小牛血去蛋白注射液对大鼠脑缺血再灌注损伤的保护作用

目的：研究小牛血去蛋白注射液(depmteinised calf blood iniection，DCBI)对大鼠脑缺血及再灌注损伤的保护作用。方法：采用SD大鼠双侧颈总动脉结扎法制作不完全性脑缺血再灌注模型，观察DCBI对脑缺血再灌注SD大鼠的脑组织水含量，脑组织丙二醛(MDA)含量的影响，并通过光镜观察其病理组织学变化。结果：缺血组与缺血再灌注组脑组织水含量、MDA含量较假手术组明显增高；DCBI组脑组织水含量、MDA含量较缺血组与缺血再灌注组下降。病理组织学检查可见缺血组与假手术组相比大脑皮层细胞出现肿胀改变。缺血再灌注组脑组织水肿加剧，细胞周围呈海绵状。用药组脑细胞仅有轻度肿胀改变。结论：DCBI对大鼠脑缺血及缺血再灌注损伤具有保护作用。     

雷公藤多苷对大鼠局灶性脑缺血再灌注损伤的保护作用

目的:观察雷公藤多苷(TwP)对局灶性脑缺血再灌注损伤大鼠的保护作用。方法:180只大鼠随机分为脑缺血再灌注组,TwP大、中、小剂量组,阳性对照组,假手术组。灌胃给药,采用大脑中动脉局灶性缺血再灌注模型,测定再灌注2 h缺血侧脑组织含水量、超氧化物歧化酶(SOD)、丙二醛含量及大脑皮层神经元细胞内[Ca2+]i水平。结果:与假手术组相比,脑缺血再灌注组脑组织丙二醛含量增加、SOD活性降低、含水量及[Ca2+]i显著升高(P<0.01);与脑缺血再灌注组相比,TwP大、中、小剂量组,阳性对照组脑组织丙二醛含量减少、SOD活性升高、[Ca2+]i显著下降(P<0.01或P<0.05),TwP大、中剂量组,阳性对照组脑组织含水量显著降低(P<0.01);TwP各剂量组间结果亦有显著差异(P<0.01或P<0.05)。结论:TwP能减轻大鼠急性脑缺血再灌注损伤引起的脑水肿、降低脑组织丙二醛含量,提高SOD活性,降低细胞内钙积累;且随着剂量增加,变化显著增强,提示雷公藤多苷对大鼠急性脑缺血再灌注损伤具有保护作用。     

脑脉通对老龄大鼠脑缺血再灌注细胞因子的影响

目的：从细胞因子方面研究脑脉通抗脑缺血损伤的作用机制。方法：以二血管阻断结合降压法建立脑缺血再灌注模型，测定脑组织病理改变、脑组织含水量变化以及血清和脑组织细胞因子水平变化。结果：脑脉通组脑组织病理损伤减轻，含水量降低。模型对照组血清及脑组织IL-1、IL-8和TNF水平较假手术对照组增高；与模型对照组比较，尼莫地平组、脑脉通大剂量组血清及脑组织IL-1、IL-8和TNF不同程度降低，脑脉通大剂量组血清IL-8水平和脑组织TNF水平降低较尼莫地平组显著。结论：脑脉通对急性脑缺血防护作用显著，其机制可能与其降低抑制IL-1、IL-8及TNF介导损害有关。     

氨基胍对大鼠局灶性缺血—再灌流后脑内MPO活性的影响

目的：探讨大鼠局灶性脑缺血后脑组织MPO活性变化规律、与脑缺血－再灌流损伤的关系以及氨基胍（AG）对其影响。方法：用线栓法制备大鼠脑中动脉缺血－再灌流模型，检测缺血3小时后再灌流不同时点脑组织中MPO活性、脑梗死体积及光镜病理学变化。结果：缺血组脑组织有MPO活性升高、中性粒细胞浸润，以再充后48、72小时最为明显，脑梗死体积、神经元变性程度随再灌流时间延长而加重，AG可以明显减轻缺血再灌流12小时后的脑组织中MPO活性、神经元变化，降低脑梗死体积，减轻脑损伤。结论：局灶性缺血脑组织中MPO活性与缺血－再灌流损伤间具有一定的关系，炎症反应是加重脑组织损伤的重要因素，AG可以减轻缺血区炎症反应，具有脑保护作用。     

