜黄素治疗组均可明显减少TUNEL染色阳性细胞数（P〈0.01），明显增加Bcl-2蛋白表达（P〈0.01），并且姜黄素治疗组Bax表达低于缺血组（P〈0．05）。结论：姜黄素可通过上调Bcl-2蛋白下调Bax蛋白表达而减少脑缺血再灌注后神经细胞凋亡．从而改善受损的神经功能．对脑缺血再灌注损伤起保护作用．     

川芎嗪对局灶性脑缺血再灌注后Bcl-2、Bax蛋白表达的影响

目的：探讨川芎嗪对大鼠局灶性脑缺血再灌注后脑组织Bcl-2和Bax蛋白表达的影响。方法：SD大鼠18只随机分为3组各6只：A组为假手术组，B组为缺血再灌注组，C组为川芎嗪干预组。采用免疫组化与医学图像分析检测大鼠大脑皮层Bcl-2和Bax蛋白阳性细胞的数目、平均灰度。结果：B组与A组比较损伤侧脑组织Bcl-2、Bax蛋白阳性细胞数目均增多，平均灰度均降低（P〈0．01）；C组与B组比较Bcl-2蛋白阳性细胞数目增多，平均灰度降低，Bax蛋白阳性细胞数目减少，平均灰度升高（P〈0．05）。结论：局灶性脑缺血再灌注后Bcl-2、Bax蛋白表达增强；川芎嗪可上调Bcl-2蛋白及下调Bax蛋白的表达，这可能是川芎嗪治疗缺血性脑血管病的机制之一。     

Inhibition of lipopolysaccharide-induced apoptosis by cilostazol in human umbilical vein endothelial cells.

This work describes the pharmacological inhibition by cilostazol and its metabolites, OPC-13015 and OPC-13213, of the apoptosis in the human umbilical vein endothelial cells (HUVECs) damaged by lipopolysaccharide (LPS) in comparison with its analog, cilostamide. Cilostazol and OPC-31213 caused a significant suppression of cell death induced by LPS (1 microg/ml) in a concentration-dependent manner but a modest suppression by cilostamide and OPC-13015. These compounds potently inhibited the 5,5-dimethyl-1-pyrroline-1-oxide (DMPO)/(*)OH adduct formation and significantly reduced the increased intracellular reactive oxygen species (ROS) and tumor necrosis factor-alpha (TNF-alpha) production induced by LPS (1 microg/ml). An apoptotic death of HUVECs by 1 microg/ml LPS (DNA ladders on electrophoresis) was strongly suppressed by all these compounds. Incubation with LPS caused a marked decrease in Bcl-2 protein, which was significantly reversed by cilostazol and its analogs. The greatly increased Bax protein expression and cytochrome c release by LPS were, in contrast, suppressed by cilostazol and, to a lesser degree, by others. In conclusion, cilostazol and its analogs exert a strong protection against apoptotic cell death by scavenging hydroxyl radicals and intracellular ROS with reduction in TNF-alpha formation and by increasing Bcl-2 protein expression and decreasing Bax protein and cytochrome c release.     

6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)quinolinone (cilostazol), a phosphodiesterase type 3 inhibitor, reduces infarct size via activation of mitochondrial Ca2+-activated K+ channels in rabbit hearts

