【作者】 杨燚；

【导师】 张祥建；

【作者基本信息】 河北医科大学 ， 神经病学， 2014， 博士

【摘要】 脑卒中严重危害人类健康，影响人们生活质量。多年来，研究人员在脑卒中的防治方面做了大量工作，如积极抗凝、溶栓、急诊介入以及微创手术等方法的开展，极大的改善了脑卒中患者的预后。尽管如此，仍有约75%的患者因病情严重或复杂、就诊不及时等原因留有不同程度的后遗症。因此，脑卒中的研究仍任重道远。临床上约30%的缺血性脑卒中患者由于溶栓治疗或栓子自发向远端推移，出现血管再通现象。血管再通将伴随缺血再灌注损伤的发生。因此，深入研究缺血再灌注损伤机制，开发具有抗缺血损伤作用的新药物或是发现传统药物抗缺血损伤的新作用，成为近年来的研究热点，以期为脑卒中的临床治疗提供新途径和理论依据。脑血管闭塞继而血管再通伴随着一系列病理生理事件，包括缺血期间ATP耗竭、乳酸堆积、酸中毒以及再灌注期间活性氧族的产生、一氧化氮的细胞毒作用等。神经元对缺血后氧、糖的改变十分敏感。针对缺血再灌注病理过程进行深入研究及干预，成为挽救缺血脑组织的关键。近年来通过对神经保护剂的研究，一些天然或合成的物质逐渐被发现和证实对缺血脑组织具有保护作用。在这些令人惊喜的作用被发掘的同时，更需要对其作用机制进行深入研究。只有通过多途径的探索，对某种具有神经保护作用的物质进行全面了解，才有望为临床新药物的开发提供有力的理论依据。血管紧张素受体AT1相关的受体蛋白（putative receptor protein relatedto the angiotensin receptor AT1, APJ）是1993年在人类基因中识别出的一种G蛋白偶联受体，当时被称为孤儿G蛋白偶联受体。1998年人类利用反向药理学方法，从牛胃分泌物中提取并纯化出APJ的内源性配体（APJendogenous ligand, apelin），命名为apelin。Apelin在人类主要存在于心脏、肾上腺、肾、肺的血管内皮和大血管内皮细胞，在肺、心肌细胞、肾、肾上腺分泌细胞、血管平滑肌细胞、神经细胞、脂肪组织和结缔组织中也有表达。apelin的前体肽源由77个氨基酸残基组成，其羧基C末端为合成肽序列，可被肽酶分解成多种相对分子量不同的成熟apelin活性肽，包括apelin-17、apelin-36、apelin-12、apelin-13等，其中apelin-13的生物活性可能最强。Apelin/APJ系统在心脏方面的研究表明，apelin/APJ对缺血再灌注心肌具有保护作用：外源性apelin-13或apelin-36能够减少心肌梗死体积；apelin-13能够抑制缺血再灌注心肌细胞内质网依赖的凋亡；另外，心肌缺血早期和缺血性心衰时内源性apelin/APJ表达增加，可以促使心肌细胞对抗缺血损伤、增加心肌收缩力。目前，apelin/APJ系统在脑血管病中的作用研究甚少，apelin对在体缺血再灌注后脑组织是否具有保护作用？Apelin作用的下游机制如何？这些问题还有待于进一步研究。本研究将分三部分对上述问题进行探讨，现将各部分内容概述如下：第一部分Apelin对局灶性脑缺血再灌注小鼠的神经保护作用目的：观察apelin-13对脑缺血再灌注小鼠的作用。方法：选用成年雄性CD-1小鼠作为研究对象，采用改良线栓法制备大脑中动脉缺血再灌注模型。将CD-1小鼠随机分为6组：假手术组（Sham）、溶剂对照组（Vehicle）、缺血再灌注组（I/R）、apelin-13小剂量组（APLN-L）、apelin-13中剂量组（APLN-M）、apelin-13大剂量组（APLN-H）。Sham组：除不插入尼龙鱼线外其余操作同缺血再灌注组。Apelin干预组：在脑缺血后45min，即再灌注前15min，脑室注射5μl apelin-13。Vehicle组：在脑缺血后45min，即再灌注前15min，脑室注射5μl2%DMSO。Apelin-13溶解于2%DMSO中，apelin-13小剂量为10μg/kg，中剂量为50μg/kg，大剂量为100μg/kg。脑缺血后24h处死动物进行观察、检测。各组在取材前进行神经功能评分、TTC染色评价脑梗死体积、干湿重法测定脑水肿、TUNEL染色观察脑组织凋亡改变；并应用RT-qPCR和Western blotting方法检测缺血脑组织Bax、Bcl-2、caspase-3mRNA和蛋白水平的变化，酶组织化学法检测cleaved caspase-3活性的变化。结果：①神经功能评分：I/R组明显高于Sham组（P＜0.05）；APLN-H组明显低于Vehicle组（P＜0.05）；APLN-M组、APLN-L组与Vehicle组差异无统计学意义（P＞0.05）。②脑组织TTC染色：Sham组为均匀一致的红色；I/R组、Vehicle组出现大范围苍白色梗死区域；APLN-L组、APLN-M组、APLN-H组梗死体积比Vehicle组减小。③脑组织含水量： I/R组（84.68±0.929）%明显高于Sham组（78.33±0.625）%（P＜0.05）；APLN-H组（82.70±1.172）%、APLN-M组（83.70±1.048）%明显低于Vehicle组（84.51±1.401）%（P＜0.05）；APLN-L（84.81±0.919）%组与Vehicle组（84.51±1.401）%差异无统计学意义（P＞0.05）。④TUNEL染色：光镜（400×）观察，Sham组脑组织切片未见明显病理学改变，细胞形态完整，未见TUNEL阳性细胞；I/R组梗死周围组织明显水肿、坏死，可见许多深染、固缩核细胞；TUNEL阳性细胞数：APLN-H组（28.00±2.280/每高倍视野）、APLN-M组（32.40±3.262/每高倍视野）明显低于Vehicle组（42.80±3.544/每高倍视野）（P＜0.05）；APLN-L组（41.40±1.200/每高倍视野）与Vehicle组（42.80±3.544/每高倍视野）差异无统计学意义（P＞0.05）。⑤RT-qPCR结果显示，各实验组Bax、Bcl-2、caspase-3mRNA均表达；I/R组Bax、Bcl-2、caspase-3表达明显高于Sham组（P＜0.05）；APLN-H组、APLN-M组、APLN-L组Bax、caspase-3表达明显低于Vehicle组（P＜0.05）；APLN-H组、APLN-M组Bcl-2明显高于Vehicle组（P＜0.05）；APLN-L组与Vehicle组Bcl-2表达差异无统计学意义（P＞0.05）。⑥Western-blotting结果显示，各实验组均有Bax、Bcl-2、caspase-3表达；I/R组Bax、Bcl-2、caspase-3表达明显高于Sham组（P＜0.05）；APLN-H组、APLN-M组、APLN-L组Bax、caspase-3表达明显低于Vehicle组，Bcl-2表达明显高于Vehicle组（P＜0.05）。⑦Western-blotting结果显示，各实验组均有cleaved caspase-3表达；I/R组cleaved caspase-3表达明显高于Sham组（P＜0.05）；APLN-H组、APLN-M组、APLN-L组cleaved caspase-3表达明显低于Vehicle组（P＜0.05）。⑧I/R组cleaved caspase-3活性明显高于Sham组（P＜0.05）；APLN-H组、APLN-M组、APLN-L组cleaved caspase-3活性明显降于Vehicle组（P＜0.05）。结论：局灶性脑缺血再灌注后，小鼠出现明显的神经功能缺损、脑水肿，apelin-13能够改善神经功能缺损，减轻脑水肿并减少梗死体积，具有神经保护作用。局灶性脑缺血再灌注后，缺血周边区脑组织细胞发生凋亡，出现大量TUNEL阳性细胞，apelin-13干预后TUNEL阳性细胞减少，抑制细胞凋亡。Apelin-13的抗凋亡作用是通过对凋亡相关因子Bax、Bcl-2、caspase-3的调节实现的，其可以下调促凋亡蛋白Bax、caspase-3表达，抑制caspase-3活性，同时上调抗凋亡蛋白Bcl-2表达。由此证明，apelin-13对缺血再灌注后脑组织具有保护作用，抗凋亡是其作用靶点之一。