Ucf-101对大鼠局灶性脑缺血再灌注后Caspase-3蛋白表达的影响

目的观察Ucf—101对大鼠脑缺血再灌注后神经元caspase-3蛋白表达及细胞凋亡的影响,研究其对缺血性脑损伤是否具有保护作用。方法将36只雄性WiStar大鼠随机分为3组：假手术组、缺血组及Ucf—101组,采用线栓法建立大鼠右侧大脑中动脉闭塞（middle cerebral artery occlusion,MCAO）2h再灌注模型,于再灌注后6h和24h断头取脑,采用TTC法测梗死体积,TUNEL法原位标记DNA片段,检测TUNEL阳性细胞的变化,免疫组化法观察脑皮质神经元caspase-3的表达。结果脑缺血再灌注后不同时间点（6h、24h）,Ucf-101组与缺血组相比梗死体积明显缩小,有显著性差异（P〈0．05）;假手术组未见梗死现象。缺血组TUNEL阳性细胞数较假手术组明显增多（P〈0．05）,脑皮质caspase-3的表达较假手术组亦显著增强（P〈0．05）,给予Ucf-101处理后,TUNEL阳性细胞数较缺血组明显减少（P〈0．05）,caspase-3的表达较缺血组亦明显减弱（P〈0．05）。结论Ucf-101能有效地抑制脑缺血再灌注损伤,下调脑皮质神经元Caspase-3蛋白的表达,抑制神经元的凋亡,发挥神经保护作用。     

The protective effects and potential mechanism of Calpain inhibitor Calpeptin against focal cerebral ischemia–reperfusion injury in rats

To demonstrate the protective effects of Calpeptin as the Calpain inhibitor against focal cerebral ischemia–reperfusion injury in rats and to explore it’s possible mechanism. 96 rats were randomly divided into four groups. The model of middle cerebral artery occlusion was used for the research of focal cerebral ischemia. Using this animal model, the effects of Calpeptin on the neurological functions, infarction volume and infarction volume percentage of brain, Caspase-3 expression and neuronal apoptosis in hippocampal CA1 sector after focal cerebral ischemia–reperfusion injury in rats were investigated. The current results confirmed that Calpeptin as the Calpain inhibitor might paly an important role for neuroprotection against focal cerebral ischemia–reperfusion injury. Calpeptin could reduce the neuronal apoptosis in hippocampal CA1 sector when the rats was subjected to the focal cerebral ischemia–reperfusion, the potential mechanism might be related to the inhibition of the expression of Caspase-3 by Calpeptin. However, it is still unknown to what the exact mechanism of Calpeptin inhibits the activation of Caspase-3 in this process. Therefore, further research needs to be done to unravel the underlying mechanisms in the future.     

Cross-talk Between Calpain and Caspase-3 in Penumbra and Core During Focal Cerebral Ischemia-reperfusion

Aims Some data have shown the functional connection between calpain and caspase-3. Here, we investigated the cross-talk between calpain and caspase-3 in penumbra and core during focal cerebral ischemia-reperfusion. Methods The activities of calpain and the levels of calpastatin, microtubule-associated protein-2 (MAP-2), and spectrin in penumbra and core at 3 or 23 h of reperfusion (R 3 h or R 23 h) after 1-h focal cerebral ischemia in rats were determined in sham- or caspase-3 inhibitor z-DEVD-CHO-treated rats.On the other hand, the determination of the activities of caspase-3 and the levels of MAP-2 and spectrin was done in sham- or calpain-inhibitor I-treated rats. Results z-DEVD-CHO (600 ng/rat, i.c.v.) markedly reduced the μ- and m-calpain activities in penumbra and the m-calpain activities in core at R 3 h and R 23 h, and enhanced the calpastatin levels in penumbra at R 3 h and in core at R 3 h and R 23 h significantly; however, it had no significant effects on the μ-calpain activities in core and the calpastatin levels in penumbra at R 23 h. Calpain inhibitor I (0.8 mg/rat, i.c.v.) markedly reduced the caspase-3 activities in core at R 3 h and R 23 h, but not in penumbra. Both calpain and caspase-3 inhibitors increased the levels of MAP-2 and spectrin in penumbra and core significantly after focal cerebral ischemia-reperfusion. Conclusions Our data provide direct evidence to demonstrate the cross-talk between calpain and caspase-3 in penumbra and core during focal cerebral ischemia-reperfusion.     