6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)quinolinone (cilostazol), a phosphodiesterase type 3 (PDE III) inhibitor, activates cAMP-dependent protein kinase A (PKA). The cAMP/PKA pathway potentiates the opening of mitochondrial Ca(2+)-activated K(+) (mitoK(Ca)) channels and confers cardioprotection. Although cilostazol has been reported to directly activate sarcolemmal large-conductance Ca(2+)-activated K(+) channels, it remains unclear whether cilostazol modulates the opening of mitoK(Ca) channels. Therefore, we tested the possibility that cilostazol opens mitoK(Ca) channels and protects hearts against ischemia/reperfusion injury. Flavoprotein fluorescence in rabbit ventricular myocytes was measured to assay mitoK(Ca) channel activity. Infarct size in the isolated perfused rabbit hearts subjected to 30-min global ischemia and 120-min reperfusion was determined by triphenyltetrazolium chloride staining. Cilostazol (1, 3, 10, and 30 microM) oxidized flavoprotein in a concentration-dependent manner. The oxidative effect of cilostazol (10 microM) was antagonized by the mitoK(Ca) channel blocker paxilline (2 microM). Activation of PKA by 8-bromoadenosine 3'5'-cyclic monophosphate (0.5 mM) potentiated the cilostazol-induced flavoprotein oxidation. Treatment with cilostazol (10 microM) for 10 min before ischemia significantly reduced the infarct size from 67.2 +/- 1.3 (control) to 33.6 +/- 5.3% (p < 0.05). This infarct size-limiting effect of cilostazol was abolished by paxilline (60.3 +/- 4.9%) but not by the PKA inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]-benzodiazocine-10-carboxylic acid hexyl ester (KT5720) (200 nM, 40.5 +/- 3.5%). On the other hand, another PDE III inhibitor, milrinone (10 microM), neither oxidized flavoprotein nor reduced infarct size. Our results suggest that cilostazol exerts a cardioprotective effect via direct activation of mitoK(Ca) channels.     

缺血预处理对大鼠冷缺血再灌注损伤的保护作用的实验研究

目的：探讨缺血预处理对大鼠肾脏冷缺血再灌注损伤的保护作用。方法：36只SD大鼠制备成肾脏冷缺血再灌注损伤模型，随机分为假手术组（A组）、冷缺血再灌注组（B组）、缺血预处理组（C组），免疫组化观察Bcl2、Bax蛋白表达，测定肾功能、肾组织超氧化物歧化酶、丙二醛，病理切片观察肾组织损伤形态学的变化。结果：B组、C组BUN、CR测定值均显著高于A组，而C组低于B组，差异具有显著性（P〈0.01）；C组大鼠肾组织超氧化物酯化酸水平较B组显著升高，而丙二醛水平显著性下降（P〈0．01）；B组、C组Bcl2、Bax蛋白较A组均明显增加，C组较B组Bcl—2表达显著增强，Bax表达明显下降（P均〈0．01），肾组织损伤的病理分级显著减轻。结论：缺血预处理对肾脏冷缺血再灌注性损伤具有保护作用，其机制可能与上调Bcl2和下调Bax蛋白表达相关。     

亚低温对大鼠局灶性脑缺血神经元细胞凋亡及调控基因Bcl-2和Bax表达的影响

目的研究不同时程的亚低温治疗对大鼠大脑中动脉闭塞(MCAO)模型的神经元细胞凋亡及Bcl-2,Bax基因表达的影响。方法健康雄性SD大鼠共40只,月龄两三个月。按抽签法随机分为5组,每组8只(n=8),分别为:假手术组(A)、对照组(B)、亚低温0.5h组(C)、亚低温1.0h组(D)、亚低温3.0h组(E)。采用Longa线栓法制作MCAO模型,缺血3.0h后再灌注。缺血后即刻分别进行不同时程(0.5,1.0,3.0h)的亚低温治疗,再灌注后24h断头取脑,连续切片作TUNEL染色及Bcl-2,Bax免疫组化染色。结果凋亡神经元细胞(TUNEL阳性细胞)主要见于梗死灶周围的纹状体及额顶部皮层。Bcl-2及Bax阳性细胞也主要见于梗死灶周围的纹状体及额顶部皮层,与该区凋亡细胞的集中分布相一致。与对照组的Bcl-2细胞阳性率相比,亚低温3.0h组增加犤(23.0±3.8)%,P<0.05)犦;与对照组的Bax细胞阳性率相比,亚低温1.0,3.0h组均降低犤分别为(45.9±4.1)%,(32.4±3.7)%,P<0.05,P<0.01犦;凋亡细胞阳性率与Bcl-2/Bax的比值呈负相关关系(r=-0.9759,P<0.01)。结论亚低温1.0h以上有抑制细胞凋亡作用;Bcl-2/Bax的比值能影响细胞凋亡的发生。     