第二部分ERK1/2信号通路与apelin-13抗凋亡作用相关性的研究目的：研究apelin-13对脑缺血再灌注损伤保护作用的机制，探讨ERK1/2信号通路是否参与apelin-13的抗凋亡机制。方法：选用成年雄性CD-1小鼠为研究对象，采用改良线栓法制备大脑中动脉缺血再灌注模型。实验1：采用随机方法将CD-1小鼠分为6组：假手术组（Sham）、缺血再灌注组（I/R）、溶剂对照组（Vehicle）、apelin-13小剂量组（APLN-L）、apelin-13中剂量组（APLN-M）、apelin-13大剂量组（APLN-H）。实验2：采用随机方法将CD-1小鼠分为5组：假手术组（Sham）、溶剂对照组（Vehicle）、apelin-13中剂量组（APLN-M）、apelin-13中剂量+PD98059组（APLN+PD）、PD98059+缺血再灌注组（PD）。Sham组：除不插入尼龙鱼线外其余操作同缺血再灌注组。Apelin-13干预组：实验1中，在脑缺血后45min，即再灌注前15min，脑室注射5μl apelin-13。实验2中，在脑缺血前15min脑室注射5μl2%DMSO，在再灌注前15min脑室注射5μl中剂量apelin-13。Vehicle组：实验1中，在脑缺血后45min，即再灌注前15min，脑室注射5μl2%DMSO。实验2中，分别在脑缺血前15min、再灌注前15min，脑室注射5μl2%DMSO。PD98059干预组：实验2中，APLN+PD组在脑缺血前15min脑室注射5μl PD98059，在再灌注前15min脑室注射5μl中剂量apelin-13。PD组在脑缺血前15min脑室注射5μl PD98059，在再灌注前15min脑室注射5μl2%DMSO。Apelin-13溶解于2%DMSO中，apelin-13小剂量为10μg/kg，中剂量为50μg/kg，大剂量为100μg/kg。PD98059溶解于2%DMSO中，浓度为2mmol/L。各组在术后24h取材，分别用RT-qPCR和Western-blotting方法检测缺血脑组织中ERK1/2、Bax、Bcl-2、caspase-3mRNA和蛋白水平的变化,酶组织化学法检测cleaved caspase-3活性改变。结果：实验1：①RT-qPCR结果显示，各实验组ERK1/2mRNA均表达，差异无统计学意义（P＞0.05）。②Western-blotting结果显示，各实验组均有ERK1/2蛋白表达，差异无统计学意义（P＞0.05）。各实验组均有p-ERK1/2蛋白表达；I/R组p-ERK1/2表达明显高于Sham组（P＜0.05）；APLN-H组、APLN-M组、APLN-L组p-ERK1/2表达明显高于Vehicle组（P＜0.05）。实验2：Apelin-13联合PD98059干预①RT-qPCR结果显示，各实验组均有ERK1/2mRNA表达，差异无统计学意义（P＞0.05）。②Western-blotting结果显示，各实验组均有ERK1/2蛋白表达，差异无统计学意义（P＞0.05）；PD组p-ERK1/2表达明显低于Vehicle组（P＜0.05）；APLN+PD组p-ERK1/2表达明显低于APLN-M组（P＜0.05）。③RT-qPCR结果显示，各实验组Bax、Bcl-2、caspase-3mRNA均表达，PD组与Vehicle组Bax、Bcl-2、caspase-3mRNA表达差异无统计学意义（P＞0.05）；APLN-M组Bax、caspase-3mRNA表达明显低于Vehicle组， Bcl-2mRNA表达高于Vehicle组（P＜0.05）；APLN+PD组Bax、caspase-3mRNA表达明显高于APLN-M组，Bcl-2mRNA表达明显低于APLN-M组（P＜0.05）；APLN+PD组Bax、Bcl-2mRNA表达与PD组差异无统计学意义（P＞0.05），caspase-3mRNA表达明显低于PD组（P＜0.05）。④Western-blotting结果显示，各实验组均有Bax、Bcl-2、caspase-3蛋白表达，PD组与Vehicle组Bax、Bcl-2、caspase-3表达差异无统计学意义（P＞0.05）；APLN-M组Bax、caspase-3表达明显低于Vehicle组，Bcl-2表达高于Vehicle组（P＜0.05）；APLN+PD组Bax、caspase-3表达明显高于APLN-M组，Bcl-2表达明显低于APLN-M组（P＜0.05）；APLN+PD组Bax、Bcl-2表达与PD组差异无统计学意义（P＞0.05），caspase-3表达明显低于PD组（P＜0.05）。⑤Western-blotting结果显示，各实验组均有cleaved caspase-3蛋白表达；PD组与Vehicle组cleaved caspase-3表达差异无统计学意义（P＞0.05）APLN-M组cleaved caspase-3表达明显低于Vehicle组（P＜0.05）；APLN+PD组cleaved caspase-3表达明显高于APLN-M组（P＜0.05）；APLN+PD组cleaved caspase-3表达与PD组差异无统计学意义（P＞0.05）。⑥APLN-M组cleaved caspase-3活性明显低于Vehicle组（P＜0.05）；APLN+PD组cleaved caspase-3活性明显高于APLN-M组（P＜0.05）；APLN+PD组cleaved caspase-3活性明显低于PD组（P＜0.05）。结论：局灶性脑缺血再灌注后，脑组织p-ERK1/2蛋白表达增高，而ERK1/2总蛋白未发生明显改变，提示ERK1/2蛋白的激活可能在缺血再灌注损伤病理机制中发挥着重要作用。Apelin-13能使局灶性脑缺血再灌注后脑组织p-ERK1/2蛋白表达进一步增高，提示apelin-13能够促进缺血脑组织ERK1/2蛋白的激活，同时提示ERK1/2信号通路可能参与了apelin-13的作用机制。应用PD98059抑制ERK1/2活化后，apelin-13对局灶性脑缺血再灌注脑组织凋亡相关因子Bax、Bcl-2、caspase-3的调节作用减弱或者消失，证明了apelin-13对Bax、Bcl-2、caspase-3的调节作用与ERK1/2的活化相关即ERK1/2信号通路参与了apelin-13的抗凋亡作用。第三部分PI-3K/Akt信号通路与apelin-13抗凋亡作用相关性的研究目的：研究apelin-13对脑缺血再灌注损伤保护作用的机制，探讨PI-3K/Akt信号通路是否参与apelin-13抗凋亡机制。方法：选用成年雄性CD-1小鼠为研究对象，采用改良线栓法制备大脑中动脉缺血再灌注模型。实验1：采用随机的方法将CD-1小鼠分为6组：假手术组（Sham）、缺血再灌注组（I/R）、溶剂对照组（Vehicle）、apelin-13小剂量组（APLN-L）、apelin-13中剂量组（APLN-M）、apelin-13大剂量组（APLN-H）。实验2：采用随机的方法将CD-1小鼠分为5组：假手术组（Sham）、溶剂对照组（Vehicle）、apelin-13中剂量组（APLN-M）、apelin-13中剂量+LY294002组（APLN+LY）、LY294002+缺血再灌注组（LY）。Sham组：除不插入尼龙鱼线外其余操作同缺血再灌注组。Apelin-13干预组：实验1中，在脑缺血后45min，即再灌注前15min脑室注射5μl apelin-13。实验2中，在脑缺血前15min脑室注射5μl2%DMSO；再灌注前15min脑室注射5μl中剂量apelin-13。Vehicle组：实验1中，在脑缺血后45min，即再灌注前15min，脑室注射5μl2%DMSO。实验2中，分别在脑缺血前15min、再灌注前15min，脑室注射5μl2%DMSO。LY294002干预组：实验2中，APLN+LY组在脑缺血前15min脑室注射5μl LY294002，在再灌注前15min脑室注射5μl中剂量apelin-13。LY组在脑缺血前15min脑室注射5μl LY294002，在再灌注前15min脑室注射5μl2%DMSO。Apelin-13溶解于2%DMSO中，apelin-13小剂量为10μg/kg，中剂量为50μg/kg，大剂量为100μg/kg。