迷迭香酸对局灶性脑缺血再灌注损伤小鼠的脑保护及作用机制研究
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摘要
随着我国人口老龄化进程的加速，脑血管病（脑卒中）已成为危害人类健康和生命的主要疾病。缺血性脑血管病最常见，且其复发率、致残率高居不下，给社会造成沉重的经济负担。
脑是一个对缺氧最敏感的器官，脑重量仅占人体重量之2％，脑耗氧量却占人体总耗氧量的20％。脑组织发生缺血后早期、及时、有效的恢复血供是治疗的关键，组织纤溶酶原激活剂（tissue plasminogenactivator，tPA）是迄今为止美国食品和药物管理局FDA批准的、用于急性缺血卒中治疗的首选药物。然而，缺血脑组织实现血流再通的同时带来的再灌注损伤一直困扰着临床的治疗和预后。脑缺血及再灌注损伤的病理生理机制复杂，认为存在多种机制相互作用形成的复杂调控网络，包括炎症损害、自由基损伤、氧化应激、兴奋性氨基酸毒性、细胞内钙超载等，直接/间接导致神经细胞凋亡/死亡，血脑屏障破坏，最终导致神经功能的缺失，甚至危及患者生命。
因此，积极寻求有效的神经保护剂是一直以来缺血性卒中研究的重点，尽管动物实验取得了一定的效果，但目前为止尚没有临床证实有明确疗效的神经保护剂。近年来在细胞实验和动物实验中应用天然植物有效成分及其制剂治疗急性卒中成为研究的热点，为新型神经保护剂的研制提供了广阔的思维空间。迷迭香酸(Rosmarinic acid，RA)是天然存在于多种植物中的一种多酚，也是丹参多酚酸的主要成分之一，具有抗氧化、抗炎、抗凋亡、抗肿瘤等多种生物活性，且在多种疾病模型中证明有效，而其在缺血卒中治疗的研究鲜见报道。因此，本课题旨在研究迷迭香酸作为新型神经保护剂的可能性及潜在的神经保护机制，为临床治疗提供新的实验依据。
本研究选用成年健康雄性CD1小鼠，建立局灶性脑缺血再灌注模型，在此模型基础上观察缺血再灌注后HO-1，Nrf2，claudin-5，MMP-9，Bcl-2和Bax的表达，应用迷迭香酸干预，评价干预前后梗死体积、脑含水量、神经功能缺损评分，TUNEL染色评价细胞凋亡情况，检测迷迭香酸对HO-1，Nrf2，claudin-5，MMP-9，Bcl-2和Bax的表达调节，以期初步探讨其在缺血再灌注损伤后的脑保护作用机制；应用HO-1活性抑制剂和PI3K/Akt信号通路抑制剂进一步探讨Nrf2/HO-1通路激活在迷迭香酸发挥神经保护作用中的重要地位。本研究分三部分，现将各部分内容概述如下。
第一部分迷迭香酸对局灶性脑缺血再灌注损伤小鼠的抗氧化及抗凋亡机制研究
目的：观察缺血再灌注小鼠脑缺血损伤后24h梗死体积、神经功能缺损评分，HO-1，Nrf2，Bcl-2和Bax的表达变化，探讨迷迭香酸对缺血再灌注损伤小鼠的抗氧化及抗凋亡机制。
方法：选用成年健康雄性CD-1小鼠为研究对象，采用改良Longa线栓法制备大脑中动脉缺血再灌注（MCAO/R）模型。实验1：观察迷迭香酸的神经保护作用。实验动物随机分为5组，假手术组（Sham），假手术后腹腔注射等量生理盐水；手术组（MCAO/R），再灌注后腹腔注射等量生理盐水；迷迭香酸小剂量组（RA-L），再灌注后立即腹腔注射迷迭香酸10mg/kg；迷迭香酸中剂量组（RA-M），再灌注后立即腹腔注射迷迭香酸20mg/kg；迷迭香酸大剂量组（RA-H），再灌注后立即腹腔注射迷迭香酸40mg/kg。缺血再灌注后24h取材前Longa评分法进行神经功能评分，TTC染色评价脑梗死体积。实验2：观察迷迭香酸的抗氧化及抗凋亡功能。检测缺血脑组织中HO-1，Nrf2，Bcl-2和Bax表达的变化情况。实验动物随机分为4组，假手术组（Sham），手术组（MCAO/R），迷迭香酸中剂量组（RA-M），迷迭香酸大剂量组（RA-H）。TUNEL染色检测缺血周围组织的细胞凋亡情况，RT-PCR方法和Western blot方法检测缺血脑组织HO-1，Nrf2，Bcl-2和Bax的mRNA和蛋白水平的变化。
结果：
1Sham组无神经功能缺损，神经功能缺损评分为0分；缺血再灌注后24h，MCAO/R组神经功能缺损评分严重，RA-L组MCAO/R组相比无明显降低神经功能缺损，差异无统计学意义（P＞0.05），RA-M和RA-H组与MCAO/R组相比均可显著降低神经功能缺损，差异有统计学意义（P＜0.05）；
2再灌注24h，RA-L组与MCAO/R组相比没有明显降低脑梗死体积，差异无统计学意义（50.36±1.81%vs.48.09±2.45%）（P＞0.05），RA-M组（50.36±1.81%vs.41.67±0.83%）和RA-H组（50.36±1.81%vs.30.86±1.79%）与MCAO/R组相比均可明显降低脑梗死体积，差异有统计学意义（P＜0.05）。且RA-H组较RA-M组在减少梗死体积的效果上作用更显著（P＜0.05）。我们可以看到迷迭香酸在20mg/kg（RA-M）和40mg/kg（RA-H）时发挥更好的治疗作用，因此，下面的研究集中在20mg/kg和40mg/kg。
3TUNEL染色结果，再灌注后24h，与MCAO/R组比较，RA-M组(51.75±2.36vs.41±1.41)和RA-H组(51.75±2.36vs.32.75±1.70)均可显著减少TUNEL阳性细胞数，且RA-H组作用更为显著（P＜0.05）。
4Western blot中，与MCAO/R组比较，RA-M和RA-H组在再灌注后24h均可明显增加HO-1，Nrf2，Bcl-2的蛋白表达和明显降低Bax蛋白的表达，差异有统计学意义（P＜0.05），且RA-H组作用更为显著（P＜0.05）。
5再灌注后24h，RT-PCR分别检测RA-M和RA-H组HO-1，Nrf2，Bcl-2和Bax的基因表达情况。与MCAO/R组相比，两个剂量组均可明显增加HO-1，Nrf2，Bcl-2的mRNA表达，明显降低Bax的mRNA表达，差异有统计学意义（P＜0.05）。RA-H组作用更为显著（P＜0.05）。
结论：迷迭香酸对局灶性脑缺血再灌注小鼠有较好的神经保护作用，可改善脑缺血损伤后的神经功能缺失，减小梗死体积，上调Bcl-2表达、下调Bax表达，诱导Nrf2活化、HO-1表达上调，因此我们推测迷迭香酸的神经保护作用可能通过激活Nrf2/HO-1通路，上调Bcl-2的表达，抑制Bax表达实现的；这些是迷迭香酸发挥其抗氧化、抗凋亡的作用，从而实现其神经保护作用。