全脑缺血再灌注损伤大鼠海马神经元凋亡调节蛋白Bcl-2和Bax的表达

背景:脑缺血再灌注对中枢神经系统的影响,除了急性期的细胞坏死,还有迟发性的神经元凋亡.目的:观察大鼠全脑缺血不同再灌注阶段海马神经元凋亡率、坏死率及凋亡调节蛋白Bcl-2和Bax的表达情况,并探讨其对全脑缺血再灌注损伤的调节作用.设计:随机对照实验.单位:首都医科大学附属北京天坛医院的神经外科和麻醉科.材料:实验于2003-01/2004-01在首都医科大学附属北京神经外科研究所完成.选择清洁级成年雄性健康Wistar大鼠33只,随机分5组,缺血再灌注24 h组7只、缺血再灌注48 h组7只、缺血再灌注72 h组7只、缺血再灌注7 d组7只和假手术对照组5只.干预:制备大鼠全脑缺血再灌注模型.分别于再灌注24,48,72 h和7 d取脑海马组织,用流式细胞仪检测细胞凋亡率,坏死率,Bcl-2和Bax蛋白在大鼠脑海马神经元中的表达情况.主要观察指标:①流式细胞仪检测各组大鼠脑海马组织细胞凋亡率和坏死率.②Bcl-2和Bax蛋白表达百分率.结果:33只大鼠全部进入结果分析.①缺血再灌注7 d组海马神经元的凋亡率最高[(24.59±0.97)%],坏死率峰值出现在缺血再灌注24 h组[(16.67±1.04)%],明显高于假手术对照组[(1.28±0.50)%,(0.90±0.38)%](P＜0.01).②假手术对照组Bcl-2表达极低[(1.07±0.27)%],但Bax有高表达[(46.09±5.37)%].③Bcl-2蛋白峰值出现在缺血再灌注后48 h[(14.41±0.67)%],而Bax蛋白峰值出现在缺血再灌注之后72 h[(77.38±1.52)%].结论:全脑缺血再灌注损伤后海马神经元凋亡率逐渐增高,凋亡调控基因Bcl-2和Bax表达异常增高,提示Bcl-2和Bax蛋白参与了全脑缺血再灌注损伤的凋亡调节.     

AMPK信号通路在异丙酚减轻老年大鼠短暂脑缺血再灌注损伤中的作用

目的探讨AMPK信号通路在异丙酚减轻老年大鼠短暂脑缺血再灌注损伤中的作用。方法健康雄性SD老龄大鼠(18²2月龄)150只,体重45022月龄)150只,体重450⁶00 g,采用随机数字表法将其分为5组(n=30):对照组(C组);假手术组(S组);缺血再灌注组(IR组);缺血再灌注组+异丙酚组(IR+P组)和缺血再灌注组+异丙酚组+AMPK激活剂组(IR+P+Y组)。采用Pusinelli四动脉阻断法建立全脑缺血模型。于缺血前10 min腹腔内注射异丙酚100 mg/kg,于夹闭动脉前时腹腔注射AMPK激活剂AICAR 500 mg/kg,假手术组仅暴露两侧翼孔和颈总动脉,对照组不行任何处理。采用Lawner等制定的评分标准神经行为学评估。于再灌注1、3和5 d时每组随机取10只处死,采用TUNEL法检测神经元的凋亡情况;采用Western blot法测定大鼠海马内AMPK、pAMPK、Bcl-2和Bax的表达。结果与C组比较,IR组、IR+P组和IR+P+Y组老年大鼠缺血/再灌注24 h后神经行为学评分升高,海马区TUNEL阳性神经元计数增加,海马AMPK、pAMPK表达水平升高,Bcl-2表达下调,Bax表达上调(P<0.05),与IR组比较,IR+P组老年大鼠缺血/再灌注24 h后神经行为学评分降低、海马区TUNEL阳性神经元计数减少、海马AMPK、pAMPK表达水平降低,Bcl-2表达上调、Bax表达下调(P<0.05),与IR+P组比较,IR+P+Y组老年大鼠缺血/再灌注24 h后神经行为学评分升高,海马区TUNEL阳性神经元计数增加,海马AMPK、p-AMPK表达水平升高,Bcl-2表达下调,Bax表达上调(P<0.05)。结论 AMPK信号通路参与了异丙酚减轻老年大鼠短暂脑缺血再灌注损伤的过程。     