LY294002溶解于2%DMSO中，浓度为10mmol/L。各组在术后24h取材，分别用RT-qPCR和Western-blotting方法检测缺血脑组织中Akt、Bax、Bcl-2、caspase-3mRNA和蛋白水平的变化,酶组织化学法检测cleaved caspase-3活性改变。结果：实验1：①RT-qPCR结果显示，各实验组均有Akt mRNA表达，差异无统计学意义（P＞0.05）。②Western-blotting结果显示，各实验组均有Akt蛋白表达，差异无统计学意义（P＞0.05）。各实验组均有p-Akt蛋白表达；I/R组p-Akt表达明显高于Sham组（P＜0.05）；APLN-H组、APLN-M组、APLN-L组p-ERK1/2表达明显高于Vehicle组（P＜0.05）。实验2：Apelin-13联合PD98059干预①RT-qPCR结果显示，各实验组均有ERK1/2mRNA表达，差异无统计学意义（P＞0.05）。②Western-blotting结果显示，各实验组均有Akt蛋白表达，差异无统计学意义（P＞0.05）；LY组p-Akt表达明显低于Vehicle组（P＜0.05）；APLN+LY组p-Akt表达明显低于APLN-M组（P＜0.05）。③RT-qPCR结果显示，各实验组Bax、Bcl-2、caspase-3mRNA均表达；LY组与Vehicle组Bax、Bcl-2、caspase-3mRNA表达差异无统计学意义（P＞0.05）；APLN-M组Bax、caspase-3mRNA表达明显低于Vehicle组， Bcl-2mRNA表达明显高于Vehicle组（P＜0.05）；APLN+LY组Bax、caspase-3mRNA表达明显高于APLN-M组，Bcl-2mRNA表达明显低于APLN-M组（P＜0.05）；APLN+LY组Bax、Bcl-2mRNA表达与LY组差异无统计学意义（P＞0.05），caspase-3mRNA表达明显低于LY组（P＜0.05）。④Western-blotting结果显示，各实验组均有Bax、Bcl-2、caspase-3蛋白表达；LY组与Vehicle组Bax、Bcl-2、caspase-3表达差异无统计学意义（P＞0.05）；APLN-M组Bax、caspase-3表达明显低于Vehicle组，Bcl-2表达明显高于Vehicle组（P＜0.05）；APLN+LY组Bax、caspase-3表达明显高于APLN-M组，Bcl-2表达明显低于APLN-M组（P＜0.05）；APLN+LY组Bcl-2表达与LY组差异无统计学意义（P＞0.05），Bax、caspase-3表达明显低于LY组（P＜0.05）。⑤Western-blotting结果显示，各实验组均有cleaved caspase-3蛋白表达；LY组与Vehicle组cleaved caspase-3表达差异无统计学意义（P＞0.05）；APLN-M组cleaved caspase-3表达明显低于Vehicle组（P＜0.05）；APLN+LY组cleaved caspase-3表达明显高于APLN-M组（P＜0.05）；APLN+LY组cleaved caspase-3表达明显低于LY组（P＜0.05）。⑥APLN-M组cleaved caspase-3活性明显低于Vehicle组（P＜0.05）；APLN+LY组cleaved caspase-3活性明显高于APLN-M组（P＜0.05）；APLN+LY组cleaved caspase-3活性明显低于LY组（P＜0.05）。结论：局灶性脑缺血再灌注后，脑组织p-Akt表达增高，提示Akt蛋白的激活可能在缺血再灌注损伤病理机制中发挥着重要作用。Apelin-13干预使局灶性脑缺血再灌注后脑组织p-Akt表达进一步增高，提示apelin-13能够促进缺血脑组织Akt蛋白的激活，Akt信号通路可能参与了apelin-13的作用机制。应用LY294002抑制Akt活化后，apelin-13对局灶性脑缺血再灌注后脑组织凋亡相关因子Bax、Bcl-2、caspase-3的调节作用减弱或者消失，证明了apelin-13对Bax、Bcl-2、caspase-3的调节作用与Akt的活化相关，即Akt信号通路参与了apelin-13的抗凋亡机制。更多还原

【Abstract】 Stroke causes serious damage to human health, and influences people’slife quality. Over the years, researchers have done a lot of work in theprevention and treatment of stroke, such as positive anticoagulation,thrombolysis, emergency intervention as well as the methods of minimallyinvasive surgery, and greatly improved the prognosis of patients with stroke.Still, about75%of the patients suffer from varying degrees of sequelaebecause of severe or complicated conditions or seeing a doctor not in time.Therefore, the study of stroke is still a long way. Recanalization happens inabout30%of ischemic stroke patients because of thrombolysis therapy orspontaneous to the distal embolus. Recanalization will be accompanied by theoccurrence of ischemia/reperfusion injury, so further study ofischemia/reperfusion injury mechanisms and development of new drugsagainst ischemic damage or finding the new role of traditional drugs againstischemic damage become a research hotspot in recent years, in order toprovide theoretical basis and clinical treatment for stroke.The events that occur subsequent to cerebral artery occlusion are termedcerebral ischemia/reperfusion denoting that these are distinct phases of cellularinjury with ATP depletion, lactate accumulation and acidosis observed duringischemia and the production of reactive oxygen and nitrogen species duringreperfusion. Neurons are very sensitive to changes in the supply of glucoseand oxygen that occurs during ischemic conditions. In-depth researchaccording to these pathological process and intervention become the key tosave the ischemic brain.In recent years, based on the research of the neuroprotective