第二部分迷迭香酸上调HO-1表达对局灶性脑缺血再灌注损伤小鼠发挥多重神经保护作用
目的：应用HO-1活性抑制剂ZnPPIX，探讨脑缺血再灌注损伤后，迷迭香酸诱导HO-1表达上调发挥抗凋亡和保护血脑屏障等多重神经保护作用
方法：选用成年健康雄性CD1小鼠为研究对象，应用改良Longa线栓法制备小鼠右侧大脑中动脉缺血再灌注（MCAO/R）模型。实验动物随机分为5组，假手术组（Sham），假手术后腹腔注射等量生理盐水；手术组（MCAO/R），再灌注后腹腔注射等量生理盐水；迷迭香酸组（RA），再灌注后立即腹腔注射迷迭香酸40mg/kg；迷迭香酸组+ZnPPIX组（RA+ZnPPIX），缺血前24h腹腔注射10mg/kg的ZnPPIX，再灌注后立即腹腔注射迷迭香酸40mg/kg；ZnPPIX组，缺血前24h腹腔注射10mg/kg的ZnPPIX。各组取材前进行神经功能评分，脑组织含水量测定，TTC染色评价脑梗死体积，用Western blot技术和RT-PCR技术分别检测缺血脑组织中claudin-5, MMP-9, Bcl-2和Bax蛋白和mRNA水平的变化。
结果：
1再灌注后24h，同实验一结果，迷迭香酸可以降低行为学评分减小梗死体积。与MCAO/R组比较，迷迭香酸亦可减轻脑水肿，且统计学有显著差异（MCAO/R组：84.09±0.60%vs. RA组：80.43±0.55%）（P＜0.05）。予以迷迭香酸药物同时在缺血前予以10mg/kg的ZnPPIX可部分抑制上述迷迭香酸的神经保护作用，即RA+ZnPPIX组与RA组比较，有统计学意义（P＜0.05）。
2Western blot中，在缺血再灌注后24h，RA组与MCAO/R相比可明显降低Bax、MMP-9的蛋白表达，增加claudin-5和Bcl-2的蛋白表达，差异有统计学意义（P＜0.05）。而ZnPPIX可部分抑制上述Bax、MMP-9、claudin-5和Bcl-2的蛋白表达的变化，即RA+ZnPPIX组与RA组比较，有统计学意义（P＜0.05）。RT-PCR的结果与Western blot结果相一致。
结论：脑缺血再灌注损伤后，缺血脑组织的Bcl-2和claudin-5的表达下降，Bax和MMP-9表达增多；迷迭香酸可以减小梗死体积、减轻脑水肿，上调claudin-5的蛋白和mRNA表达，下调MMP-9的蛋白和mRNA表达，保护血脑屏障，且这一保护机制被ZnPPIX部分逆转，结合第一部分实验结果，提示迷迭香酸干预后经Nrf2诱导上调的HO-1通过调整claudin-5和MMP-9的表达发挥保护血脑屏障的作用；迷迭香酸抑制缺血再灌注后的细胞凋亡，调节Bcl-2和Bax的表达，且这一有益的调节作用被ZnPPIX部分抑制，提示迷迭香酸上调的HO-1同时通过调节凋亡相关蛋白Bcl-2和Bax的表达发挥有效的抗凋亡保护作用。
第三部分迷迭香酸通过PI3K/Akt信号通路激活Nrf2/HO-1对局灶性脑缺血再灌注损伤小鼠发挥神经保护作用
目的：应用PI3K/Akt信号通路抑制剂LY294002，探讨脑缺血再灌注损伤后予以迷迭香酸激活Nrf2/HO-1的信号转导通路。
方法：选用健康成年雄性CD1小鼠为研究对象，应用改良Longa线栓法制备小鼠右侧大脑中动脉缺血再灌注模型（MCAO/R）。实验动物随机分为5组，假手术组（Sham），假手术后腹腔注射等量生理盐水；手术组（MCAO/R），再灌注后腹腔注射等量生理盐水；迷迭香酸组（RA），再灌注后立即腹腔注射迷迭香酸40mg/kg；迷迭香酸组+LY294002组（RA+LY294002），缺血前15min左侧侧脑室注射10mmol/L LY294002共5μL，再灌注后立即腹腔注射迷迭香酸40mg/kg；DMSO组，缺血前15min侧脑室注射5μL2%DMSO。各组取材前进行神经功能评分，TTC染色评价脑梗死体积，用Western blot分别检测缺血脑组织中p-Akt、Akt、Nrf2和HO-1蛋白水平的变化。
结果：
1再灌注后24h，与MCAO/R组相比较，迷迭香酸干预后p-Akt蛋白表达明显上升，差异具有统计学意义（P＜0.05），若同时予以PI3K/Akt通路的抑制剂LY294002，表达上调的p-Akt明显降低，即RA+LY294002与RA组比较具有显著差异（P＜0.05），而MCAO/R与DMSO组比较，无统计学差异（P＞0.05）。Akt蛋白表达各组间无显著差异（P＞0.05）。
2MCAO/R组的HO-1、Nrf2蛋白表达水平较Sham组上调（P＜0.05），迷迭香酸组HO-1、Nrf2蛋白明显上调，且与MCAO/R组比较，具有统计学差异（P＜0.05）。若同时予以PI3K/Akt通路的抑制剂LY294002， HO-1、Nrf2蛋白水平上调受到了抑制，即RA+LY294002与RA组比较具有显著差异（P＜0.05）。MCAO/R与DMSO组比较，无统计学差异，提示侧脑室注射DMSO对实验结果无影响（P＞0.05）。
结论：局灶性脑缺血再灌注损伤后，予以迷迭香酸干预，p-Akt表达上升，而侧脑室注射PI3K/Akt抑制剂后p-Akt表达下降，提示脑缺血再灌注后，迷迭香酸可激活PI3K/Akt信号通路；LY294002可下调Nrf2和HO-1蛋白水平，提示迷迭香酸可通过PI3K/Akt信号通路激活Nrf2诱导HO-1表达上调对局灶性脑缺血再灌注损伤小鼠发挥多重神经保护作用。
With the rapid development of the national economy and the arrival of anaging population, cerebrovascular disease(CVD) has become the main diseaseendangering people’s health and life. Ischemic stroke has a high incidence,mortality, morbidity and high recurrence rate, which results in heavy socialand economic burden.