The serine protease Omi/HtrA2 is involved in XIAP cleavage and in neuronal cell death following focal cerebral ischemia/reperfusion

Omi/HtrA2 is a pro-apoptotic mitochondrial serine protease involved in both forms of apoptosis, caspase-dependent as well as caspase-independent cell death. However, the impact of Omi/HtrA2 in the apoptotic cell machinery that takes place in vivo under pathological conditions such as cerebral ischemia remains unknown. The present study was monitored in order to examine whether Omi/HtrA2 plays a decisive role in apoptosis observed after focal cerebral ischemia in rats. Male adult rats were subjected to 90min of focal cerebral ischemia followed by reperfusion and treated with vehicle or ucf-101, a novel and specific Omi/HtrA2 inhibitor, prior reperfusion. Focal cerebral ischemia/reperfusion induced a mitochondrial up-regulation of Omi/HtrA2 and significantly increased cytosolic accumulation of Omi/HtrA2. Furthermore, ischemia led to activation of caspase-3 and degradation X-linked inhibitor of apoptosis protein (XIAP). Treatment of animals prior ischemia with ucf-101, the specific inhibitor of Omi/HtrA2, was able to (1) reduce the number of TUNEL-positive cells, to (2) attenuate the XIAP-breakdown and to (3) reduce the infarct size. This study shows for the first time that focal cerebral ischemia in rats results in Omi/HtrA2 translocation from the mitochondria to the cytosol, where it participates in neuronal cell death. Blocking the proteolytic activity of Omi/HtrA2 with specific inhibitors, such as the ucf-101, could be a novel way to afford neuroprotection and minimize cellular damage in cerebral ischemia/reperfusion.     

Free radical spin trap alpha-phenyl-N-tert-butyl-nitron inhibits caspase-3 activation and reduces brain damage following a severe forebrain ischemic injury.

It has been documented that alpha-phenyl-N-tert-butyl-nitron (PBN) possesses a potent neuroprotective effect when administered after transient focal cerebral ischemia. However, contradicting results were reported regarding its effect in transient global ischemia. To further elucidate the mechanism of PBN action, we have studied the effect of PBN on animal survival, histopathological outcome, and activation of caspase-3 following 30 min of global ischemia in vehicle- and PBN-treated rats. The results showed that 30 min of global ischemia was such a severe insult that no animal could survive beyond 2 d of reperfusion. Histopathological evaluation showed severe tissue edema and microinfarct foci in the neocortex and thalamus. Close to 100% damage was observed in the stratum and hippocampal CA1, CA3, and dentate gyrus subregions. Postischemic PBN treatment significantly enhanced animal survival and reduced damage in the neocortex, thalamus, and hippocampus. Immunohistochemistry demonstrated that caspase-3 was activated following ischemia in the striatum and the neocortex. PBN suppressed the activation of caspase-3 in both structures. It is concluded that PBN is a potent neuroprotectant against both focal and global ischemia; besides its function as a free radical scavenger, PBN may reduce ischemic brain damage by blocking cell death pathways that involve caspase-3 activation.     

Intranasal Delivery of a Caspase-1 Inhibitor in the Treatment of Global Cerebral Ischemia