三七总皂甙对缺血再灌注鼠脑组织Ca2+和兴奋性氨基酸的影响

目的：探讨Ca^2 ，兴奋性氨基酸在缺血再灌注鼠脑损伤中的作用及三七总皂甙对其影响。方法：对缺血再灌注鼠脑组织Ca^2 ，氨基酸及血清氨基酸分别进行测定，并观察三七总皂甙对其影响。结果：模型组脑组织Ca^2 ，脑和血清谷氨酸（Glu),天门冬氨酸（Asp)，甘氨酸（Gly)γ－氨基丁酸（GABA）高于正常动物组，三七总皂甙组低于模型组。结论：Ca^2 ，氨基酸参予再灌注脑损伤的病理过程，而三七总皂甙可能通过降低脑内Ca^2 ，降低兴奋性氨基酸（EAA）来保护脑组织。     

抗血小板溶栓素对局灶性脑缺血/再灌注大鼠脑损伤的作用观察

目的:观察抗血小板溶栓素(anti-platelet thrombolysin,APT)对缺血性脑损伤的保护作用并初步探讨其作用机制。方法:SD大鼠,随机分为假手术组(Sham)、模型组(缺血再灌注组)、阳性组(依达拉奉注射液)、APT高、中、低组。双侧颈动脉结扎,建立脑缺血模型,测定大鼠脑含水量,HE染色观察脑组织病理改变;线栓法建立大鼠脑缺血/再灌注模型,进行行为学评分,酶联免疫法(ELISA)及免疫组化测定脑组织中Toll样受体4(TLR4)、c-Jun氨基末端激酶(JNK)、Bax蛋白含量。结果:与模型组比较,APT高、中剂量组可明显改善缺血再灌注后大鼠神经功能障碍症状,降低脑水肿程度,改善缺血后脑组织的病理改变,明显降低缺血后脑组织中TLR4、JNK、Bax蛋白表达。结论:APT对缺血性脑损伤有较好的保护作用,其机制可能与抑制脑组织中TLR4/JNK/Bax信号通路表达,从而抑制细胞凋亡有关。     

参芎葡萄糖注射液对急性不完全性脑缺血的保护作用

目的研究参芎葡萄糖注射液对脑缺血的保护及治疗作用,为其临床应用提供科学依据。方法雄性SD大鼠随机分成6组:假手术组(等容量生理盐水),脑缺血模型组(等容量生理盐水),参芎葡萄糖注射液低、中、高剂量组(12、24、36 mg·kg-1),香丹注射液阳性对照组(3 mL·kg-1)。每日腹腔注射给药1次,连续给药3 d。检测脑指数及脑含水量、脑组织丙二醛(MDA)含量、乳酸脱氢酶(LDH)活力、观察脑组织形态学变化。结果参芎葡萄糖注射液能减轻缺血引起的脑细胞损伤,降低脑含水量及脑指数,降低缺血脑组织丙二醛含量及提高乳酸脱氢酶活力。结论参芎葡萄糖注射液对脑缺血有保护作用。     

针药结合对反复短暂脑缺血再灌注大鼠行为学及脑损伤的影响

目的：探讨针刺对脑缺血再灌注损伤的神经保护作用及对脑组织丙二醛(MDA)和谷胱甘肽(GSH)、血清降钙素基因相关肽(CGRP)影响。方法：观察大鼠颈动脉反复缺血的动物模型的行为学改变和MDA、GSH、CGRP的变化。结果：大鼠反复脑缺血再灌注3d后与模型组比较，针刺干预组和针剌加药组无论在学习和记忆方面，其时间和错误次数均有显著性差异。MDA降低，有显著性差畀。GSH升高，有显著性差异和极显著性差异。尼莫地平组和针剌加药组血清CGRP含量有极显著性差异，而针刺干预组有显著性差异。结论：针剌可以发挥对脑缺血再灌注损伤的神经保护作用。针刺与药物的结合作用较好。临床上对缺血性脑血管病应该进行综合治疗。对预防缺血性脑血管病亦有重要意义。     