Brain is an organ most sensitive to hypoxia and brain weight accounts foronly2%of body weight. The brain oxygen consumption accounts for20%oftotal body oxygen consumption. When the ischemic stroke occurs, the keytreatment is to recover the blood supply timely and effectively. Recombinanttissue plasminogen activator (tPA), targeting the occlusion and inducingreperfusion, is the only FDA-approved stroke medication currently. However,recanalization following ischemia paradoxically causes severe cerebralischemia-reperfusion (I/R) injury, which always plagues clinical treatment andprognosis. Cerebral I/R injury is a complex pathophysiologic process and isthought to be a complex regulatory networks formed by the interaction of avariety of mechanisms, including inflammatory damage, free radical damage,oxidative stress, excitatory amino acid toxicity, intracellular calcium overload,etc. Cerebral I/R injury directly or indirectly leads to neuronal apoptosis/death,BBB damage, eventually leading to neurological deficit, and even endangerthe lives of patients. Therefore, seeking effective neuroprotective agent toischemic stroke has been the focus of the study. Nowadays, the use ofmedicinal plants is becoming an increasingly attractive approach as acomplement and an alternative for treating acute stroke.
Rosmarinic acid (RA), is a naturally occurring hydroxylated polyphenoliccompound, and it is commonly found in various plant families. It has been reported that RA elicits multiple promising biological activities such asanti-oxidative, anti-inflammatory, antiviral and anticancer abilities. Abundantstudies have demonstrated the beneficial effects of RA in various diseases.However, whether RA could confer the neurorescue effects in ischemic strokein vivo has not been well investigated. Therefore, we designed this study toevaluate the role and possible mechanisms of RA on I/R brain injury.
Male, healthy CD1mice were used and subjected to focal cerebralischemia/reperfusion by right MCA occlusion as described by Longapreviously. RA was used to treat ischemic stroke and its neuroprotective effectwas analyzed. Neurological deficits, brain water content and infarct volumewere evaluated. TUNEL staining was used to detect apoptosis. The expressionof HO-1, Nrf2, Bcl-2, Bax, MMP-9and claudin-5were detected. Furthermore,ZnPPIX (a common used HO-1activity inhibitor) and LY294002(a typicalPI3K/Akt pathway inhibitor) were used to explore the important role ofNrf2/HO-1pathway in the neuroprotection of RA against I/R brain injury.
PartⅠ Anti-oxidative and Anti-apoptotic effects of RA against cerebralI/R injury
Objective：The aim of this study was to evaluate the neuroprotectiveeffects of RA against focal cerebral ischemia reperfusion. Neurologicaldeficits, infarct volume, the expression of HO-1, Nrf2, Bcl-2and Bax weremeasured.
Methods：Male, healthy CD1mice were subjected to transcient middlecerebral artery occlusion (MCAO/R). Experiment1was used to analyze RA’sneuroprotective effect. Neurological deficits, and infarct volume wereevaluated at24h after reperfusion. Mice were randomly assigned to fivegroups: Sham-operated group (Sham): animals received sham operation andequal volume of saline; MCAO/R group: animals received transcient middlecerebral artery occlusion and equal volume of saline; and RA groups: animalsreceived transcient middle cerebral artery occlusion and treated with low doseof RA10mg/kg (RA-L), middle dose of RA20mg/kg (RA-M) and high doseof RA40mg/kg (RA-H). RA was administered immediately at the onset of reperfusion. Experiment2was used to analyze RA’s anti-oxidative andanti-apoptotic effects. TUNEL staining was used to detect apoptosis. Theexpression of HO-1, Nrf2, Bcl-2and Bax were detected at24h afterreperfusion using western blot and RT-PCR. Mice were randomly assigned tofour groups: Sham-operated group (Sham): animals received sham operationand equal volume of saline; MCAO/R group: animals received transcientmiddle cerebral artery occlusion and equal volume of saline; and RA groups:animals received transcient middle cerebral artery occlusion and treated withmiddle dose of RA20mg/kg (RA-M) and high dose of RA40mg/kg (RA-H).RA was administered immediately at the onset of reperfusion.
Results：
1Mice in Sham group had no palsy and score was zero. Mice inMCAO/R group, low dose group, middle dose group and high dose groupperformed significant neurological deficits. Compared with MCAO/R group,there was a significant improvement in neurological deficit scores in the RA-Hgroup and RA-M group at24h after reperfusion (P <0.05). By contrast, therewas no significant effect in RA-L group compared with MCAO/R group (P>0.05).