agent, somenatural or synthetic materials have been gradually discovered and confirmedto be protective against cerebral ischemia injury. Though these roles were amazing, more in-depth studies of their mechanism of action need to be done.Only by way of exploration and comprehensive understanding of theseneuroprotective agents, we can provide strong theoretical basis for thedevelopment of new drugs.Putative receptor protein related to the angiotensin receptor AT1(APJ) isa G protein coupled receptor first identified in human genes in1993, andknown as orphans G protein coupled receptor. In1998, human extracted andpurified APJ endogenous ligand from bovine stomach secretions using thereverse pharmacology method, and called it apelin. In humans, apelin mainlyexists in the heart, adrenal glands, kidney, lung vascular endothelium and largevascular endothelial cells. It is also expressed in the lung, heart muscle cells,kidney, adrenal gland cells, vascular smooth muscle cells, nerve cells, adiposetissue and connective tissue. Preproapelin is composed of77amino acidresidues, and its carboxyl C end is sequence for synthetic peptide and can bebroken down into a variety of relative molecular weight of different matureapelin active peptides by peptide enzyme, including apelin-17, apelin-36,apelin-12and apelin-13. Biological activity of apelin-13may be the strongest.More recent studies demonstrated that apelin-13is protective againstischemia-induced injury in cardiomyocytes. Apelin/APJ has been proved to bemyocardial protective against ischemia/reperfusion injury. Exogenousapelin-13or apelin-36could reduce myocardial infarction size. Apelin-13inhibited myocardial cells endoplasmic apoptosis induced byischemia/reperfusion. In addition, early myocardial ischemia and ischemicendogenous induced apelin/APJ expression increase, which protected theischemic myocardial cell damage and increased myocardial contraction force.Currently, research on apelin/APJ’s role in cerebrovascular disease is very few.Does apelin play a protective role against cerebral ischemia/reperfusion invivo? What is the mechanism of apelin? These problems need to further study.Therefore we evaluated the role of apelin in cerebral ischemia and its potentialmechanism. Part Ⅰ Apelin’s effect against focal cerebral ischemia/reperfusion injuryin miceObjective：This study is to evaluate the effect of apelin on cerebralischemia/reperfusion injury in mice.Methods：Focal transient cerebral ischemia was induced in male ICRmice using modified suture occlusion technique. The mice were randomlydivided into6groups: Sham group, Vehicle group, ischemia/reperfusion (I/R)group, apelin-low dose (APLN-L) group, apelin-middle dose (APLN-M)group and apelin-high dose (APLN-H) group.Sham group: with the same operation to I/R group except not insertingnylon line. Apelin-13treatment groups:5μl apelin-13was injected into ateralcerebral ventricle45min after ischemia, namely,15min before reperfusion.Vehicle group: equal volume of2%DMSO was injected into ateralcerebral ventricle45min after ischemia, namely,15min before reperfusion.Apelin-13concentration was10μg/kg in APLN-L group,50μg/kg inAPLN-M group and100μg/kg in APLN-H group. Mice were reanesthetizedand killed at24h after stroke, and neurological function, infarct volume, brainedema and apoptosis were measured. RT-qPCR and western blotting wereused to analyze the expression of Bax, Bcl-2, caspase-3and cleaved caspase-3.Cleaved caspase-3activity was also tested.Results：①Neurological function score of I/R group was higher than that of Shamgroup. Neurological function scores were significant reduced in APLN-Hgroup compared with Vehicle group (P＜0.05). There was no significantdifference between APLN-M and Vehicle group and between APLN-L groupand Vehicle group (P>0.05).