二十二碳六烯酸乙酯对脑缺血再灌注氧化损伤及脑水肿的影响

目的 研究二十二碳六烯酸乙酯(ethyl docosahexaenoate，E-DHA)对沙土鼠脑缺血再灌注引起的氧化损伤和脑水肿的保护作用。方法 采用沙土鼠双侧颈总动脉阻断法制作全脑缺血再灌注损伤模型。检测E-DHA对缺血再灌注后脑中丙二醛(MDA)和谷胱甘肽(GSH)含量；谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、ATP酶活性；水及Na^ 、Ca^2 含量的影响．并进行了病理检查。结果 缺血前E-DHA预防给药10周有效阻止了脑缺血再灌注引起的MDA升高，GsH降低，GsH-Px、CAT活性降低．ATP酶活性降低，水及Ca^2 、Na^ 含量升高。结论 E-DHA对沙土鼠脑缺血再灌注具有保护作用，其机理与清除自由基和减轻脑水肿有关。     

丹皮酚抑制大鼠局灶性脑缺血再灌注损伤脑组织细胞间黏附分子-1和血管细胞黏附分子-1的表达

目的观察丹皮酚对大鼠脑缺血再灌注损伤后脑组织细胞间黏附分子-1(ICAM-1)和血管细胞黏附分子-1(VCAM-1)表达的影响,以探讨其脑保护的作用机制。方法线栓法制备大鼠局灶性脑缺血再灌注损伤模型,大鼠脑缺血2 h再灌注24 h,采用Western blot及RT-PCR法观察大鼠脑组织ICAM-1、VCAM-1的蛋白及mRNA表达。结果丹皮酚明显降低缺血区脑组织ICAM-1、VCAM-1的蛋白及mRNA的表达。结论丹皮酚可能通过降低缺血区脑组织黏附分子表达发挥脑保护作用。     

Hot topics in phospholipase A2 field

As detailed in previous reviews, phospholipase A2 (PLA2) enzymes belonging to the secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2), and Ca2+-independent PLA2 (iPLA2) families may play specific physiologic and pathologic roles. In the past two years, there have been considerable advances in the understanding of the regulatory functions of individual PLA2s. This short article focuses on the latest topics in the PLA2 field, which have offered new insights into this intriguing enzyme family. Specifically, I describe a novel cellular action and unexplored in vivo functions of sPLA2, expanding regulatory aspects of cPLA2, and unique functional roles of iPLA2 in apoptosis, Ca2+ homeostasis, and myocardial ischemia.     

Effects of an endothelin B receptor agonist on secretory phospholipase A2-IIA-induced apoptosis in cortical neurons

Endothelin (ET), a vasoconstrictive peptide, acts as an anti-apoptotic factor, and endothelin receptor B (ETB receptor) is associated with neuronal survival in the brain. Human group IIA secretory phospholipase A2 (sPLA2-IIA) is expressed in the cerebral cortex after brain ischemia and causes neuronal cell death via apoptosis. In primary cultures of rat cortical neurons, we investigated the effects of an ETB receptor agonist, ET-3, on sPLA2-IIA-induced cell death. sPLA2-IIA caused neuronal cell death in a concentration- and time-dependent manner. ET-3 significantly prevented neurons from undergoing sPLA2-IIA-induced cell death. These agonists reversed sPLA2-IIA-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Before cell death, sPLA2-IIA potentiated the influx of Ca2+ into neurons. Blockers of the L-type voltage-dependent calcium channel (L-VSCC) not only suppressed the Ca2+ influx, but also exhibited neuroprotective effects. As well as L-VSCC blockers, ET-3 significantly prevented neurons from sPLA2-IIA-induced Ca2+ influx. An ETB receptor antagonist, BQ788, inhibited the effects of ET-3. The present cortical cultures contained few non-neuronal cells, indicating that the ETB receptor agonist affected the survival of neurons directly, but not indirectly via non-neuronal cells. In conclusion, we demonstrate that the ETB receptor agonist rescues cortical neurons from sPLA2-IIA-induced apoptosis. Furthermore, the present study suggests that the inhibition of L-VSCC contributes to the neuroprotective effects of the ETB receptor agonist.     

羟基红花黄色素A对脑缺血所致大鼠脑线粒体损伤的保护作用

目的研究羟基红花黄色素A对脑缺血所致大鼠脑线粒体损伤的保护作用。方法用栓线法制作大鼠大脑中动脉缺血(MCAO)模型，测定线粒体肿胀度、膜流动性、膜磷脂含量、呼吸功能、线粒体呼吸酶、超氧化物歧化酶(SOD)、丙二醛(MDA)、Ca²⁺等。结果羟基红花黄色素A(10，20 mg·kg^-1)能明显抑制缺血脑线粒体膜流动性的降低，膜磷脂降解，减少脑缺血引起的线粒体肿胀，抑制NADH脱氢酶、琥珀酸脱氢酶和细胞色素c氧化酶活性的降低，改善线粒体呼吸功能；同时羟基红花黄色素A能明显降低中风大鼠脑细胞线粒体MDA含量、升高SOD活性、抑制Ca²⁺过多摄入。结论羟基红花黄色素A对缺血脑细胞线粒体的损伤有明显的保护作用，该作用可能与清除氧自由基、抑制脂质过氧化、拮抗Ca²⁺有关。     

