紫草素对局灶性脑缺血再灌注损伤小鼠的脑保护作用及其相关机制研究
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摘要
缺血性脑血管病是临床常见病、多发病，具有致残率高、复发率高、恢复缓慢等特点。国际卫生组织统计在全球范围内平均每400个人中存在一个缺血性脑血管病患者；而在我国每年新发生的250万脑血管病患者中，大约有75%以上为缺血性脑血管病。缺血性脑血管病的死亡率仅次于心脏疾病，是人类主要的死亡原因之一，而在所有的缺血性脑血管病患者中约有30%患者由于溶栓治疗或栓子自发向远端推移出现血管再通，伴随着缺血再灌注损伤的发生。
缺血再灌注损伤是导致患者病情加重的重要原因，近年来随着现代免疫学和分子生物学的迅速发展，对于脑缺血再灌注损伤病理生理机制的研究已取得了重大进步，认为其病理生理机制存在损伤级联反应，主要包括炎症损害、氧化应激、兴奋性氨基酸毒性、凋亡、细胞内钙超载、自由基损伤等几种因素相互作用形成了复杂的调控网络，造成一系列病理级联反应，可直接或者间接导致神经细胞凋亡/死亡。在脑缺血后复杂的病理损伤机制中，炎症反应、氧化应激是脑缺血再灌注损伤的主要原因，是目前神经科学研究的热点，抗氧化应激、减轻炎症损伤成为治疗缺血性脑血管病的主要途径之一。
尽管已经有许多合成或天然物质的神经保护剂经过实验研究，证明具有抗炎、抗氧化、抗凋亡等作用，但是由于动物与人类生理机制及耐受性等方面的差异以及脑缺血后级联反应的复杂性，单一阻断某一病理环节的药物可能难以奏效，多数神经保护剂未能在临床得到广泛应用，出现临床和理论的严重脱节，所以在目前的临床治疗中，理想的神经保护剂应该具备针对缺血再灌注损伤多个损伤环节发挥保护作用的特性。
紫草素(Shikonin)是存在于天然植物中的一种脂溶性萘醌类化合物，分子式为：C16H16O5，主要存在于紫草科植物紫草的根中，具有多种药理活性，研究表明紫草素具有抗氧化、抗肿瘤、抗炎、抗凋亡、抗病毒等生物学作用，并且毒副作用低，具有良好的临床应用潜力。
本研究选用雄性CD-1小鼠为研究对象，采用线栓法建立脑缺血再灌注模型，在此模型基础上对脑缺血后炎症反应代表性因子TLR4，P38MAPK，NF-κB，TNF-α，氧化应激代表性因子HO-1，Nrf2，PI3K，AKT和MMP-9，claudin-5为代表的血脑屏障损伤程度进行观察，测定大脑皮层梗塞灶超氧化物歧化酶（Superoxide Dismutase，SOD）和丙二醛（Malondiadehyde，MDA）的含量。确定缺血后与炎症反应、氧化损伤的关系，通过选用萘醌类化合物紫草素进行干预，对干预前后梗死体积、脑含水量、神经功能缺损进行评估，观察紫草素对TLR4，P38MAPK，NF-κB，TNF-α，PI3K，AKT，HO-1，Nrf2，MMP-9及Claudin-5信号通路的作用，初步探讨其在缺血再灌注损伤后的脑保护作用机制，并应用PI3K特异性拮抗剂LY294002进行干预，观察上述各因子的变化，评价神经功能缺失、脑水肿和梗死体积情况。本研究分三部分，现将各部分内容叙述如下。
第一部分紫草素对局灶性脑缺血再灌注损伤小鼠的抗氧化作用及其相关机制研究
目的：观察脑缺血再灌注损伤后梗死体积、脑含水量、神经功能缺损评分、PI3K、AKT、HO-1、Nrf2的表达变化以及SOD活性和MDA含量的变化，研究紫草素对脑缺血再灌注损伤所致的氧化应激反应的作用，探讨紫草素抗氧化应激作用的相关机制。
方法：选用成年雄性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模型。试验1：随机将CD-1小鼠分为2组：假手术组(Sham)，手术组(MCAO)：根据不同的时间点又分为3h、6h、24h、48h、72h五个亚组。试验2：随机将CD-1小鼠分为4组：假手术组(Sham)、手术组(Vehicle)、低剂量紫草素干预组(L-Shi)(10mg/Kg)、高剂量紫草素干预组(H-Shi)(25mg/Kg)。试验3：采用随机的方法分别将CD-1小鼠分为4组：假手术组（Sham)、溶剂对照组（Vehicle)、大剂量紫草素干预组（H-Shi)及紫草素+LY294002组（Shikonin+LY294002)。Sham组：假手术前以灌胃方式给予等量PBS+1%DMSO，每日一次，连续三天，Vehicle组：缺血再灌注前以灌胃方式给予等量PBS+1%DMSO，每日一次，连续三天，L-Shi组：缺血再灌注前以灌胃方式给予紫草素10mg/Kg，每日一次，连续三天，H-Shi组：缺血再灌注前以灌胃方式给予紫草素25mg/Kg，每日一次，连续三天，紫草素+LY294002组：缺血再灌注前以灌胃方式给予以灌胃方式给予紫草素25mg/Kg，每日一次，连续三天，并于术前15分钟脑室注射30μg LY-294002。各组取材前进行神经功能评分，干湿重法测定脑组织含水量，TTC染色评价脑梗死体积，检测超氧化物歧化酶（SOD）的活性，丙二醛（MDA）含量的变化情况，分别用免疫组化，Western-blotting和RT-qPCR的方法检测缺血脑组织中PI3K、AKT、HO-1、Nrf2蛋白和mRNA水平的变化。