②After TTC staining, the normal tissue was stained deep red while theinfarct area pale gray. No infarction was observed in Sham group. Apelin-13treated mice demonstrated smaller infarct volumes compared with Vehicle andI/R mice.③Percentage of brain water content of I/R group (84.68±0.929)%was higher than that of Sham group (78.33±0.625)%(P＜0.05). Percentage ofbrain water content of APLN-H group (82.70±1.172)%and APLN-M group(83.70±1.048)%decreased compared with that Vehicle group (84.51±1.401)%. There was no significant difference between APLN-L group (84.81±0.919)%and Vehicle group (84.51±1.401)%(P＞0.05).④TUNEL staining assays demonstrated that I/R group and Vehicle groupexhibited many stained, condensed nuclei, either isolated or within thecytoplasm of cells (400×light microscopy). Apoptotic cells decreased inAPLN-H group (28.00±2.280per field) and APLN-M group (32.40±3.262per field) compared with Vehicle group (42.80±3.544per field)(P<0.05).There was no significant difference between APLN-L group (41.40±1.200per field) and Vehicle group (42.80±3.544per field)(P＞0.05).⑤RT-qPCR results showed that elevations in Bax, Bcl-2and caspase-3mRNA expression were seen in stroke brains with minimal changes seen insham brains (P＜0.05). I/R group presented increase of Bax, Bcl-2andcaspase-3compared with Sham group (P<0.05). Bax and caspase-3mRNAexpression of APLN-H,-M,-L groups was lower than that of Vehicle group.Bcl-2mRNA expression of APLN-H,-M groups was higher than that ofVehicle group (P＜0.05). There was no significant difference of Bcl-2mRNAexpression between APLN-L group and Vehicle group (P＞0.05).⑥Western-blotting results showed that elevations in Bax, Bcl-2andcaspase-3were seen in stroke brains with minimal changes seen in shambrains (P＜0.05). APLN-H,-M and-L groups presented decrease of Bax, andcaspase-3and increase of Bcl-2compared with Vehicle group (P <0.05).⑦Western-blotting results showed that elevations in cleaved caspase-3were seen in stroke brains with minimal changes seen in sham brains (P＜0.05). APLN-H,-M,-L groups presented decrease of cleaved caspase-3compared with Vehicle group (P <0.05).⑧Cleaved caspase-3activity was obviously lower in APLN-L,-M and-H groups than that in Vehicle group (P <0.05).Conclusions： Mice appeared obvious neurologic deficits and brain edema after focal cerebral ischemia/reperfusion. Apelin-13treatmenteffectively ameliorated neurological defect, brain infarct volume and brainedema. Apelin-13also significantly attenuated apoptosis by reducingTUNEL-positive cells, down-regulating Bax, caspase-3and cleaved caspase-3and up-regulating Bcl-2. It provided the evidence that apelin-13protectedneurons against cerebral ischemia insults and anti-apoptosis was one of itsactions.Part Ⅱ Research in correlation between ERK1/2signal pathway andapelin-13’s anti-apoptosis effectObjective： This study is to detect the mechanism of apelin-13’sneuroprotection, and evaluate whether ERK1/2signal pathway was involvedin it.Methods：Focal transient cerebral ischemia was induced in male ICRmice using modified suture occlusion technique. Experiment Ⅰ: The micewere randomly divided into6groups: Sham group, Vehicle group, I/R group,APLN-L group, APLN-M group and APLN-H