丁基苯酞对大脑中动脉阻断后皮层组织中花生四烯酸释放及磷脂酶A2基因表达的影响

目的　研究丁基苯酞 (dl NBP) ,d NBP和l NBP对大脑中动脉阻断 (MCAO) 6h后缺血区皮层中花生四烯酸 (AA)释放及磷脂酶A2 (PLA2 )基因表达的影响。方法　阻断大脑中动脉起始部造成局灶性脑缺血模型。HPLC检测AA。Northernblot检测皮层中PLA2 基因表达。结果　MCAO后 6h ,皮层中AA释放明显增加。于脑缺血后 5min和 12 0min ,给dl NBP(10或 2 0mg·kg- 1)和尼莫地平 (0 5mg·kg- 1)可显著抑制AA的释放。d NBP和l NBP作用比较 ,显示d NBP有与dl NBP相似的作用 ,而l NBP则无明显影响。Northern印迹结果表明 ,脑缺血 6h ,皮层中PLA2 的基因表达增强。dl NBP和d NBP(10 ,2 0mg·kg- 1,ip)皆可使表达降低 ,而l NBP对缺血脑组织中PLA2 的基因表达的升高无明显影响。结论　dl NBP和d NBP可抑制MCAO后脑组织中AA释放和PLA2 的基因表达。     

Identification of the phospholipase A(2) isoforms that contribute to arachidonic acid release in hypoxic endothelial cells: limits of phospholipase A(2) inhibitors

Changes in endothelium functions during ischemia are thought to be of importance in numerous pathological conditions, with, for instance, an increase in the release of inflammatory mediators like prostaglandins. Here, we showed that hypoxia increases phospholipase A(2) (PLA(2)) activity in human umbilical vein endothelial cells. Both basal PLA(2) activity and PG synthesis are sensitive to BEL and AACOCF3, respectively, inhibitors of calcium-independent PLA(2) (iPLA(2)) and cytosolic PLA(2) (cPLA(2)), while OPC, an inhibitor of soluble PLA(2) (sPLA(2)) only inhibited the hypoxia-induced AA release and PGF(2alpha) synthesis. Hypoxia does not alter expression of iPLA(2), sPLA(2) and cPLA(2) and cycloheximide did not inhibit PLA(2) activation, indicating that hypoxia-induced increase in PLA(2) activity is due to activation rather than induction. However, mRNA levels for sPLA(2) displayed a 2-fold increase after 2 hr incubation under hypoxia. BAPTA, an intracellular calcium chelator, partially inhibited the AA release in normoxia and in hypoxia. Direct assays of specific PLA(2) activity showed an increase in sPLA(2) activity but not in cPLA(2) activity after 2hr hypoxia. Taken together, these results indicate that the hypoxia-induced increase in PLA(2) activity is mostly due to the activation of sPLA(2).     

Phospholipase A2 inhibitors in development

To date, three isoforms of phospholipase A2 (PLA2) have been identified. Of these, the two Ca2+-dependent isoforms, secretory (sPLA2) and cytosolic phospholipase A2 (cPLA2), are targets for new anti-inflammatory drugs. The catalytic mechanisms and functions of the third isoform, Ca2+-independent cytosolic phospholipase A2 (iPLA2), are unknown at present. sPLA2 and cPLA2 are both implicated in the release of arachidonic acid and prophlogistic lipid mediators. However, recent findings provide evidence that cPLA2 is the dominant isoform in various kinds of inflammation, such as T-cell-mediated experimental arthritis. A triple function of PLA2-derived lipid mediators has been suggested: causing immediate inflammatory signs, involvement in secondary processes, e.g., superoxide free radical (O2) generation, apoptosis, or tumour necrosis factor-alpha (TNF-alpha)-cytotoxicity, and controlling the expression and activation of pivotal proteins implicated in inflammation and cell development, e.g., cytokines, adhesion proteins, proteinases, NF-kappaB, fos/jun/AP-1, c-Myc, or p21ras. In the past, research predominantly focused on the development of sPLA2 inhibitors; however, present techniques enable discrimination of cPLA2, sPLA2, and iPLA2, and specific inhibitors of each of the three isoforms are likely to appear soon. Over the last decade, between 40 and 50 sPLA2 inhibitors have been described; and the list is growing. However, of these, few have the potential for clinical success, and those that do are predominantly active site-directed inhibitors, e.g., BMS-181162, LY311727, ARL-67974, FPL67047, SB-203347, Ro-23-9358, YM-26734, and IS-741. At present, there are no likely clinical candidates emerging from the ranks of cPLA2 and iPLA2 inhibitors in development. Indications for which PLA2 inhibitors are being pursued include, sepsis, acute pancreatitis, inflammatory skin and bowel diseases, asthma, and rheumatoid arthritis. The three main obstacles to the successful development of PLA2 inhibitors include, insufficient oral bioavailability, low affinity for the enzyme corresponding to low in vivo efficacy and insufficient selectivity.     