结果：Sham组中未见小鼠神经功能缺损；Vehicle组在24h和72h神经功能缺损评分均较严重，L-Shi组在24h和72h与Vehicle组相比可降低神经功能缺损，但差异无统计学意义（P＞0.05），H-Shi组在24h和72h与Vehicle组相比可明显降低神经功能缺损，结果有统计学差异（P＜0.05）；与Vehicle组相比，L-Shi组在24h和72h可降低脑含水量，但是差异无统计学意义（P＞0.05），而H-Shi组在24h和72h与Vehicle组相比可明显降低脑含水量，差异有统计学意义（P＜0.05）；L-Shi组在24h和72h可减小脑梗死体积，但是差异无统计学意义（P＞0.05），H-Shi组在24h和72h与Vehicle组相比可明显降低脑梗死体积，差异有统计学意义（P＜0.05），但是H-Shi对神经功能缺损、脑水肿、脑梗死体积这种改善作用可被PI3K抑制剂LY294002阻断，差异有统计学意义（P＜0.05）。
免疫组化结果显示，与Vehicle组相比，缺血后24h和72h，H-Shi组在24h和72h均可明显升高p-PI3K、p-AKT、HO-1、Nrf2的阳性细胞数，差异有统计学意义（P＜0.05）；L-Shi组在24h和72h可升高PI3K、AKT、HO-1、Nrf2的阳性细胞数，但是差异无统计学意义（P＞0.05），H-Shi对PI3K、AKT、HO-1、Nrf2阳性细胞数的升高作用可被PI3K抑制剂LY294002阻断，差异有统计学意义（P＜0.05）。Western blot结果显示，H-Shi在24h和72h可明显升高p-PI3K、p-AKT、HO-1、Nrf2的蛋白表达，差异有统计学意义（P＜0.05）；L-Shi组在24h和72h可升高PI3K、AKT、HO-1、Nrf2的蛋白表达，但差异无统计学意义（P＞0.05），H-Shi对PI3K、AKT、HO-1、Nrf2蛋白的升高作用可被PI3K抑制剂LY294002阻断，差异有统计学意义（P＜0.05）。与免疫组化和Western blot结果一致，H-Shi在24h和72h可升高HO-1、Nrf2的mRNA表达，差异有统计学意义（P＜0.05）；L-Shi组在24h和72h可升高HO-1、Nrf2的mRNA表达，但差异无统计学意义（P＞0.05）。与Vehicle组相比，H-Shi可明显增加SOD，减低MDA的含量，差异有统计学意义（P＜0.05）；L-Shi组可增加SOD，减低MDA的含量，但差异无统计学意义（P＜0.05）。
结论：缺血小鼠脑组织中的HO-1水平出现动态表达的变化过程，紫草素可通过改善脑缺血再灌注损伤后的神经功能缺失，减轻脑水肿，减小脑梗死体积，诱导HO-1、Nrf2、p-PI3K及p-AKT的表达，使SOD含量明显增加，MDA含量明显减少，从而对局灶性缺血再灌注脑损伤小鼠发挥其神经保护作用，同时这种保护作用可被PI3K特异性拮抗剂LY294002阻断，因此我们推测紫草素的神经保护可能是通过PI3K/HO-1通路的激活实现其神经保护作用。
第二部分紫草素对局灶性脑缺血再灌注损伤小鼠的抗炎作用及其相关机制研究
目的：观察脑缺血再灌注损伤后TLR4/MAPKs/NF-κB/TNF-α的表达变化，研究紫草素对脑缺血再灌注损伤所致的炎症反应的作用，并探讨紫草素抗炎相关作用机制。
方法：选用成年雄性CD-1小鼠为研究对象，应用改良线栓法制备大脑中动脉缺血再灌注模型。试验1：将CD-1小鼠随机分为2组：假手术组(Sham)，手术组(MCAO)，根据不同的时间点又分为3h、6h、24h、48h、72h五个亚组。试验2：将CD-1小鼠随机分为4组：假手术组(Sham)、手术组(Vehicle)、低剂量紫草素干预组(L-Shi)(10mg/Kg)、高剂量紫草素干预组(H-Shi)(25mg/Kg)。Sham组：假手术前以灌胃方式给予等量PBS+1%DMSO，每日一次，连续三天，Vehicle组：缺血再灌注前以灌胃方式给予等量PBS+1%DMSO，每日一次，连续三天，L-Shi组：缺血再灌注前以灌胃方式给予紫草素10mg/Kg，每日一次，连续三天，H-Shi组：缺血再灌注前以灌胃方式给予紫草素25mg/Kg，每日一次，连续三天。各组取材前进行神经功能评分，分别用免疫组化，Western-blotting和RT-qPCR的方法检测缺血脑组织中TLR4，P38MAPK，NF-κB，TNF-αmRNA和蛋白水平的变化。
结果：在缺血再灌注早期TLR4、p-p38MAPK的表达呈逐渐增高的趋势，在24h达到高峰，可持续到48h，于72h逐渐开始下降。两种剂量紫草素干预后均可以使脑缺血再灌注后缺血脑组织炎症反应降低，TLR4/MAPKs/NF-κB/TNF-α mRNA表达下降。基因水平表达统计结果显示，H-Shi可明显减轻TLR4，p-p38MAPK，NF-κB，TNF-α表达，差异有统计学意义（P＜0.05），而L-Shi组虽可减轻缺血后上调的TLR4，p-p38MAPK，NF-κB，TNF-α表达，但结果无统计学差异（P＞0.05）。免疫组化和Western blot与基因水平表达统计结果一致。
结论：紫草素对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，减轻脑水肿，减小脑梗死体积，同时缺血再灌注损伤后的脑组织的TLR4，p-p38MAPK，NF-κB，TNF-α的表达变化与脑组织损伤程度一致，提示脑缺血再灌注后TLR4/MAPKs/NF-κB通路的参与了缺血后损伤；紫草素可下调TLR4，p-p38MAPK，TNF-α，抑制NF-κB的核转位，从而减轻缺血后的过度炎症反应，实现缺血后的神经保护作用。