group. Experiment Ⅱ: Themice were randomly divided into5groups: Sham group, Vehicle group,APLN-M group, APLN-M+PD98059(APLN+PD) group and PD98059+I/R(PD) group.Sham group: with the same operation to I/R group except not insertingnylon line.Apelin-13treatment groups: in experiment Ⅰ,5μl of apelin-13wasinjected into ateral cerebral ventricle45min after ischemia, namely,15minbefore reperfusion; in experiment Ⅱ,5μl of2%DMSO was injected intoateral cerebral ventricle15min before ischemia and5μl of apelin-13wasinjected into ateral cerebral ventricle15min before reperfusion.Vehicle group: in experiment Ⅰ,5μl of2%DMSO was injected intoateral cerebral ventricle45min after ischemia, namely,15min beforereperfusion; in experiment Ⅱ:5μl of2%DMSO was injected into ateralcerebral ventricle15min before ischemia and15min before reperfusionrespectively. PD98059treatment groups: in experiment Ⅱ in APLN+PD group,5μlof PD98059was injected into ateral cerebral ventricle15min before ischemia,and then5μl of apelin-13was injected into ateral cerebral ventricle15minbefore reperfusion; in PD group,5μl of PD98059was injected into ateralcerebral ventricle15min before ischemia, and then5μl of2%DMSO wasinjected into ateral cerebral ventricle15min before reperfusion.Mice were reanesthetized and killed at24h after stroke. RT-qPCR andwestern blotting were used to analyze the expression of Bax, Bcl-2, caspase-3,cleaved caspase-3and ERK1/2. Cleaved caspase-3activity was also tested.Results：Experiment Ⅰ①RT-qPCR results showed that ERK1/2mRNA expression was seen inall groups. There was no significant difference among all groups (P＞0.05).②Western-blotting results showed that ERK1/2protein expression wasseen in all groups. There was no significant difference among all groups (P＞0.05). P-ERK1/2protein expression was seen in all groups. P-ERK1/2increased in I/R group compared with Sham group (P＜0.05). P-ERK1/2increased APLN-H,-M,-L groups compared with Vehicle group (P＜0.05).Experiment Ⅱ①RT-qPCR results showed that ERK1/2mRNA expression was seen inall groups. There was no significant difference among all groups (P＞0.05).②Western-blotting results showed that ERK1/2protein expression wasseen in all groups. There was no significant difference among all groups (P＞0.05). P-ERK1/2protein expression was seen in all groups. P-ERK1/2decreased in PD group compared with Vehicle group(P＜0.05). P-ERK1/2decreased in APLN+PD group compared with APLN-M group (P＜0.05).③RT-qPCR results showed that Bax, Bcl-2and caspase-3mRNAexpression were seen in all groups. There was no significant differencebetween PD group and Vehicle group (P＞0.05). Bax and caspase-3mRNAdecreased and Bcl-2mRNA increased in APLN-M group compared withVehicle group (P＜0.05). Bax and caspase-3mRNA increased and Bcl-2 mRNA decreased in APLN+PD group compared with APLN-M group (P＜0.05). There was no significant difference of Bax、Bcl-2mRNA betweenAPLN+PD group and PD group (P＞0.05). Caspase-3mRNA decreased inAPLN+PD group compared with PD group (P＜0.05).④Western-blotting results showed that Bax, Bcl-2and caspase-3proteinexpression was seen in all groups. There was no significant difference betweenPD group and Vehicle group (P＞0.05). Bax and caspase-3decreased andBcl-2increased in APLN-M group compared with Vehicle group (P＜0.05).Bax and caspase-3increased and Bcl-2decreased in APLN+PD groupcompared with APLN-M group (P＜0.05). There was no significant differenceof Bax、Bcl-2between APLN+PD group and PD group (P＞0.05). Caspase-3decreased in APLN+PD group compared with PD group (P＜0.05).