2Compared with MCAO/R group, RA-H and RA-M group reduced theinfarct volume significantly at24h after reperfusion(P <0.05). RA-L grouphad no effect on infarct volume (P>0.05). Based on the results above, wedemonstrated that RA delivered at middle dose (20mg/kg) and high dose (40mg/kg) have a better therapeutic effect after reperfusion, and therefore wefocused on the RA treatment at middle dose (RA-M) and high dose (RA-H)for subsequent molecular and biochemical analysis.
3After reperfusion, TUNEL-positive cells were numerous in theMCAO/R group. Treatment with RA, the number of TUNEL-positive cellswas significantly decreased and the effect was more obvious in the RA-Hgroup(P <0.05).
4Compared with MCAO/R group, RA-H group significantlyup-regulated the expression of Nrf2, HO-1, Bcl-2and down-regulated the expression of Bax at24h after reperfusion in protein and gene levels (P <0.05). A similar but less marked effect was observed in the RA-M groupcompared with the RA-H group (P <0.05).
Conclusions：Systemic administration of RA is effective which candecrease the infarct volume, ameliorate the neurological deficits, and reducedTUNEL-positive cells. RA significantly increased the expression of Nrf2andHO-1, which suggested the Nrf2/HO-1pathway was activated and activationof Nrf2with RA results in upregulation of HO-1. Meanwhile, RAup-regulated the expression of Bcl-2and down-regulated the expression ofBax. Taken together, the expression changes of Nrf2, HO-1, Bcl-2and Baxwere involved in the underlying mechanism of anti-oxidantive andanti-apoptotic effects of RA against cerebral I/R injury.
PartⅡ Multiple neuroprotection of RA against cerebral I/R Injury isattibutable to upregulation of HO-1
Objective：This study is to evaluate the upregulation of HO-1after RAtreatment in multiple neuroprotection against cerebral I/R Injury by using acommonly used specific HO-1inhibitor ZnPPIX.
Methods：Male, healthy CD1mice were subjected to transcient middlecerebral artery occlusion (MCAO/R).Mice were randomly assigned to fivegroups: Sham-oprated group (Sham): animals received sham operation andequal volume of saline; MCAO/R group: animals received transcient middlecerebral artery occlusion and equal volume of saline; RA groups: animalsreceived transcient middle cerebral artery occlusion and treated with RA (40mg/kg); RA+ZnPPIX group: Mice were pretreated with ZnPPIX(10mg/Kg)via intraperitoneal injection24h before operation and treated with RA at doseof40mg/kg after reperfusion. ZnPPIX group: Mice were pretreated withZnPPIX (10mg/Kg) via intraperitoneal injection24h before operation.Neurological deficits, brain water content and infarct volume were evaluatedat24h after reperfusion. The expressions of Bax, Bcl-2, MMP-9andclaudin-5were detected to evaluated the important role of HO-1upregulatedwith RA treatment on alleviating apoptosis and blood-brain barrier(BBB) permeability.
Results：
1Compared with MCAO/R group, RA decreased the neurological deficitscore, infarct volume and brain water content significantly at24h afterreperfusion (P <0.05). These protective role of RA was partly reversed withZnppIX adminiatration (P <0.05).
2Compared with MCAO/R group, RA significantly up-regulated theexpression of claudin-5, Bcl-2and down-regulated the expression of MMP-9,Bax at24h after reperfusion in protein and mRNA levels(P <0.05). Theregulation of claudin5, MMP-9, Bcl-2and Bax expression was partly inhibitedwith ZnppIX applyment and there was a significant difference between RAgroup and ZnppIX co-treatment group.
Conclusions：RA treatment reduced brain edema, increased the claudin-5expression and decreased the MMP-9expression, which suggested that RAalleviated BBB permeability. But the positive effect was reversed by ZnppIX.Futhermore, ZnppIX co-treatment partly abolished the regulation of Bcl-2andBax by RA. Taken together, the above results showed that the anti-apoptosisand alleviating BBB permeability abilities of RA were attributable toupregulation of HO-1.
PartⅢ RA induces upregulation of Nrf2and HO-1expression involvingPI3K/Akt signal pathway in a mice model of focal cerebralischemia reperfusion
Objective: This study is to evaluate the involvement of PI3K/Akt signalpathway in upregulating Nrf2and HO-1expression induced by RA after focalcerebral ischemia reperfusion.
Methods：Male, healthy CD1mice were subjected to transcient middlecerebral artery occlusion (MCAO/R).Mice were randomly assigned to fivegroups: Sham-oprated group (Sham): animals received sham operation andequal volume of saline; MCAO/R group: animals received transcient middlecerebral artery occlusion and equal volume of saline; RA groups: animalsreceived transcient middle cerebral artery occlusion and treated with RA (40 mg/kg); RA+LY294002group: Mice were treated with LY294002viaintracerebroventricular injection15min before ischemia and treated with RAat dose of40mg/kg after reperfusion. DMSO group: Mice were treated withequal volume of DMSO via intracerebroventricular injection15min beforeischemia. Neurological deficits and infarct volume were evaluated at24hafter reperfusion. The expressions of p-Akt, Akt, HO-1and Nrf2weredetected to evaluated the important role of PI3K/Akt signal pathway inup-regulating Nrf2and HO-1expression.
Results：
1Compared with MCAO/R group, RA increased the expression of p-Aktin ischemic cortex significantly at24h after reperfusion (P <0.05).Intracerebroventricular injection of LY294002reversed the positive effectpartly and there was a great significance between RA group and LY294002co-treatment group (P <0.05). By contrast, there was no significant change inexpression of p-Akt between MCAO/R group and DMSO group(P>0.05).
2The expression of HO-1and Nrf2was increased with RA treatment (P<0.05). Treated with LY294002via intracerebroventricular injection15minbefore ischemia partly abolished the induction of HO-1and Nrf2expression.There was a great significance between RA group and LY294002co-treatmentgroup (P <0.05). Meanwhile, there was no significant change in expression ofHO-1and Nrf2between MCAO/R group and DMSO group (P>0.05).
Conclusions：RA increased the expression of p-Akt in ischemic cortexsignificantly after reperfusion and this positive effect was reversed byLY294002, which suggested that PI3K/Akt signal pathway was activated withRA treatment after focal cerebral ischemia reperfusion. The up-regulatedexpression of HO-1and Nrf2after RA administration was inhibited partly byLY294002, which suggested that the regulation of HO-1and Nrf2expressioninvolved in PI3K/Akt signal pathway.