Caspase-1 is an enzyme implicated in neuroinflammation, a critical component of many diseases that affect neuronal degeneration. However, it is unknown whether a caspase-1 inhibitor can modify apoptotic neuronal damage incurred during transient global cerebral ischemia (GCI) and whether intranasal administration of a caspase-1 inhibitor is an effective treatment following GCI. The present study was conducted to examine the potential efficiency of post-ischemic intranasal administration of the caspase-1 inhibitor Boc-D-CMK in a 4-vessel occlusion model of GCI in the rat. Herein, we show that intranasal Boc-D-CMK readily penetrated the central nervous system, subsequently inhibiting caspase-1 activity, decreasing mitochondrial dysfunction, and attenuating caspase-3-dependent apoptotic pathway in ischemia-vulnerable hippocampal CA1 region. Further investigation regarding the mechanisms underlying Boc-D-CMK’s neuroprotective effects revealed marked inhibition of reactive gliosis, as well as reduction of the neuroinflammatory response via inhibition of the downstream pro-inflammatory cytokine production. Intranasal Boc-D-CMK post-treatment also significantly enhanced the numbers of NeuN-positive cells while simultaneously decreasing the numbers of TUNEL-positive and PARP1-positive cells in hippocampal CA1. Correspondingly, behavioral tests showed that deteriorations in spatial learning and memory performance, and long-term recognition memory following GCI were significantly improved in the Boc-D-CMK post-treated animals. In summary, the current study demonstrates that the caspase-1 inhibitor Boc-D-CMK coordinates anti-inflammatory and anti-apoptotic actions to attenuate neuronal death in the hippocampal CA1 region following GCI. Furthermore, our data suggest that pharmacological inhibition of caspase-1 is a promising neuroprotective strategy to target ischemic neuronal injury and functional deficits following transient GCI.     

The Effects of Middle Cerebral Artery Occlusion on Central Nervous System Apoptotic Events in Normal and Diabetic Rats

Apoptosis and neural degeneration are characteristics of cerebral ischemia and brain damage. Diabetes is associated with worsening of brain damage following ischemic events. In this study, the authors characterize the influence of focal cerebral ischemia, induced by middle cerebral artery occlusion, on 2 indexes of apoptosis,TUNEL(terminal deoxynucleotidyl transferase–mediated deoxyuridine 5-triphosphate nick end-labeling) staining and caspase- 3 immunohistochemistry. Diabetes was induced in normal rats using streptozotocin and maintained for 5 to 6 weeks. The middle cerebral artery of both normal and diabetic rats was occluded and maintained from 24 or 48 hours. Sham-operated normal and diabetic animals served as controls. Following 24 to 48 hours of occlusion, the animals were sacrificed and the brains were removed, sectioned, and processed for TUNEL staining or caspase-3 immunohistochemistry. Middle cerebral artery occlusion in normal rats was associated with an increase in the number of both TUNEL-positive and caspase-3– positive cells in selected brain regions (hypothalamic preoptic area, piriform cortex, and parietal cortex) when compared to nonoccluded controls. Diabetic rats without occlusion showed significant increases in both TUNEL-positive and caspase-3–positive cells compared to normal controls. Middle cerebral artery occlusion in diabetic rats resulted in increases in TUNEL-positive as well as caspase-3–positive cells in selected regions, above those seen in nonoccluded diabetic rats. Both TUNEL staining and caspase-3 immunohistochemistry revealed that the number of apoptotic cells in diabetic animals tended to be greatest in the preoptic area and parietal cortex. The authors conclude that focal cerebral ischemia is associated with a significant increase in apoptosis in nondiabetic rats, and that diabetes alone or diabetes plus focal ischemia are associated with significant increases in apoptotic cells.     

Is neuroprotective efficacy of nNOS inhibitor 7-NI dependent on ischemic intracellular pH?

The purpose of this study was to test the hypothesis that the efficacy of 7-nitroindazole (7-NI), a selective neuronal nitric oxide (NO) synthase (NOS) inhibitor, is pH dependent in vivo during focal cerebral ischemia. Wistar rats underwent 2 h of focal cerebral ischemia under 1% halothane anesthesia. 7-NI, 10 and 100 mg/kg in 0.1 ml/kg DMSO, was administered 30 min before occlusion. Ischemic brain acidosis was manipulated by altering serum glucose concentrations. Confirmation of the effects of these serum glucose manipulations on brain intracellular pH (pH(i)) was confirmed in a group of acute experiments utilizing umbelliferone fluorescence. The animals were euthanized at 72 h for histology. 7-NI significantly (P < 0.05) reduced infarction volume in both the normoglycemic by 93.3% and hyperglycemic animals by 27.5%. In the moderate hypoglycemic animals, the reduction in infarction volume did not reach significance because moderate hypoglycemia in itself dramatically reduced infarction volume. We hypothesize that a mechanism to explain the published discrepancies on the effects of neuronal NOS inhibitors in vivo may be due to the effects by differences in ischemic brain acidosis on the production of NO.     