大鼠局灶性脑缺血再灌注时c－fos、bcl－2蛋白的动态变化

目的:探讨细胞凋亡在脑缺血再灌注时脑损伤过程中的作用。方法 :采用免疫组化法检测大鼠脑缺血再灌注不同时间内c -fos、bcl -2蛋白表达的水平。结果 :(1)脑缺血再灌注时在缺血侧皮层和基底节区可见c -fos阳性表达 ,并于缺血30min再灌注1h时阳性表达达高峰 ;(2)脑缺血再灌注时缺血侧皮层和基底节区均有bcl-2阳性表达 ,并随再灌注时间不同 ,其阳性表达率亦不同。于缺血2h再灌注3h时阳性表达达高峰。结论 :c -fos和bcl-2参与大鼠脑缺血再灌注时缺血性细胞损伤(包括细胞凋亡)的发生     

神经生长因子预处理对沙土鼠全脑缺血/再灌注损伤脑神经细胞凋亡及Bcl-2和Bax蛋白表达的影响

目的探讨神经生长因子(NGF)预处理对沙土鼠全脑缺血/再灌注(I/R)损伤的脑保护作用的可能机制及最佳给药时间窗。方法采用夹闭沙土鼠双侧颈总动脉造成全脑I/R损伤模型。采用NGF侧脑室注射法进行预处理。沙土鼠30只随机分为5组,每组6只:假手术组(A组)、I/R损伤组(B组)、NGF预处理12、24和48h组(C、D、E组),除A组外各组分别于脑缺血20min、再灌注72h后处死取标本。用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法(TUNEL)检测沙土鼠全脑I/R损伤后脑皮质及海马CA1区凋亡神经细胞,用免疫组化法检测凋亡相关调控基因Bcl-2、Bax蛋白的表达。结果与B组比较,NGF预处理各组可显著减少沙土鼠全脑I/R损伤后脑皮质及海马CA1区神经细胞凋亡数目(P均<0.05),诱导Bcl-2蛋白及抑制Bax蛋白的表达(P均<0.05),其中以NGF预处理48h时脑皮质及海马CA1区细胞凋亡指数和Bax蛋白表达的阳性细胞指数最低,Bcl-2蛋白表达的阳性细胞指数最高。结论NGF预处理能明显减轻沙土鼠全脑I/R损伤引起的神经细胞凋亡,而以NGF预处理48h对脑保护效果最好;其抑制神经细胞凋亡的机制可能是通过调节凋亡相关调控基因Bcl-2及Bax的不同表达来发挥作用。     

脑缺血/再灌注后缺血核心区皮质内MCP-1mRNA和蛋白质表达

目的　观察大鼠脑缺血 /再灌注后缺血核心区皮质内单核细胞趋化蛋白 (MCP) - 1mRNA和蛋白质表达情况。方法　用半定量的逆转录PCR(RT -PCR)法测定缺血 2h再灌注不同时间缺血核心区脑皮质内MCP - 1mRNA的表达 ,ELISA法测定MCP - 1含量的变化。结果　缺血核心区脑皮质内MCP - 1的mRNA表达于缺血 2h明显升高 ,再灌注后 16h达高峰(均值是缺血 2h组的 2 2倍 ,与缺血 2h组相比差异显著 ,P <0 0 1) ,此后仍维持较高水平的表达直至再灌注后 4 8h(与假手术组相比 ,P <0 0 5 ) ;MCP - 1含量于再灌注后 6h开始升高 (均值 2 2 5ng/g组织 ,是假手术组的 17 0倍 ,P <0 0 5 ) ,此后逐渐升高 ,4 8h达到高峰 (均值 110 9ng/g组织 ,是假手术组的 83 7倍 ,P <0 0 1)。结论　脑缺血 /再灌注后缺血核心区皮质内MCP - 1的mRNA和蛋白质表达均明显增加 ,提示MCP - 1在脑缺血 /再灌注损伤中发挥重要作用     