脑是一个对缺氧最敏感的器官，脑重量仅占人体重量之2％，脑耗氧量却占人体总耗氧量的20％。脑组织发生缺血后早期、及时、有效的恢复血供是治疗的关键，组织纤溶酶原激活剂（tissue plasminogenactivator，tPA）是迄今为止美国食品和药物管理局FDA批准的、用于急性缺血卒中治疗的首选药物。然而，缺血脑组织实现血流再通的同时带来的再灌注损伤一直困扰着临床的治疗和预后。脑缺血及再灌注损伤的病理生理机制复杂，认为存在多种机制相互作用形成的复杂调控网络，包括炎症损害、自由基损伤、氧化应激、兴奋性氨基酸毒性、细胞内钙超载等，直接/间接导致神经细胞凋亡/死亡，血脑屏障破坏，最终导致神经功能的缺失，甚至危及患者生命。
因此，积极寻求有效的神经保护剂是一直以来缺血性卒中研究的重点，尽管动物实验取得了一定的效果，但目前为止尚没有临床证实有明确疗效的神经保护剂。近年来在细胞实验和动物实验中应用天然植物有效成分及其制剂治疗急性卒中成为研究的热点，为新型神经保护剂的研制提供了广阔的思维空间。迷迭香酸(Rosmarinic acid，RA)是天然存在于多种植物中的一种多酚，也是丹参多酚酸的主要成分之一，具有抗氧化、抗炎、抗凋亡、抗肿瘤等多种生物活性，且在多种疾病模型中证明有效，而其在缺血卒中治疗的研究鲜见报道。因此，本课题旨在研究迷迭香酸作为新型神经保护剂的可能性及潜在的神经保护机制，为临床治疗提供新的实验依据。
本研究选用成年健康雄性CD1小鼠，建立局灶性脑缺血再灌注模型，在此模型基础上观察缺血再灌注后HO-1，Nrf2，claudin-5，MMP-9，Bcl-2和Bax的表达，应用迷迭香酸干预，评价干预前后梗死体积、脑含水量、神经功能缺损评分，TUNEL染色评价细胞凋亡情况，检测迷迭香酸对HO-1，Nrf2，claudin-5，MMP-9，Bcl-2和Bax的表达调节，以期初步探讨其在缺血再灌注损伤后的脑保护作用机制；应用HO-1活性抑制剂和PI3K/Akt信号通路抑制剂进一步探讨Nrf2/HO-1通路激活在迷迭香酸发挥神经保护作用中的重要地位。本研究分三部分，现将各部分内容概述如下。
第一部分迷迭香酸对局灶性脑缺血再灌注损伤小鼠的抗氧化及抗凋亡机制研究
目的：观察缺血再灌注小鼠脑缺血损伤后24h梗死体积、神经功能缺损评分，HO-1，Nrf2，Bcl-2和Bax的表达变化，探讨迷迭香酸对缺血再灌注损伤小鼠的抗氧化及抗凋亡机制。
方法：选用成年健康雄性CD-1小鼠为研究对象，采用改良Longa线栓法制备大脑中动脉缺血再灌注（MCAO/R）模型。实验1：观察迷迭香酸的神经保护作用。实验动物随机分为5组，假手术组（Sham），假手术后腹腔注射等量生理盐水；手术组（MCAO/R），再灌注后腹腔注射等量生理盐水；迷迭香酸小剂量组（RA-L），再灌注后立即腹腔注射迷迭香酸10mg/kg；迷迭香酸中剂量组（RA-M），再灌注后立即腹腔注射迷迭香酸20mg/kg；迷迭香酸大剂量组（RA-H），再灌注后立即腹腔注射迷迭香酸40mg/kg。缺血再灌注后24h取材前Longa评分法进行神经功能评分，TTC染色评价脑梗死体积。实验2：观察迷迭香酸的抗氧化及抗凋亡功能。检测缺血脑组织中HO-1，Nrf2，Bcl-2和Bax表达的变化情况。实验动物随机分为4组，假手术组（Sham），手术组（MCAO/R），迷迭香酸中剂量组（RA-M），迷迭香酸大剂量组（RA-H）。TUNEL染色检测缺血周围组织的细胞凋亡情况，RT-PCR方法和Western blot方法检测缺血脑组织HO-1，Nrf2，Bcl-2和Bax的mRNA和蛋白水平的变化。
结果：
1Sham组无神经功能缺损，神经功能缺损评分为0分；缺血再灌注后24h，MCAO/R组神经功能缺损评分严重，RA-L组MCAO/R组相比无明显降低神经功能缺损，差异无统计学意义（P＞0.05），RA-M和RA-H组与MCAO/R组相比均可显著降低神经功能缺损，差异有统计学意义（P＜0.05）；
2再灌注24h，RA-L组与MCAO/R组相比没有明显降低脑梗死体积，差异无统计学意义（50.36±1.81%vs.48.09±2.45%）（P＞0.05），RA-M组（50.36±1.81%vs.41.67±0.83%）和RA-H组（50.36±1.81%vs.30.86±1.79%）与MCAO/R组相比均可明显降低脑梗死体积，差异有统计学意义（P＜0.05）。且RA-H组较RA-M组在减少梗死体积的效果上作用更显著（P＜0.05）。我们可以看到迷迭香酸在20mg/kg（RA-M）和40mg/kg（RA-H）时发挥更好的治疗作用，因此，下面的研究集中在20mg/kg和40mg/kg。
3TUNEL染色结果，再灌注后24h，与MCAO/R组比较，RA-M组(51.75±2.36vs.41±1.41)和RA-H组(51.75±2.36vs.32.75±1.70)均可显著减少TUNEL阳性细胞数，且RA-H组作用更为显著（P＜0.05）。
4Western blot中，与MCAO/R组比较，RA-M和RA-H组在再灌注后24h均可明显增加HO-1，Nrf2，Bcl-2的蛋白表达和明显降低Bax蛋白的表达，差异有统计学意义（P＜0.05），且RA-H组作用更为显著（P＜0.05）。
5再灌注后24h，RT-PCR分别检测RA-M和RA-H组HO-1，Nrf2，Bcl-2和Bax的基因表达情况。与MCAO/R组相比，两个剂量组均可明显增加HO-1，Nrf2，Bcl-2的mRNA表达，明显降低Bax的mRNA表达，差异有统计学意义（P＜0.05）。RA-H组作用更为显著（P＜0.05）。
结论：迷迭香酸对局灶性脑缺血再灌注小鼠有较好的神经保护作用，可改善脑缺血损伤后的神经功能缺失，减小梗死体积，上调Bcl-2表达、下调Bax表达，诱导Nrf2活化、HO-1表达上调，因此我们推测迷迭香酸的神经保护作用可能通过激活Nrf2/HO-1通路，上调Bcl-2的表达，抑制Bax表达实现的；这些是迷迭香酸发挥其抗氧化、抗凋亡的作用，从而实现其神经保护作用。