第三部分紫草素对局灶性脑缺血再灌注损伤小鼠的血脑屏障保护作用及其相关机制研究
目的：观察脑缺血再灌注损伤后血脑屏障通透性变化，探讨紫草素对血脑屏障的保护作用及相关机制。
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。试验1：随机将CD-1小鼠分为2组：假手术组(Sham)(n=30），手术组(MCAO)，每组又根据不同的时间点分为3h、6h、24h、48h、72h五个亚组（n=90）。试验2：随机将CD-1小鼠分为4组：假手术组(Sham)、手术组(Vehicle)、小剂量紫草素组(L-Shi)(10mg/Kg)、大剂量紫草素组(H-Shi)(25mg/Kg)。Sham组：假手术前以灌胃方式给予等量PBS+1%DMSO，每日一次，连续三天，Vehicle组：缺血再灌注前以灌胃方式给予等量PBS+1%DMSO，每日一次，连续三天，L-Shi组：缺血再灌注前以灌胃方式给予紫草素10mg/Kg，每日一次，连续三天，H-Shi组：缺血再灌注前以灌胃方式给予紫草素25mg/Kg，每日一次，连续三天。各组取材前进行神经功能评分，血脑屏障通透性通过检测血管内皮细胞紧密连接蛋白成分Claudin-5进行评价。将各组小鼠分别于相应时间点进行神经功能评分后断头处死，采用免疫组化、western blot和qR-TPCR来观察脑缺血后Claudin-5以及MMP-9的mRNA和蛋白水平动态变化，通过Evans Blue的渗出情况评估血脑屏障的完整性。
结果：在缺血再灌注早期MMP-9表达升高（P＜0.05），至24h达到高峰，可持续至72h，与正常组比较，差异有统计学意义；两种剂量紫草素干预后均可以上调脑缺血再灌注后小鼠的Claudin-5表达，同时使MMP-9表达减少，H-Shi可明显诱导Claudin-5表达增加，减少MMP-9表达，差异有统计学意义，L-Shi组可上调诱导Claudin-5表达增加，减少MMP-9，但差异无统计学意义（P＞0.05）。Claudin-5、MMP-9mRNA表达以CT值表示，Sham组、Vehicle组、L-Shi组、H-Shi组Claudin-5的CT值分别为：Claudin-51.75±0.03，0.19±0.10，0.63±0.28，1.63±0.38；MMP-90.25±0.06，1.87±0.23，1.62±0.10，1.16±0.15。Claudin-5、MMP-9蛋白的表达变化与基因水平改变类似。再灌注24h后，Evans Blue染色的结果显示，与Vehicle组相比，H-Shi能够明显减少Evans Blue的渗出，维持血脑屏障的完整性。
结论：血脑屏障通透性显著增加是导致明显的神经功能缺失、脑水肿形成的直接原因。局灶性脑缺血再灌注后，缺血脑组织中Claudin-5、MMP-9表达的变化过程与脑组织损伤程度一致；紫草素通过抑制MMP-9、上调Claudin-5的表达，保护了血脑屏障的完整性，因此改善了脑缺血的神经功能缺失、脑水肿，减小了梗死体积。
Ischemic cerebrovascular disease is the most common type of disease,which is the first leading cause of death and the most frequent cause ofpermanent disability in adults. Ischemic stroke is one of the main diseasesendangering people’s health in our state, have a high incidence, mortality,morbidity, high recurrence rate, and the characteristics of the slow recovery.The worldwide average of one in every400people suffer a stroke in anischemic cerebrovascular disease according to the World Health Organisation.There are250million patients with cerebrovascular disease each year in China,equating to more than75%with ischemic brain disease. Stroke is aslo aleading cause of mortality after heart disease, equating to9%of total deathseach year. Ischaemic stroke accounts for approximately80–85%of all cases,with approximately30%of the former undergoing haemorrhagictransformation.