Combinatorial effect of probucol and cilostazol in focal ischemic mice with hypercholesterolemia

Hypercholesterolemia may increase stroke risk by accelerating atherosclerosis, narrowing the luminal diameter in cerebral vessels, and disrupting both vascular endothelial and smooth muscle function. In the present study, we investigated the beneficial effects of combinatorial therapy with probucol and cilostazol on focal cerebral ischemia with hypercholesterolemia. Apolipoprotein E (ApoE) knockout (KO) mice were fed a high-fat diet with or without 0.5% probucol and/or 0.2% cilostazol for 10 weeks. Probucol alone and probucol and cilostazol significantly decreased total, low-density lipoprotein, and high-density lipoprotein cholesterol, whereas cilostazol did not affect the plasma cholesterol levels in ApoE KO mice. Administration of probucol alone and cilostazol alone significantly decreased atherosclerotic lesion area in the aorta, with a significant decrease evident using combinatorial administration. Middle cerebral artery occlusion resulted in significantly larger infarct volumes in ApoE KO mice fed 10 weeks of high-fat diet compared with those in ApoE KO mice fed a regular diet. The infarct volume was reduced significantly using probucol alone or cilostazol alone and even was reduced significantly by their combinatorial administration. Consistent with a larger infarct size, the combinatorial therapy prominently improved neurological function. The combinatorial administration increased cerebral blood flow during ischemia. Expression of endothelial nitric oxide synthase and adiponectin in the cortex were decreased by high-fat diet but were elevated by combinatorial treatment. Adiponectin expression colocalized within the cerebral vascular endothelium. The data suggest that the combination of probucol and cilostazol prevents cerebrovascular damage in focal cerebral ischemic mice with hypercholesterolemia by up-regulation of endothelial nitric oxide synthase and adiponectin.     

丙泊酚对大鼠肾缺血再灌注损伤细胞凋亡信号通路的影响

背景:肾移植中肾缺血再灌注损伤能引发凋亡程序的执行,丙泊酚可能的肾保护作用及其与细胞凋亡通路间的关系,有助于探讨丙泊酚的作用机制.目的:探讨丙泊酚对大鼠肾缺血再灌注损伤细胞凋亡通路的影响及可能的作用机制.设计,时间及地点:动物实验,细胞形态学观察,于2004/2005在北京友谊医院泌尿外科研究所实验室共同设计和完成.材料:选取封闭群SD成年雄性大鼠99只.兔抗鼠Bcl-2、Bax、caspase3、cytochrome C均为武汉博士德生物工程有限公司产品.方法:将动物随机分为对照组26只、缺血再灌注组35只、缺血再灌注+丙泊酚组38只.建立大鼠肾缺血再灌注损伤模型,术前单次静脉缓慢输注乳酸林格液5 mL/kg,逐层正中切口开骏,切除右肾,用无创血管夹夹闭左侧肾蒂,缺血时间45 min,松夹再灌注后全层缝合腹壁,取再灌注0,3,12,24,72 h左侧肾脏,取肾同时采血处死动物.缺血再灌注+丙泊酚组在缺血前15 min用药,丙泊酚1 mg/(kg·min)直至再灌注后30 min,共用药75 min.对照组和缺血再灌注组以乳酸林格液等量输注.主要观察指标:观察损伤肾的形态学改变,用免疫组织化学观察细胞凋亡相关蛋白Bcl-2、Bax、caspase 3和细胞色素C的表达情况.结果:光镜可观察到肾缺血再灌注引起的肾损害,程度依次为近曲小管、远曲小管、集合管、肾小球;免疫组化图像分析观察到缺血再灌注组与对照组相比,Bax、caspase 3,细胞色素C表达增加,Bcl-2表达未见明显变化;缺血再灌注+丙泊酚组与对照组比较,前者Bax、caspase 3和细胞色素C表达下降,Bcl-2表达增高(P＜0.05).结论:丙泊酚对肾缺血再灌注损伤引起的细胞凋亡可能具有保护作用,可能机制是抑制促凋亡蛋白Bax、caspase 3和细胞色素C的表达,对Bcl-2的表达无影响,与细胞凋亡的线粒体通路途径有关.     