第二部分迷迭香酸上调HO-1表达对局灶性脑缺血再灌注损伤小鼠发挥多重神经保护作用
目的：应用HO-1活性抑制剂ZnPPIX，探讨脑缺血再灌注损伤后，迷迭香酸诱导HO-1表达上调发挥抗凋亡和保护血脑屏障等多重神经保护作用
方法：选用成年健康雄性CD1小鼠为研究对象，应用改良Longa线栓法制备小鼠右侧大脑中动脉缺血再灌注（MCAO/R）模型。实验动物随机分为5组，假手术组（Sham），假手术后腹腔注射等量生理盐水；手术组（MCAO/R），再灌注后腹腔注射等量生理盐水；迷迭香酸组（RA），再灌注后立即腹腔注射迷迭香酸40mg/kg；迷迭香酸组+ZnPPIX组（RA+ZnPPIX），缺血前24h腹腔注射10mg/kg的ZnPPIX，再灌注后立即腹腔注射迷迭香酸40mg/kg；ZnPPIX组，缺血前24h腹腔注射10mg/kg的ZnPPIX。各组取材前进行神经功能评分，脑组织含水量测定，TTC染色评价脑梗死体积，用Western blot技术和RT-PCR技术分别检测缺血脑组织中claudin-5, MMP-9, Bcl-2和Bax蛋白和mRNA水平的变化。
结果：
1再灌注后24h，同实验一结果，迷迭香酸可以降低行为学评分减小梗死体积。与MCAO/R组比较，迷迭香酸亦可减轻脑水肿，且统计学有显著差异（MCAO/R组：84.09±0.60%vs. RA组：80.43±0.55%）（P＜0.05）。予以迷迭香酸药物同时在缺血前予以10mg/kg的ZnPPIX可部分抑制上述迷迭香酸的神经保护作用，即RA+ZnPPIX组与RA组比较，有统计学意义（P＜0.05）。
2Western blot中，在缺血再灌注后24h，RA组与MCAO/R相比可明显降低Bax、MMP-9的蛋白表达，增加claudin-5和Bcl-2的蛋白表达，差异有统计学意义（P＜0.05）。而ZnPPIX可部分抑制上述Bax、MMP-9、claudin-5和Bcl-2的蛋白表达的变化，即RA+ZnPPIX组与RA组比较，有统计学意义（P＜0.05）。RT-PCR的结果与Western blot结果相一致。
结论：脑缺血再灌注损伤后，缺血脑组织的Bcl-2和claudin-5的表达下降，Bax和MMP-9表达增多；迷迭香酸可以减小梗死体积、减轻脑水肿，上调claudin-5的蛋白和mRNA表达，下调MMP-9的蛋白和mRNA表达，保护血脑屏障，且这一保护机制被ZnPPIX部分逆转，结合第一部分实验结果，提示迷迭香酸干预后经Nrf2诱导上调的HO-1通过调整claudin-5和MMP-9的表达发挥保护血脑屏障的作用；迷迭香酸抑制缺血再灌注后的细胞凋亡，调节Bcl-2和Bax的表达，且这一有益的调节作用被ZnPPIX部分抑制，提示迷迭香酸上调的HO-1同时通过调节凋亡相关蛋白Bcl-2和Bax的表达发挥有效的抗凋亡保护作用。
第三部分迷迭香酸通过PI3K/Akt信号通路激活Nrf2/HO-1对局灶性脑缺血再灌注损伤小鼠发挥神经保护作用
目的：应用PI3K/Akt信号通路抑制剂LY294002，探讨脑缺血再灌注损伤后予以迷迭香酸激活Nrf2/HO-1的信号转导通路。
方法：选用健康成年雄性CD1小鼠为研究对象，应用改良Longa线栓法制备小鼠右侧大脑中动脉缺血再灌注模型（MCAO/R）。实验动物随机分为5组，假手术组（Sham），假手术后腹腔注射等量生理盐水；手术组（MCAO/R），再灌注后腹腔注射等量生理盐水；迷迭香酸组（RA），再灌注后立即腹腔注射迷迭香酸40mg/kg；迷迭香酸组+LY294002组（RA+LY294002），缺血前15min左侧侧脑室注射10mmol/L LY294002共5μL，再灌注后立即腹腔注射迷迭香酸40mg/kg；DMSO组，缺血前15min侧脑室注射5μL2%DMSO。各组取材前进行神经功能评分，TTC染色评价脑梗死体积，用Western blot分别检测缺血脑组织中p-Akt、Akt、Nrf2和HO-1蛋白水平的变化。
结果：
1再灌注后24h，与MCAO/R组相比较，迷迭香酸干预后p-Akt蛋白表达明显上升，差异具有统计学意义（P＜0.05），若同时予以PI3K/Akt通路的抑制剂LY294002，表达上调的p-Akt明显降低，即RA+LY294002与RA组比较具有显著差异（P＜0.05），而MCAO/R与DMSO组比较，无统计学差异（P＞0.05）。Akt蛋白表达各组间无显著差异（P＞0.05）。
2MCAO/R组的HO-1、Nrf2蛋白表达水平较Sham组上调（P＜0.05），迷迭香酸组HO-1、Nrf2蛋白明显上调，且与MCAO/R组比较，具有统计学差异（P＜0.05）。若同时予以PI3K/Akt通路的抑制剂LY294002， HO-1、Nrf2蛋白水平上调受到了抑制，即RA+LY294002与RA组比较具有显著差异（P＜0.05）。MCAO/R与DMSO组比较，无统计学差异，提示侧脑室注射DMSO对实验结果无影响（P＞0.05）。
结论：局灶性脑缺血再灌注损伤后，予以迷迭香酸干预，p-Akt表达上升，而侧脑室注射PI3K/Akt抑制剂后p-Akt表达下降，提示脑缺血再灌注后，迷迭香酸可激活PI3K/Akt信号通路；LY294002可下调Nrf2和HO-1蛋白水平，提示迷迭香酸可通过PI3K/Akt信号通路激活Nrf2诱导HO-1表达上调对局灶性脑缺血再灌注损伤小鼠发挥多重神经保护作用。
With the rapid development of the national economy and the arrival of anaging population, cerebrovascular disease(CVD) has become the main diseaseendangering people’s health and life. Ischemic stroke has a high incidence,mortality, morbidity and high recurrence rate, which results in heavy socialand economic burden.