Brain tissue secondary injuries to ischemia often leads to an aggravatedillness after cerebral ischemia/reperfusion. Cerebral ischemia/reperfusioninjury is aslo a complex pathophysiologic process which is not been clarifiednow. With the rapid development of modern immunology and molecularbiology, recent studies found that the injuries caused by cerebral blood flowcessation and reperfusion are a rapid cascade reaction which includesinflammatory damage, oxidative stress, excitatory amino acid toxicity,apoptosis, intracellular calcium overload, free radical damage and so on.These pathophysiologic processes overlap and intercommunicate then form avicious cycle which direct/indirect resulting in blood-brain barrier disruptionand brain edema formation, neurological deficit, as well as cell apoptosis ornecrosis.
Nowadays, beyond only a restricted number of hospitalized patients profiting from thrombolytic therapy, the vast majority of patients sufferingfrom stroke resulted in disability even death owing to the limited therapies.Delaying the development of brain damage and protecting neuron areremained. There a large number of agents have been proved to displayanti-inflammation, anti-oxidation properties but never employed in treatingcerebral ischemia. It is a focus in neuroscience that looking for the idealneuroprotective agents that can block ischemic cascade reaction.
Shikonin is one of the major naphthoquinone pigment extracted from atraditional herbal medicine Lithospermum erythrorhizon. Evidence has shownthat shikonin possesses a wide range of biological effects, such asantimicrobial, anticancer, antithrombotic, and anti-inflammatory activities totreat thermal edema, arthritis, atherosclerosis. However, the molecular targetsand mechanisms underlying shikonin are not completely characterized, andthe effect of the shikonin in acute stroke is still unknown. In the present study,we demonstrated the unexplored potential of shikonin for the treatment ofcerebral ischemic damage and its potential mechanism.
ICR mice were induced into focal cerebral ischemiaby transient middlecerebral artery occlusion (MCAO), and received shikonin treatmentimmediately after MCAO. The present study examined the ability of shikonin,the major constituents of Chinese herb Lithospermum erythrorhizon, to induceexpression of HO-1, p85(PI3-kinase) and AKT phospholation, NF-E2-relatedfactor-2(Nrf2) and analyzed its signaling mechanism in mice brain. Theneurological deficits, brain water content, infarct volume and the expression ofTLR4, p-p38MAPK, NF-κB, TNF-α, MMP-9and claudin-5were measured at24h and72h after cerebral ischemia/reperfusion. The activities of superoxidedismutase (SOD) and malondialdehyde (MDA) content in ischemic corticaltissue were aslo detected to examine the oxidative response at24h and72hafter ischemia.
The study was divided into three part list as below.
PartⅠAnti-oxidative effect of shikonin on focal cerebralischemia/reperfusion injury in mice and its mechanism of signal transduction pathway
Objective: This study is to evaluate the time course expression regularityof HO-1, Nrf2, p85(PI3-kinase) and p-AKT, and estimate the anti-oxidativeeffects of shikonin on focal cerebral ischemia/reperfusion injury in mice andexplore its possible mechanisms and related signal transduction pathways.