Human group IIA secretory phospholipase A2 induces neuronal cell death via apoptosis.

Expression of group IIA secretory phospholipase A2 (sPLA2-IIA) is documented in the cerebral cortex (CTX) after ischemia, suggesting that sPLA2-IIA is associated with neurodegeneration. However, how sPLA2-IIA is involved in the neurodegeneration remains obscure. To clarify the pathologic role of sPLA2-IIA, we examined its neurotoxicity in rats that had the middle cerebral artery occluded and in primary cultures of cortical neurons. After occlusion, sPLA2 activity was increased in the CTX. An sPLA2 inhibitor, indoxam, significantly ameliorated not only the elevated activity of the sPLA2 but also the neurodegeneration in the CTX. The neuroprotective effect of indoxam was observed even when it was administered after occlusion. In primary cultures, sPLA2-IIA caused marked neuronal cell death. Morphologic and ultrastructural characteristics of neuronal cell death by sPLA2-IIA were apoptotic, as evidenced by condensed chromatin and fragmented DNA. Before apoptosis, sPLA2-IIA liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2), an AA metabolite, from neurons. Indoxam significantly suppressed not only AA release, but also PGD2 generation. Indoxam prevented neurons from sPLA2-IIA-induced neuronal cell death. The neuroprotective effect of indoxam was observed even when it was administered after sPLA2-IIA treatment. Furthermore, a cyclooxygenase-2 inhibitor significantly prevented neurons from sPLA2-IIA-induced PGD2 generation and neuronal cell death. In conclusion, sPLA2-IIA induces neuronal cell death via apoptosis, which might be associated with AA metabolites, especially PGD2. Furthermore, sPLA2 contributes to neurodegeneration in the ischemic brain, highlighting the therapeutic potential of sPLA2-IIA inhibitors for stroke.     

S-2474, a novel nonsteroidal anti-inflammatory drug, rescues cortical neurons from human group IIA secretory phospholipase A(2)-induced apoptosis

The elevated level of group IIA secretory phospholipase A(2) (sPLA(2)-IIA) activity contributes to neurodegeneration in the cerebral cortex after ischemia. The up-regulation of cyclooxygenase-2 (COX-2) is also relevant to cerebral ischemia in humans. Studies of ischemia with COX-2 inhibitors suggest a clinical benefit. In the present study, we investigated effects of S-2474 on sPLA(2)-IIA-induced cell death in primary cultures of rat cortical neurons, which was established as an in vitro model of brain ischemia. S-2474 is a novel nonsteroidal anti-inflammatory drug (NSAID), which inhibits COX-2 and contains the di-tert-butylphenol antioxidant moiety. S-2474 significantly prevented neurons from undergoing sPLA(2)-IIA-induced cell death. S-2474 completely ameliorated sPLA(2)-IIA-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. sPLA(2) also generated neurotoxic prostaglandin D(2) (PGD(2)) and free radicals from neurons before cell death. S-2474 significantly inhibited the sPLA(2)-IIA-induced generation of PGD(2). The present cortical cultures contained few non-neuronal cells, indicating that S-2474 affected neuronal survival directly, but not indirectly via non-neuronal cells. The inhibitory effect of S-2474 on COX-2 might contribute to its neuroprotective effect. In conclusion, S-2474 exhibits neuroprotective effects against sPLA(2)-IIA. Furthermore, the present study suggests that S-2474 may possess therapeutic potential for stroke via ameliorating neurodegeneration.