Sevoflurane reduces ischemic brain injury in rats with diet and streptozotocin-induced diabetes

Abstract

To investigate the role of S100B, oxidative stress and the apoptosis signaling pathways in the sevoflurane induced neuroprotective effect on stroke. The brain injury, molecular and cellular damage, and functional recovery were investigated upon ischemic brain injury followed by sevoflurane treatment. Longa rodent stroke scales was used to quantify neurological deficits. TTC staining was used to measure infarct volume of brain tissue. Absolute brain water content was measured by wet/dry weight method. The neuronal morphological change was assessed by H and E staining. The spatial learning and memory ability were measured by water maze test. Serum proteins including S100B, GSH-PX, SOD, Bcl-2, Bax, Caspase-3 were measured by ELISA. The level of NOS and NO in serum was determined by colorimetric method. Compared with control, the serum proteins including S100B, Bax, NO, Caspase-3, and NOS activity in cerebral infarction rats increased significantly while SOD, GSH-PX, and Bcl-2 decreased significantly. Diabetic mellitus complicated with cerebral infarction rats showed more dramatic increase for S100B, Bax, NO, Caspase-3, and NOS activity and dramatic decrease for SOD, GSH-PX, and Bcl-2. Interestingly, sevoflurane reduced the changes significantly. The S100B level positively correlated with brain damage, NO, Bax, caspase-3, and NOS activity but negatively correlated with SOD, Bax, and GSH-PX. Brain damage in sevoflurane groups decreased while behavior outcomes including Longa neurologic score, learning, and memory increased significantly. The neuroprotective effect of sevoflurane is associated with defense mechanisms against free radical-induced oxidative stress and inhibition of apoptosis. S100B protein correlated with oxidative stress and the apoptosis signaling pathways.     

Bcl-2 overexpression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome c translocation and caspase-3 activity

Bcl-2 protects against both apoptotic and necrotic death induced by several cerebral insults. We and others have previously demonstrated that defective herpes simplex virus vectors expressing Bcl-2 protect against various insults in vitro and in vivo, including cerebral ischemia. Because the infarct margin may be a region that is most amenable to treatment, we first determined whether gene transfer to the infarct margin is possible using a focal ischemia model. Since ischemic injury with and without reperfusion may occur by different mechanisms, we also determined whether Bcl-2 protects against focal cerebral ischemic injury either with or without reperfusion in rats. Bax expression, cytochrome c translocation and activated caspase-3 expression were also assessed. Viral vectors overexpressing Bcl-2 were delivered to the infarct margin. Reperfusion resulted in larger infarcts than permanent occlusion. Bcl-2 overexpression significantly improved neuron survival in both ischemia models. Bcl-2 overexpression did not alter overall Bax expression, but inhibited cytosolic accumulation of cytochrome c and caspase-3 activation. Thus, we provide the first evidence that gene transfer to the infarct margin is feasible, that overexpression of Bcl-2 protects against damage to the infarct margin induced by ischemia with and without reperfusion, and that Bcl-2 overexpression using gene therapy attenuates apoptosis-related proteins. This suggests a potential therapeutic strategy for stroke.     

Specific Alterations of the HtrA2/HAX-1 Ratio in the Penumbra Upon Focal Cerebral Ischemia in Mice

HAX-1 is a mitochondrial protein which acts as an antiapoptotic protein in HeLa- and Jurkat cells after Fas-treatment, irradiation or serum deprivation. This underlines the evidence that HAX-1 might be involved in several apoptotic pathways. In this context, it is known that cell death executed by cerebral ischemia involves both receptor- and mitochondrial apoptotic mechanisms. In this study, we performed focal cerebral ischemia in mice and investigated principally the dynamic changes of HAX-1 expression and other apoptotic agents such as HtrA2, AIF and caspase-3. Western blot and immunohistochemistry analysis revealed that HAX-1 was expressed at very low levels under normal conditions. Focal cerebral ischemia significantly decreased cytosolic accumulation of HAX-1, induced an upregulation of HtrA2, an upregulation of AIF and activation of caspase-3. Taken together, these results suggested that HAX-1 is probably involved in the pathophysiology of cell death induced by focal ischemia.     