Methods: CD-1mice were subjected to transient focal cerebralischemia/reperfusion model was established by middle cerebral arteryocclusion using modified suture occlusion technique. Experiment1: The micewere randomly divided into Sham group(n=30), MCAO group(n=90), Thetime course expression of HO-1in the brain tissue after MCAO. Two groupswere studied, including Sham group, MCAO group. The last two groupincluded3h,6h,24h,48h,72h sub-groups. Experiment2: Shikonin’sneuroprotection against damage from cerebral ischemia/reperfusion. Shikoninwas injected intraperitoneally after MCAO. Mice were reanesthetized andkilled at24h and72h after MCAO. In this part, mice were individed into4groups randomly. Shikonin was injected intraperitoneally immediately afterMCAO. Group1: Sham-operated group (Sham): animals received shamoperation; group2: Vehicle controls (Vehicle): animals received transientMCAO and equal volume PBS including1%DMSO respectively once a dayfor three times before surgery; group3: Shikonin low dose group (L-Shi):animals received transient MCAO and10mg/kg of Shikonin respectively oncea day for three times before surgery; group4: MCAO-Shikonin high dosegroup (H-Shi): animals received transient MCAO and25mg/kg of Shikoninrespectively once a day for three times before surgery. Drug or solvent wasadministered before MCAO, then once daily thereafter. Experiment3:Shikonin was administered before MCAO. Sham-operated group (Sham):animals received sham operation; vehicle controls (Vehicle): animals receivedtransient MCAO and equal volume PBS including1%DMSO; Shikonin lowdose group (L-Shi): animals received transient MCAO and10mg/kg ofShikonin respectively once a day for three times before surgery; MCAO-Shikonin high dose group (H-Shi): animals received transient MCAO and25 mg/kg of Shikonin respectively once a day for three times before surgery;Shikonin+LY-294002group: animals received transient MCAO and25mg/kg of Shikonin respectively once a day for three times before surgery andLY-294002(10μL10mM dissolved in3%DMSO). Infarct volume wasmeasured by TTC staining and morphologic changes were observed by H.E.we measured superoxide dismutase (SOD) and malondialdehyde (MDA) ofcerebral tissue to evaluate antioxidation activitis of shikonin.Immunohistochemistry, RT-qPCR and Western blot were used to analyse theexpression of HO-1, p85(PI3-kinase) and p-AKT and Nrf2.
Results: Compared with Sham group, HO-1were upregulated at geneand protein level in ischemia/reperfusion brain, beginning at6h and peakingat24h after MCAO (P＜0.05). Treatment of shikonin for indicated timeperiods increased up-regulation of HO-1in ischemic brain after cerebralischemia. In addition, treatment of various concentrations of shikonin for24halso increased HO-1expression in a concentration-dependent manner.Treatment with shikonin for indicated time periods, or with variousconcentrations of shikonin also increased HO-1mRNA expression in atime-dependent or a concentration dependent manners. Taken together, thesefindings demonstrate that shikonin increases HO-1protein and mRNAexpression. Shikonin high dose (25mg/kg) upregulated Nrf2and HO-1inMCAO-affected brain tissue, The Nrf2CT of Vehicle group, MCAO group,L-Shi group, H-Shi group were (0.22±0.27,0.85±0.10,0.70±0.05,0.53±0.06). Shikonin reduced infarct volume (P＜0.05), and behavioral deficitscaused by MCAO. And also, shikonin significantly increased the activities ofSOD and decreased the production of MDA at24h and72h after ischemia inH-Shi group. Phospho-PI3K and phospho-Akt were examined withimmunohistochemistry, Western blot and RT-qPCR. Few cells were stainedwith phospho-PI3K and phospho-Akt in the cortex in sham group byimmunohistochemistry. In vehicle group, the number of positive cells ofphospho-PI3K and phospho-Akt significantly decreased in the ischemic cortex.In H-Shi group, the number of positive cells of phospho-PI3K and phospho-Akt was significantly increased compared with vehicle group (P <0.05). However, there were no significant differences about the positive cellsbetween vehicle group and L-Shi group. In agreement with the results ofwestern blotting, the mRNA and protein expression of phospho-PI3K andphospho-Akt were down-regulated in Vehicle group compared with Shamgroup (P <0.05). The expression of those factors was significantly increasedin H-Shi group (P<0.05). Whereas L-Shi group did not display changes ofphospho-PI3K and phospho-Akt expression compared with Vehicle group. Tofurther investigate whether the PI3K/Akt pathway mediates theneuroprotection of shikonin in cerebral I/R injury, we treated the mice with aPI3K inhibitor LY-294002(i.c.v.,10μL10mM LY-294002dissolved in3%DMSO) at15min before ischemia. Western blot analysis showed that thePI3K inhibitor LY-294002inhibited the increase of p-Akt level induced byshikonin treatment and almost restored p-Akt level to basal level. In addition,shikonin-induced HO-1expression was antagonized by treatment with PI-3kinase inhibitors LY294002.
Conclusions: Nrf2, HO-1were induced at the early stage after MCAO.Shikonin reduce cerebral infarct volume and improve neurologic impairmentand protected the brain from damage caused by MCAO, we found thatshikonin increased HO-1mRNA and protein expression time-dependently. Inaddition, shikonin-induced HO-1expression was attenuated by PI3-kinase(phosphatidylinositol3-kinase) inhibitors LY294002. Treatment of mice withshikonin also induced p85(PI3-kinase) and AKT phospholation. Shikoninalso increased NF-E2-related factor-2(Nrf2) accumulation in the nucleus.Moreover, shikonin-induced increase of Nrf2was reduced by PI3-kinaseinhibitors. Thus, shikonin may be useful as a therapeutic agent for thetreatment of cerebral ischemic-associated disorders. The neuroprotection ofshikonin was accomplished by antioxidation. These findings suggest thatshikonin-increased HO-1expression is mediated by Nrf2activation throughthe PI3-kinase/AKT pathway.