Brain is an organ most sensitive to hypoxia and brain weight accounts foronly2%of body weight. The brain oxygen consumption accounts for20%oftotal body oxygen consumption. When the ischemic stroke occurs, the keytreatment is to recover the blood supply timely and effectively. Recombinanttissue plasminogen activator (tPA), targeting the occlusion and inducingreperfusion, is the only FDA-approved stroke medication currently. However,recanalization following ischemia paradoxically causes severe cerebralischemia-reperfusion (I/R) injury, which always plagues clinical treatment andprognosis. Cerebral I/R injury is a complex pathophysiologic process and isthought to be a complex regulatory networks formed by the interaction of avariety of mechanisms, including inflammatory damage, free radical damage,oxidative stress, excitatory amino acid toxicity, intracellular calcium overload,etc. Cerebral I/R injury directly or indirectly leads to neuronal apoptosis/death,BBB damage, eventually leading to neurological deficit, and even endangerthe lives of patients. Therefore, seeking effective neuroprotective agent toischemic stroke has been the focus of the study. Nowadays, the use ofmedicinal plants is becoming an increasingly attractive approach as acomplement and an alternative for treating acute stroke.
Rosmarinic acid (RA), is a naturally occurring hydroxylated polyphenoliccompound, and it is commonly found in various plant families. It has been reported that RA elicits multiple promising biological activities such asanti-oxidative, anti-inflammatory, antiviral and anticancer abilities. Abundantstudies have demonstrated the beneficial effects of RA in various diseases.However, whether RA could confer the neurorescue effects in ischemic strokein vivo has not been well investigated. Therefore, we designed this study toevaluate the role and possible mechanisms of RA on I/R brain injury.
Male, healthy CD1mice were used and subjected to focal cerebralischemia/reperfusion by right MCA occlusion as described by Longapreviously. RA was used to treat ischemic stroke and its neuroprotective effectwas analyzed. Neurological deficits, brain water content and infarct volumewere evaluated. TUNEL staining was used to detect apoptosis. The expressionof HO-1, Nrf2, Bcl-2, Bax, MMP-9and claudin-5were detected. Furthermore,ZnPPIX (a common used HO-1activity inhibitor) and LY294002(a typicalPI3K/Akt pathway inhibitor) were used to explore the important role ofNrf2/HO-1pathway in the neuroprotection of RA against I/R brain injury.
PartⅠ Anti-oxidative and Anti-apoptotic effects of RA against cerebralI/R injury
Objective：The aim of this study was to evaluate the neuroprotectiveeffects of RA against focal cerebral ischemia reperfusion. Neurologicaldeficits, infarct volume, the expression of HO-1, Nrf2, Bcl-2and Bax weremeasured.
Methods：Male, healthy CD1mice were subjected to transcient middlecerebral artery occlusion (MCAO/R). Experiment1was used to analyze RA’sneuroprotective effect. Neurological deficits, and infarct volume wereevaluated at24h after reperfusion. Mice were randomly assigned to fivegroups: Sham-operated group (Sham): animals received sham operation andequal volume of saline; MCAO/R group: animals received transcient middlecerebral artery occlusion and equal volume of saline; and RA groups: animalsreceived transcient middle cerebral artery occlusion and treated with low doseof RA10mg/kg (RA-L), middle dose of RA20mg/kg (RA-M) and high doseof RA40mg/kg (RA-H). RA was administered immediately at the onset of reperfusion. Experiment2was used to analyze RA’s anti-oxidative andanti-apoptotic effects. TUNEL staining was used to detect apoptosis. Theexpression of HO-1, Nrf2, Bcl-2and Bax were detected at24h afterreperfusion using western blot and RT-PCR. Mice were randomly assigned tofour groups: Sham-operated group (Sham): animals received sham operationand equal volume of saline; MCAO/R group: animals received transcientmiddle cerebral artery occlusion and equal volume of saline; and RA groups:animals received transcient middle cerebral artery occlusion and treated withmiddle dose of RA20mg/kg (RA-M) and high dose of RA40mg/kg (RA-H).RA was administered immediately at the onset of reperfusion.
Results：
1Mice in Sham group had no palsy and score was zero. Mice inMCAO/R group, low dose group, middle dose group and high dose groupperformed significant neurological deficits. Compared with MCAO/R group,there was a significant improvement in neurological deficit scores in the RA-Hgroup and RA-M group at24h after reperfusion (P <0.05). By contrast, therewas no significant effect in RA-L group compared with MCAO/R group (P>0.05).
2Compared with MCAO/R group, RA-H and RA-M group reduced theinfarct volume significantly at24h after reperfusion(P <0.05). RA-L grouphad no effect on infarct volume (P>0.05). Based on the results above, wedemonstrated that RA delivered at middle dose (20mg/kg) and high dose (40mg/kg) have a better therapeutic effect after reperfusion, and therefore wefocused on the RA treatment at middle dose (RA-M) and high dose (RA-H)for subsequent molecular and biochemical analysis.