CEREBRAL METABOLIC AND VASCULAR EFFECTS OF BARBITURATE THERAPY FOLLOWING COMPLETE GLOBAL ISCHEMIA

Complete global cerebral ischemia was induced in dogs by temporary ligation of the ascending aorta for 10min. Prior to the ischemic period, half of the animals were given pentobarbital 30-38 mg/kg, a maneuver previously reported to prevent or attenuate cerebral damage in this same model. Cerebral blood flow (CBF) and cerebral metabolic rate (CMR_O2) were followed from prior to the ischemic period to 6 h post-ischemia. At varying time intervals following ischemia, brain biopsies were obtained and analyzed for cerebral metabolites to determine the cerebral energy state. Only a few differences were observed between pentobarbital-treated and untreated animals. Post-ischemic CMR_O2, stabilized at a significantly lower level in treated than in untreated animals. However, CBF was proportionately lower and thus O₂ delivery relative to O₂ needs in the two groups was comparable. Also in both groups, the CBF and CMR_O2 stabilized at levels significantly below pre-ischemia controls. Cerebral energy stores in both groups were depleted after 10min of ischemia but were restored to near normal within 4min post-ischemia. Total restoration of the adenine nucleotide pool and ATP were delayed as was the return of brain lactate to normal. A 10min period of post-ischemic hyperemia was observed in all animals and in the initial 4min post-ischemia CMR_O2 was also increased. The latter is probably accounted for by the O₂ needs for restoration of cerebral energy and O₂ stores. We conclude that cerebral protection as provided by barbiturates following complete global ischemia cannot be accounted for by any measurable effect on CBF, CMR_O2, or the cerebral energy stores during the initial 6 h post-ischemia.     

Caspase-3 activation is an early event and initiates apoptotic damage in a human leukemia cell line

Many apoptotic pathways culminate in the activation of caspase cascades usually triggered by the apical caspases-8 or -9. We describe a paradigm where apoptosis is initiated by the effector caspase-3. Diethylmaleate (DEM)-induced apoptotic damage in Jurkat cells was blocked by the anti-apoptotic protein Bcl-2, whereas, a peptide inhibitor of caspase-3 but not caspase-9 blocked DEM-induced mitochondrial damage. Isogenic Jurkat cell lines deficient for caspase-8 or the adaptor FADD (Fas associated death domain) were not protected from DEM-induced apoptosis. Caspase-3 activation preceded that of caspase-9 and initial processing of caspase-3 was regulated independent of caspase-9 and Bcl-2. However, inhibitors of caspase-9 or caspase-6 regulated caspase-3 later in the pathway. We explored the mechanism by which caspase-3 processing is regulated in this system. DEM triggered a loss of Erk-1/2 phosphorylation and XIAP (X-linked inhibitor of apoptosis protein) expression. The phorbol ester PMA activated a MEK-dependent pathway to block caspase-3 processing and cell death. Constitutively active MEK-1 (CA-MEK) upregulated XIAP expression and exogenous XIAP inhibited DEM-induced apoptotic damage. Thus, we describe a pathway where caspase-3 functions to initiate apoptotic damage and caspase-9 and caspase-6 amplify the apoptotic cascade. Further, we show that MEK may regulate caspase-3 activation via the regulation of XIAP expression in these cells.     

Green tea polyphenol (−)-epigallocatechin gallate reduces matrix metalloproteinase-9 activity following transient focal cerebral ischemia