PartⅡ Anti-inflammatory effect of shikonin on focal cerebral ischemia/reperfusion injury in mice and its mechanism of signaltransduction pathway
Objective: This study is to evaluate the expression regularity of TLR4,MAPKs, NF-κB, TNF-α and estimate the anti-inflammatory effects ofshikonin on focal cerebral ischemia/reperfusion injury in mice and explore itspossible mechanisms and related signal transduction pathways.
Methods: CD-1mice were subjected to transient focal cerebralischemia/reperfusion model in rats was established by middle cerebral arteryocclusion using modified suture occlusion technique. Experiment1: The micewere randomly divided into Sham group(n=30), MCAO group(n=90), Thetime course expression of TLR4, MAPKs, NF-κB, TNF-α in the brain tissueafter MCAO. Two groups were studied, including Sham group, MCAO group.The last two group included3h,6h,24h,48h and72h sub-groups.Experiment2: Shikonin’s neuroprotection against damage from cerebralischemia/reperfusion. In this part, mice were individed into4groups randomly.Shikonin (98%) was purchased from Santa Cruz Biotechnology; it wasdissolved in dimethyl sulfoxide (DMSO) as a10mmol/L stock solution andstored at–20℃. For all experiments the final concentration of the testedcompound was prepared by diluting the stock with PBS including1%DMSO.Shikonin was administered before MCAO. Mice were reanesthetized andkilled at24h and72h after MCAO. In this part, mice were individed into4groups randomly. Group1: Sham-operated group (Sham): animals receivedsham operation; group2: Vehicle controls (Vehicle): animals receivedtransient MCAO and equal volume PBS including1%DMSO respectivelyonce a day for three times before surgery; group3: Shikonin low dose group(L-Shi): animals received transient MCAO and10mg/kg of Shikoninrespectively once a day for three times before surgery; group4: MCAO-Shikonin high dose group (H-Shi): animals received transient MCAO and25mg/kg of Shikonin respectively once a day for three times before surgery.Drug or solvent was administered before MCAO, then once daily thereafter.Immunohistochemistry, RT-qPCR and Western blot were used to analyse the expression of TLR4, MAPKs, NF-κB, TNF-α.
Results: TLR4, p-p38MAPK, NF-κB and TNF-α were examined withimmunohistochemistry, Western blot and RT-qPCR. Few cells were stainedwith TLR4, p-p38MAPK, NF-κB and TNF-α in the cortex in sham group byimmunohistochemistry. In vehicle group, the number of positive cells of TLR4,p-p38MAPK, NF-κB and TNF-α significantly increased in the ischemic cortex,among which the location of NF-κB is in nucleus. In H-Shi group, the numberof positive cells of TLR4, p-p38MAPK, NF-κB and TNF-α was significantlydecreased compared with vehicle group (P <0.05). Moreover, positive nucleiof NF-κB were also reduced and lots of cells labeled by NF-κB were stainedonly in cytoplasm (P <0.05). However, there were no significant differencesabout the positive cells between vehicle group and L-Shi group.We found the protein and mRNA levels of TLR4, p-p38MAPK, NF-κB andTNF-α in ischemic tissue were upregulated at24h and72h after MCAO. Wefirst analyzed the protein levels of nuclear NF-κB p65and total TLR4,p-p38MAPK, TNF-α and MMP-9by western blot. The NF-κB p65was rich incytosolic fractions but poor in nuclear extracts in brain tissue of Sham group.In contrast, the protein level of NF-κB in Vehicle group was significantlyenhanced in nuclear fraction and concurrently decreased in cytosol at24h and72h after ischemia, indicating the translocation of these NF-κB subunits fromthe cytosol to the nucleus. High dose of shikonin significantly decreased theexpressions TLR4, p-p38MAPK, NF-κB and TNF-α at24h and72h afterMCAO. However, there were no significant differences in the protein levels ofTLR4, p-p38MAPK, NF-κB and TNF-α between Vehicle group and L-Shigroup. Another parallel set of samples treated with L-Shi and H-Shi werealso evaluated for mRNA expression of TLR4, p-p38MAPK, NF-κB andTNF-α by RT-qPCR. In agreement with the results of western blotting, themRNA expression of TLR4, p-p38MAPK, NF-κB and TNF-α wereup-regulated in Vehicle group compared with Sham group (P <0.05). Theover-expression of those factors was significantly decreased in H-Shi group(P<0.05). Whereas L-Shi group did not display changes of TLR4, p-p38MAPK, NF-κB and TNF-α expression compared with Vehicle group.
Conclusions: The expression of TLR4, p-p38MAPK, NF-κB andTNF-α were up-regulated after ischemia. Systemic administration of shikoninis effective which can decrease the expression of TLR4, p-p38MAPK, NF-κBand TNF-α. Therefore, the excessive inflammation of the brain ischemia wasalleviated.