西洛他唑对慢性脑缺血大鼠皮层HIF-1α、HO-1影响

目的观察西洛他唑对慢性脑缺血大鼠皮层中低氧诱导因子-1α（HIF-1α）和HO-1表达的影响并探讨其发病机制。方法采用大鼠双侧颈总动脉永久性阻断法（2-VO）建立动物模型,Wistar大鼠随机分为假手术组、2-VO组和西洛他唑组,选取9周（n=10）时间点,应用Morris水迷宫实验测定大鼠的学习记忆能力,采用免疫组化法检测大鼠皮层HIF-1α和HO-I蛋白的表达。结果 2VO组和西洛他唑组大鼠逃避潜伏期和游泳路程均显著增加,但西洛他唑组大鼠的学习记忆成绩显著优于2VO组;2VO组和西洛他唑组大鼠皮层HIF-1α和HO-1的表达均较假手术组明显增高,但西洛他唑组两种蛋白的表达均较2VO组明显降低。结论西洛他唑可提高缺血脑组织细胞内氧浓度,改善学习记忆功能,对慢性脑缺血具有确切的保护作用。     

p38 MAPK在大鼠局灶性脑缺血再灌注损伤中的作用

目的探讨p38 MAPK在大鼠脑缺血再灌注损伤中的作用及可能机制。方法雄性SD大鼠随机分为5组:空白对照组、假手术组、缺血再灌注组、给药组及溶媒组。除对照组及假手术组外各组采用线栓法建立大鼠大脑中动脉缺血再灌注损伤模型。给药组和溶媒组分别侧脑室注射p38 MAPK特异性抑制剂SB202190及1%DMSO。每组分别进行行为学评分和检测Bcl-2、Bax表达的变化。结果①行为学结果:空白对照组及假手术组,缺血再灌注后24 h大鼠神经功能评分降低,给药组大鼠神经功能评分高于缺血再灌注组,有统计学差异(P<0.01)。②免疫印迹检测结果:空白对照组及假手术组可见少量Bcl-2、Bax蛋白表达,两组间无统计学差异(P>0.05);缺血再灌注组再灌注后24 h可见Bcl-2表达减少,但与空白对照组及假手术组相比无统计学差异(P>0.05),而再灌注后24 h可见Bax蛋白表达明显增加,与空白对照组及假手术组相比有统计学差异(P<0.05);给药组与缺血再灌注组相比再灌注后24 h可见Bax蛋白表达明显减少(P<0.05),而Bcl-2蛋白表达明显增加(P<0.05)。结论抑制p38 MAPK激活可以减轻大鼠脑缺血再灌注时的脑神经元的凋亡。     

西洛他唑对慢性脑缺血大鼠学习记忆能力的影响

目的观察慢性脑缺血致大鼠学习记忆功能及西洛他唑对其的影响。方法结扎大鼠双侧颈总动脉（2VO）建立慢性脑缺血模型。雄性Wistar大鼠随机分为假手术组、缺血组和西洛他唑组,每组又分为3、6、9周三个时间点;应用Morris水迷宫评定不同时期大鼠认知功能,通过HE染色观察神经细胞形态学改变。结果Morris水迷宫表明,各时间点西洛他唑组和假手术组大鼠游迷宫平均潜伏期均明显低于缺血组（P〈0.05）;HE染色显示假手术组和西洛他唑组皮层、海马CA1区神经元缺血性改变较缺血组改变轻,6、9周较3周给药组缺血改变明显。结论西洛他唑能改善慢性脑缺血大鼠的学习记忆能力,可能通过减轻神经元损伤作用改善慢性脑缺血大鼠学习记忆障碍。