Green tea polyphenol (?)-epigallocatechin gallate (EGCG) has been reported to reduce neuronal damage after cerebral ischemic insult. EGCG is known to reduce matrix metalloproteinase (MMP) activity. MMP can play an important role in the pathophysiology of neurological disorders including cerebral ischemia. The purpose of the current study was to investigate whether EGCG shows an inhibitory effect on MMP activity and neural tissue damage following transient focal cerebral ischemia. In the present study, C57BL/6 mice were subjected to 80 min of focal ischemia induced by middle cerebral artery occlusion (MCAO). Animals were killed 24 h after ischemia. EGCG (50 mg/kg) was administered intraperitoneally immediately after ischemia. Gelatin gel zymography showed an increase in the active form of MMP-9 after ischemia. EGCG reduced ischemia-induced up-regulation of the active form of MMP-9. In in situ zymography, EGCG reduced up-regulation of gelatinase activity induced by cerebral ischemia. Co-incubation with EGCG reduced gelatinase activity directly in postischemic brain section. In 2,3,5-triphenyltetrazolium chloride (TTC) assay, brain infarction was remarkable in the middle cerebral artery territory after focal cerebral ischemia. In EGCG-treated mice, infarct volume was significantly reduced compared with vehicle-treated mice. These results demonstrate that EGCG, a green tea polyphenol, may reduce up-regulation of MMP-9 activity and neuronal damage following transient focal cerebral ischemia. In addition to its antioxidant effect, MMP-9 inhibition might be a possible mechanism potentially involved in the neuroprotective effect of a green tea polyphenol, EGCG.     

Degradation of the type I inositol 1,4,5-trisphosphate receptor by caspase-3 in SH-SY5Y neuroblastoma cells undergoing apoptosis

The type I inositol 1,4,5-trisphosphate (IP(3)) receptor is selectively down-regulated in several neurodegenerative diseases, including Alzheimer's disease, Huntington's chorea, and ischemia, all conditions in which apoptotic neuronal loss occurs. In the present study, we used a neuronal cell line, human neuroblastoma SH-SY5Y cells, to investigate whether the levels of IP(3) receptor are changed during apoptosis in these cells. Following induction of apoptosis by staurosporine, the immunoreactivity of the type I IP(3) receptor in microsome preparations from SH-SY5Y cells was reduced within 2 h, with a further reduction during subsequent hours. Immunoblot analyses, using antibodies to poly(ADP-ribose) polymerase and spectrin breakdown products, revealed proteolysis of these caspase-3 substrates within 3 h, confirming that IP(3) receptor cleavage is an early consequence of apoptosis. In vitro incubation of SH-SY5Y microsomes or immunopurified IP(3) receptor from rat cerebellum with recombinant caspase-3 led to generation of immunoreactive breakdown products similar to those observed in intact cells, suggesting that the type I IP(3) receptor is a potential substrate for caspase-3. Preincubation of the neuroblastoma cells with the caspase-3 inhibitor Z-Asp-Glu-Val-Asp-fluoromethyl ketone prevented IP(3) receptor degradation. These results show that the type I IP(3) receptor is a substrate for caspase-3 in neuronal cells and indicate that apoptotic down-regulation of IP(3) receptor levels may contribute to the pathology of neurodegenerative conditions.     

Quercetin Attenuates Cell Apoptosis in Focal Cerebral Ischemia Rat Brain Via Activation of BDNF–TrkB–PI3K/Akt Signaling Pathway

Many studies have demonstrated that apoptosis play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. Neuroprotective effect of quercetin has been shown in a variety of brain injury models including ischemia/reperfusion. It is not clear whether BDNF?CTrkB?CPI3K/Akt signaling pathway mediates the neuroprotection of quercetin, though there has been some reports on the quercetin increased brain-derived neurotrophic factor (BDNF) level in brain injury models. We therefore first examined the neurological function, infarct volume and cell apoptosis in quercetin treated middle cerebral artery occlusion (MCAO) rats. Then the protein expression of BDNF, cleaved caspase-3 and p-Akt were evaluated in either the absence or presence of PI3K inhibitor (LY294002) or tropomyosin receptor kinase B (TrkB) receptor antagonist (K252a) by immunohistochemistry staining and western blotting. Quercetin significantly improved neurological function, while it decreased the infarct volume and the number of TdT mediated dUTP nick end labeling positive cells in MCAO rats. The protein expression of BDNF, TrkB and p-Akt also increased in the quercetin treated rats. However, treatment with LY294002 or K252a reversed the quercetin-induced increase of BDNF and p-Akt proteins and decrease of cleaved caspase-3 protein in focal cerebral ischemia rats. These results demonstrate that quercetin can decrease cell apoptosis in the focal cerebral ischemia rat brain and the mechanism may be related to the activation of BDNF?CTrkB?CPI3K/Akt signaling pathway.     