⑤Western-blotting results showed that cleaved caspase-3proteinexpression was seen in all groups. There was no significant difference betweenPD group and Vehicle group (P＞0.05). Cleaved caspase-3decreased inAPLN-M group compared with Vehicle group (P＜0.05). Cleaved caspase-3increased in APLN+PD group compared with APLN-M group (P＜0.05).There was no significant difference between APLN+PD group and PD group(P＞0.05).⑥Cleaved caspase-3activity decreased in APLN-M group comparedwith Vehicle group (P<0.05). There was no significant difference between PDgroup and Vehicle group (P＞0.05). Cleaved caspase-3activity increased inAPLN+PD group compared with APLN-M group (P<0.05). Cleavedcaspase-3activity decreased in APLN+PD group compared with PD group (P<0.05).Conclusions：P-ERK1/2was up-regulated while total ERK has no change after focalcerebral ischemia/reperfusion, which prompted ERK1/2protein activationmay play an important role in ischemia/reperfusion mechanism. Apelin-13treatment enhanced the phosphorylation level of ERK1/2, which promptedthat apelin-13promoted the activation of ERK1/2protein, and ERK1/2 signaling pathway might be involved in the apelin-13’s action. Afterapplication of PD98059to inhibit ERK1/2activation, apelin-13’s effect onBax, Bcl-2and caspase-3was attenuated or disappeared. It proved thatapelin-13’s effect on Bax, Bcl-2and caspase-3associated with the activationof ERK1/2, namely, ERK1/2signal pathway was involved in the apelin-13anti-apoptosis effect in cerebral ischemia/reperfusion.Part Ⅲ Research in correlation between PI-3K/Akt signal pathway andapelin-13’s anti-apoptosis effectObjective： This study is to detect the mechanism of apelin-13’sneuroprotection, and evaluate whether PI-3K/Akt signal pathway wasinvolved in it.Methods：Focal transient cerebral ischemia was induced in male ICRmice using modified suture occlusion technique. Experiment Ⅰ: The micewere randomly divided into6groups: Sham group, Vehicle group, I/R group,APLN-L group, APLN-M group and APLN-H group. Experiment Ⅱ: Themice were randomly divided into5groups: Sham group, Vehicle group,APLN-M group, APLN-M+LY294002(APLN+LY) group and LY294002+I/R (LY) group.Sham group: with the same operation to I/R group except not insertingnylon line.Apelin-13treatment groups: in experiment Ⅰ,5μl of apelin-13wasinjected into ateral cerebral ventricle45min after ischemia, namely,15minbefore reperfusion; in experiment Ⅱ,5μl of2%DMSO was injected intoateral cerebral ventricle15min before ischemia and5μl of apelin-13wasinjected into ateral cerebral ventricle15min before reperfusion.Vehicle group: in experiment Ⅰ,5μl of2%DMSO was injected intoateral cerebral ventricle45min after ischemia, namely,15min beforereperfusion; in experiment Ⅱ:5μl of2%DMSO was injected into ateralcerebral ventricle15min before ischemia and15min before reperfusionrespectively.LY294002treatment groups: in experiment Ⅱ in APLN+LY group,5μl of LY294002was injected into ateral cerebral ventricle15min beforeischemia, and then5μl of apelin-13was injected into ateral cerebral ventricle15min before reperfusion; in LY group,5μl of LY294002was injected intoateral cerebral ventricle15min before ischemia, and then5μl of2%DMSOwas injected into ateral cerebral ventricle15min before reperfusion.Mice were reanesthetized and killed at24h after stroke. RT-qPCR andwestern blotting were used to analyze the expression of Akt, Bax, Bcl-2,caspase-3and cleaved caspase-3. Cleaved caspase-3activity was also tested.Results：Experiment Ⅰ①RT-qPCR results showed that Akt mRNA expression was seen in allgroups. There was no significant difference among all groups (P＞0.05).