Part Ⅲ The permeability of blood-brain barrier after focal cerebralischemia in rats and the protection of shikonin
Objective: This study is to evaluate the time course expression ofClaudin-5and MMP-9and explore the underling regulation mechanisms ofshikonin.
Methods: CD-1mice were subjected to transient focal cerebralischemia/reperfusion model in rats was established by middle cerebral arteryocclusion using modified suture occlusion technique. Experiment1: The micewere randomly divided into Sham group(n=30), MCAO group(n=90), Thetime course expression of Claudin-5and MMP-9in the brain tissue afterMCAO. Two groups were studied, including Sham group, MCAO group. Thelast two group included3h,6h,24h,48h and72h sub-groups. Experiment2:shikonin’s neuroprotection against damage from cerebral ischemia/reperfusion.shikonin was administered before MCAO. Mice were reanesthetized andkilled at24h and72h after MCAO. In this part, mice were individed into4groups randomly. Group1: Sham-operated group (Sham): animals receivedsham operation; group2: Vehicle controls (Vehicle): animals receivedtransient MCAO and equal volume PBS including1%DMSO respectivelyonce a day for three times before surgery; group3: Shikonin low dose group(L-Shi): animals received transient MCAO and10mg/kg of Shikoninrespectively once a day for three times before surgery; group4: MCAO-Shikonin high dose group (H-Shi): animals received transient MCAO and25mg/kg of Shikonin respectively once a day for three times before surgery.Drug or solvent was administered before MCAO, then once daily thereafter.RT-qPCR and Western blot were used to analyse the expression of Claudin-5 and MMP-9. The loss of BBB integrity was assessed by leakage of Evans bluefrom microvessels after intravenous injection.
Results: There was considerable leak of Evans blue into the ischemicbrain after MCAO, showing that the BBB had been disrupted in the ischemichemisphere. Compared with Vehicle group, Evans blue evasion of braintissues was significantly decreased in H-Shi group (P<0.05). Whileclaudin-5's expression was increased in both L-Shi and H-Shi groups (P<0.01) at gene and protein levels by western blotting and RT-qPCR at24h and72h, but low dose of shikonin did not display significant level. At the sametime, the MMP-9expression was obviously decreased in H-Shi group (P<0.05), relative to that in normal group.
Conclusions: The blood-brain barrier was dramatically disrupted at theearly stage of focal cerebral ischemia, which was responsible for the brainedema. Shikonin normalizing Claudin-5by inhibiting MMP-9couldcontribute to the alleviated infarct volume, neurological deficits and brainedema in the study.