原花青素对脑缺血再灌损伤大鼠模型的影响

目的研究原花青素对脑缺血/再灌损伤(ischemia/reperfusion,I/R)大鼠神经功能评分(neurologicaldeficit score,NDS)、脑梗死体积、脑含水量等指标的药理作用。方法采用大鼠大脑中动脉阻断(middle cerebralartery occlusion,MCAO)法复制类似人类缺血性卒中的I/R损伤模型。结果该模型各时间点内均有程度不同的神经功能缺失,原花青素给药组神经功能评分明显低于对照组(P0.05),假手术组大鼠均无神经功能缺失,脑水肿情况均较对照组明显改善(P0.05),脑梗死体积与盐水对照组相比差异有显著性(P0.05),而假手术组均未见有梗死灶。结论原花青素具有一定的保护大鼠I/R后受损脑组织的作用,可供后续研究,并可为缺血性卒中使用原花青素治疗提供确凿的理论依据。     

细胞周期抑制剂对大鼠局灶性脑缺血后神经元的保护作用

目的通过观察选择性细胞周期抑制剂olomoucine对局灶性脑缺血边缘区神经元凋亡的影响,以探讨细胞周期调控与神经元细胞凋亡的关系。方法建立光化学法诱导大鼠局灶性脑缺血模型,随机分为脑缺血组(对照组和干预组)和假手术组,采用HE染色显示梗死灶并测定其面积;应用免疫荧光化学法检测梗死灶周围神经元核心抗原(NeuN)的表达及通过TUNEL方法检测神经元凋亡;免疫印迹(Western blot)观察损伤侧皮层NeuN、周期素蛋白A(cyclin A)和周期素蛋白B1(cyclin B1)蛋白的表达。结果缺血后3d对照组梗死灶面积占脑片面积百分比值的平均值明显大于干预组(P<0.05);缺血后缺血边缘区NeuN表达减弱,对照组NeuN表达明显弱于干预组(P<0.05);缺血后梗死灶周围可见大量TUNEL阳性染色细胞,而且对照组数量明显多于干预组(P<0.05);干预组大鼠NeuN(TUNAL双标阳性表达明显弱于对照组大鼠(P<0.05);NeuN的蛋白量的表达,干预组较对照组明显增加(P<0.05),而对照组cyclin A和cyclin B1蛋白量的表达明显高于干预组(P<0.05)。结论通过对细胞周期的调控,可减少神经元凋亡和脑梗死体积,从而为缺血性脑损伤后的神经元提供一个保护作用。     

A Synergic Role of Caspase-6 and Caspase-3 in Tau Truncation at D421 Induced by H2O2

Tau truncation is widely detected in Alzheimer’s disease brain. Caspases activation is suggested to play a significant role in tau truncation at Aspartate 421 (D421) according to their ability to cleave recombinant tau in vitro. Ample evidence has shown that caspase-6 is involved in cognitive impairment and expressed in AD brain. Reactive oxygen species (ROS) can lead to caspase-6 activation and correlate with AD. Here, we transfected human embryonic kidney 293 (HEK 293) cells with Tau 441 plasmid and investigated the role of caspase-6 and caspase-3 in ROS-mediated tau truncation. Our data demonstrated that H₂O₂ induced oxidative stress and increased tau truncation. Caspase-6 and caspase-3 activity also increased in a dose-dependent manner in HEK 293/Tau cells during H₂O₂ insult. When cells were treated with an ROS inhibitor N-acetyl-l-cysteine, tau truncation was significantly suppressed. Compared with H₂O₂ (100 μM)/non-inhibitor group or single-inhibitor groups (z-VEID-fmk, caspase-6 inhibitor or z-DEVD-fmk, and caspase-3 inhibitor), tau truncation induced by H₂O₂ was effectively reduced in the combinative inhibitors group. Similar results were shown when cells were transfected with specific caspase-3 and caspase-6 siRNA. Inhibition of caspase-6 led to decline of caspase-3 activation. Taken together, our results suggest that the combination of caspase-6 and caspase-3 aggravates tau truncation at D421 induced by H₂O₂. Caspase-6 may play an important part in activating caspase-3. Further investigation of how the synergic role of caspase-6 and caspase-3 affects tau truncation may provide new visions for potential AD therapies.