②Western-blotting results showed that Akt protein expression was seenin all groups. There was no significant difference among all groups (P＞0.05).P-Akt protein expression was seen in all groups. P-Akt increased in I/R groupcompared with Sham group (P＜0.05). P-Akt increased APLN-H,-M,-Lgroups compared with Vehicle group (P＜0.05).Experiment Ⅱ①RT-qPCR results showed that Akt mRNA expression was seen in allgroups. There was no significant difference among all groups (P＞0.05).②Western-blotting results showed that Akt protein expression was seenin all groups. There was no significant difference among all groups (P＞0.05).P-Akt decreased in LY group compared with Vehicle group(P＜0.05). P-Aktdecreased in APLN+LY group compared with APLN-M group (P＜0.05).③RT-qPCR results showed that Bax, Bcl-2and caspase-3mRNAexpression were seen in all groups. There was no significant differencebetween LY group and Vehicle group (P＞0.05). Bax and caspase-3mRNAdecreased and Bcl-2mRNA increased in APLN-M group compared withVehicle group (P＜0.05). Bax and caspase-3mRNA increased and Bcl-2mRNA decreased in APLN+LY group compared with APLN-M group (P＜0.05). There was no significant difference of Bax、Bcl-2mRNA between APLN+LY group and LY group (P＞0.05). Caspase-3mRNA decreased inAPLN+LY group compard with LY group (P＜0.05).④Western-blotting results showed that Bax, Bcl-2and caspase-3proteinexpression was seen in all groups. There was no significant difference betweenLY group and Vehicle group (P＞0.05). Bax and caspase-3decreased andBcl-2increased in APLN-M group compared with Vehicle group (P＜0.05).Bax and caspase-3increased and Bcl-2decreased in APLN+LY groupcompared with APLN-M group (P＜0.05). There was no significant differenceof Bcl-2between APLN+LY group and LY group (P＞0.05). Bax andcaspase-3decreased in APLN+LY group compard with LY group (P＜0.05).⑤Western-blotting results showed that cleaved caspase-3proteinexpression was seen in all groups. There was no significant difference betweenLY group and Vehicle group (P＞0.05). Cleaved caspase-3decreased inAPLN-M group compared with Vehicle group (P＜0.05). Cleaved caspase-3increased in APLN+LY group compared with APLN-M group (P＜0.05).Cleaved caspase-3decreased in APLN+LY group compared with LY group (P＜0.05).⑥Cleaved caspase-3activity was lower in APLN-M group than that inVehicle group (P＜0.05). There was no significant difference between LYgroup and Vehicle group (P＞0.05). Cleaved caspase-3activity increased inAPLN+LY group compared with APLN-M group (P <0.05). Cleavedcaspase-3activity decreased in APLN+LY group compared with LY group (P＜0.05).Conclusions：P-Akt was up-regulated while total Akt has no change after focal cerebralischemia/reperfusion, which prompted Akt protein activation may play animportant role in ischemia/reperfusion mechanism. Apelin-13treatmentenhanced the phosphorylation level of Akt, which prompted that apelin-13promoted the activation of Akt protein, and PI-3K/Akt signaling pathwaymight be involved in the apelin-13’s action. After application of LY294002toinhibit Akt activation, apelin-13’s effect on Bax, Bcl-2and caspase-3was attenuated or disappeared. It proved that apelin-13’s effect on Bax, Bcl-2andcaspase-3associated with the activation of Akt, namely, PI-3K/Akt signalpathway was involved in the apelin-13anti-apoptosis effect in cerebralischemia/reperfusion.更多还原