3After reperfusion, TUNEL-positive cells were numerous in theMCAO/R group. Treatment with RA, the number of TUNEL-positive cellswas significantly decreased and the effect was more obvious in the RA-Hgroup(P <0.05).
4Compared with MCAO/R group, RA-H group significantlyup-regulated the expression of Nrf2, HO-1, Bcl-2and down-regulated the expression of Bax at24h after reperfusion in protein and gene levels (P <0.05). A similar but less marked effect was observed in the RA-M groupcompared with the RA-H group (P <0.05).
Conclusions：Systemic administration of RA is effective which candecrease the infarct volume, ameliorate the neurological deficits, and reducedTUNEL-positive cells. RA significantly increased the expression of Nrf2andHO-1, which suggested the Nrf2/HO-1pathway was activated and activationof Nrf2with RA results in upregulation of HO-1. Meanwhile, RAup-regulated the expression of Bcl-2and down-regulated the expression ofBax. Taken together, the expression changes of Nrf2, HO-1, Bcl-2and Baxwere involved in the underlying mechanism of anti-oxidantive andanti-apoptotic effects of RA against cerebral I/R injury.
PartⅡ Multiple neuroprotection of RA against cerebral I/R Injury isattibutable to upregulation of HO-1
Objective：This study is to evaluate the upregulation of HO-1after RAtreatment in multiple neuroprotection against cerebral I/R Injury by using acommonly used specific HO-1inhibitor ZnPPIX.
Methods：Male, healthy CD1mice were subjected to transcient middlecerebral artery occlusion (MCAO/R).Mice were randomly assigned to fivegroups: Sham-oprated group (Sham): animals received sham operation andequal volume of saline; MCAO/R group: animals received transcient middlecerebral artery occlusion and equal volume of saline; RA groups: animalsreceived transcient middle cerebral artery occlusion and treated with RA (40mg/kg); RA+ZnPPIX group: Mice were pretreated with ZnPPIX(10mg/Kg)via intraperitoneal injection24h before operation and treated with RA at doseof40mg/kg after reperfusion. ZnPPIX group: Mice were pretreated withZnPPIX (10mg/Kg) via intraperitoneal injection24h before operation.Neurological deficits, brain water content and infarct volume were evaluatedat24h after reperfusion. The expressions of Bax, Bcl-2, MMP-9andclaudin-5were detected to evaluated the important role of HO-1upregulatedwith RA treatment on alleviating apoptosis and blood-brain barrier(BBB) permeability.
Results：
1Compared with MCAO/R group, RA decreased the neurological deficitscore, infarct volume and brain water content significantly at24h afterreperfusion (P <0.05). These protective role of RA was partly reversed withZnppIX adminiatration (P <0.05).
2Compared with MCAO/R group, RA significantly up-regulated theexpression of claudin-5, Bcl-2and down-regulated the expression of MMP-9,Bax at24h after reperfusion in protein and mRNA levels(P <0.05). Theregulation of claudin5, MMP-9, Bcl-2and Bax expression was partly inhibitedwith ZnppIX applyment and there was a significant difference between RAgroup and ZnppIX co-treatment group.
Conclusions：RA treatment reduced brain edema, increased the claudin-5expression and decreased the MMP-9expression, which suggested that RAalleviated BBB permeability. But the positive effect was reversed by ZnppIX.Futhermore, ZnppIX co-treatment partly abolished the regulation of Bcl-2andBax by RA. Taken together, the above results showed that the anti-apoptosisand alleviating BBB permeability abilities of RA were attributable toupregulation of HO-1.
PartⅢ RA induces upregulation of Nrf2and HO-1expression involvingPI3K/Akt signal pathway in a mice model of focal cerebralischemia reperfusion
Objective: This study is to evaluate the involvement of PI3K/Akt signalpathway in upregulating Nrf2and HO-1expression induced by RA after focalcerebral ischemia reperfusion.
Methods：Male, healthy CD1mice were subjected to transcient middlecerebral artery occlusion (MCAO/R).Mice were randomly assigned to fivegroups: Sham-oprated group (Sham): animals received sham operation andequal volume of saline; MCAO/R group: animals received transcient middlecerebral artery occlusion and equal volume of saline; RA groups: animalsreceived transcient middle cerebral artery occlusion and treated with RA (40 mg/kg); RA+LY294002group: Mice were treated with LY294002viaintracerebroventricular injection15min before ischemia and treated with RAat dose of40mg/kg after reperfusion. DMSO group: Mice were treated withequal volume of DMSO via intracerebroventricular injection15min beforeischemia. Neurological deficits and infarct volume were evaluated at24hafter reperfusion. The expressions of p-Akt, Akt, HO-1and Nrf2weredetected to evaluated the important role of PI3K/Akt signal pathway inup-regulating Nrf2and HO-1expression.
Results：
1Compared with MCAO/R group, RA increased the expression of p-Aktin ischemic cortex significantly at24h after reperfusion (P <0.05).Intracerebroventricular injection of LY294002reversed the positive effectpartly and there was a great significance between RA group and LY294002co-treatment group (P <0.05). By contrast, there was no significant change inexpression of p-Akt between MCAO/R group and DMSO group(P>0.05).
2The expression of HO-1and Nrf2was increased with RA treatment (P<0.05). Treated with LY294002via intracerebroventricular injection15minbefore ischemia partly abolished the induction of HO-1and Nrf2expression.There was a great significance between RA group and LY294002co-treatmentgroup (P <0.05). Meanwhile, there was no significant change in expression ofHO-1and Nrf2between MCAO/R group and DMSO group (P>0.05).
Conclusions：RA increased the expression of p-Akt in ischemic cortexsignificantly after reperfusion and this positive effect was reversed byLY294002, which suggested that PI3K/Akt signal pathway was activated withRA treatment after focal cerebral ischemia reperfusion. The up-regulatedexpression of HO-1and Nrf2after RA administration was inhibited partly byLY294002, which suggested that the regulation of HO-1and Nrf2expressioninvolved in PI3K/Akt signal pathway.
引文
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