缺血再灌注损伤是导致患者病情加重的重要原因，近年来随着现代免疫学和分子生物学的迅速发展，对于脑缺血再灌注损伤病理生理机制的研究已取得了重大进步，认为其病理生理机制存在损伤级联反应，主要包括炎症损害、氧化应激、兴奋性氨基酸毒性、凋亡、细胞内钙超载、自由基损伤等几种因素相互作用形成了复杂的调控网络，造成一系列病理级联反应，可直接或者间接导致神经细胞凋亡/死亡。在脑缺血后复杂的病理损伤机制中，炎症反应、氧化应激是脑缺血再灌注损伤的主要原因，是目前神经科学研究的热点，抗氧化应激、减轻炎症损伤成为治疗缺血性脑血管病的主要途径之一。
尽管已经有许多合成或天然物质的神经保护剂经过实验研究，证明具有抗炎、抗氧化、抗凋亡等作用，但是由于动物与人类生理机制及耐受性等方面的差异以及脑缺血后级联反应的复杂性，单一阻断某一病理环节的药物可能难以奏效，多数神经保护剂未能在临床得到广泛应用，出现临床和理论的严重脱节，所以在目前的临床治疗中，理想的神经保护剂应该具备针对缺血再灌注损伤多个损伤环节发挥保护作用的特性。
紫草素(Shikonin)是存在于天然植物中的一种脂溶性萘醌类化合物，分子式为：C16H16O5，主要存在于紫草科植物紫草的根中，具有多种药理活性，研究表明紫草素具有抗氧化、抗肿瘤、抗炎、抗凋亡、抗病毒等生物学作用，并且毒副作用低，具有良好的临床应用潜力。
本研究选用雄性CD-1小鼠为研究对象，采用线栓法建立脑缺血再灌注模型，在此模型基础上对脑缺血后炎症反应代表性因子TLR4，P38MAPK，NF-κB，TNF-α，氧化应激代表性因子HO-1，Nrf2，PI3K，AKT和MMP-9，claudin-5为代表的血脑屏障损伤程度进行观察，测定大脑皮层梗塞灶超氧化物歧化酶（Superoxide Dismutase，SOD）和丙二醛（Malondiadehyde，MDA）的含量。确定缺血后与炎症反应、氧化损伤的关系，通过选用萘醌类化合物紫草素进行干预，对干预前后梗死体积、脑含水量、神经功能缺损进行评估，观察紫草素对TLR4，P38MAPK，NF-κB，TNF-α，PI3K，AKT，HO-1，Nrf2，MMP-9及Claudin-5信号通路的作用，初步探讨其在缺血再灌注损伤后的脑保护作用机制，并应用PI3K特异性拮抗剂LY294002进行干预，观察上述各因子的变化，评价神经功能缺失、脑水肿和梗死体积情况。本研究分三部分，现将各部分内容叙述如下。
第一部分紫草素对局灶性脑缺血再灌注损伤小鼠的抗氧化作用及其相关机制研究
目的：观察脑缺血再灌注损伤后梗死体积、脑含水量、神经功能缺损评分、PI3K、AKT、HO-1、Nrf2的表达变化以及SOD活性和MDA含量的变化，研究紫草素对脑缺血再灌注损伤所致的氧化应激反应的作用，探讨紫草素抗氧化应激作用的相关机制。
方法：选用成年雄性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模型。试验1：随机将CD-1小鼠分为2组：假手术组(Sham)，手术组(MCAO)：根据不同的时间点又分为3h、6h、24h、48h、72h五个亚组。试验2：随机将CD-1小鼠分为4组：假手术组(Sham)、手术组(Vehicle)、低剂量紫草素干预组(L-Shi)(10mg/Kg)、高剂量紫草素干预组(H-Shi)(25mg/Kg)。试验3：采用随机的方法分别将CD-1小鼠分为4组：假手术组（Sham)、溶剂对照组（Vehicle)、大剂量紫草素干预组（H-Shi)及紫草素+LY294002组（Shikonin+LY294002)。Sham组：假手术前以灌胃方式给予等量PBS+1%DMSO，每日一次，连续三天，Vehicle组：缺血再灌注前以灌胃方式给予等量PBS+1%DMSO，每日一次，连续三天，L-Shi组：缺血再灌注前以灌胃方式给予紫草素10mg/Kg，每日一次，连续三天，H-Shi组：缺血再灌注前以灌胃方式给予紫草素25mg/Kg，每日一次，连续三天，紫草素+LY294002组：缺血再灌注前以灌胃方式给予以灌胃方式给予紫草素25mg/Kg，每日一次，连续三天，并于术前15分钟脑室注射30μg LY-294002。各组取材前进行神经功能评分，干湿重法测定脑组织含水量，TTC染色评价脑梗死体积，检测超氧化物歧化酶（SOD）的活性，丙二醛（MDA）含量的变化情况，分别用免疫组化，Western-blotting和RT-qPCR的方法检测缺血脑组织中PI3K、AKT、HO-1、Nrf2蛋白和mRNA水平的变化。
结果：Sham组中未见小鼠神经功能缺损；Vehicle组在24h和72h神经功能缺损评分均较严重，L-Shi组在24h和72h与Vehicle组相比可降低神经功能缺损，但差异无统计学意义（P＞0.05），H-Shi组在24h和72h与Vehicle组相比可明显降低神经功能缺损，结果有统计学差异（P＜0.05）；与Vehicle组相比，L-Shi组在24h和72h可降低脑含水量，但是差异无统计学意义（P＞0.05），而H-Shi组在24h和72h与Vehicle组相比可明显降低脑含水量，差异有统计学意义（P＜0.05）；L-Shi组在24h和72h可减小脑梗死体积，但是差异无统计学意义（P＞0.05），H-Shi组在24h和72h与Vehicle组相比可明显降低脑梗死体积，差异有统计学意义（P＜0.05），但是H-Shi对神经功能缺损、脑水肿、脑梗死体积这种改善作用可被PI3K抑制剂LY294002阻断，差异有统计学意义（P＜0.05）。
免疫组化结果显示，与Vehicle组相比，缺血后24h和72h，H-Shi组在24h和72h均可明显升高p-PI3K、p-AKT、HO-1、Nrf2的阳性细胞数，差异有统计学意义（P＜0.05）；L-Shi组在24h和72h可升高PI3K、AKT、HO-1、Nrf2的阳性细胞数，但是差异无统计学意义（P＞0.05），H-Shi对PI3K、AKT、HO-1、Nrf2阳性细胞数的升高作用可被PI3K抑制剂LY294002阻断，差异有统计学意义（P＜0.05）。Western blot结果显示，H-Shi在24h和72h可明显升高p-PI3K、p-AKT、HO-1、Nrf2的蛋白表达，差异有统计学意义（P＜0.05）；L-Shi组在24h和72h可升高PI3K、AKT、HO-1、Nrf2的蛋白表达，但差异无统计学意义（P＞0.05），H-Shi对PI3K、AKT、HO-1、Nrf2蛋白的升高作用可被PI3K抑制剂LY294002阻断，差异有统计学意义（P＜0.05）。与免疫组化和Western blot结果一致，H-Shi在24h和72h可升高HO-1、Nrf2的mRNA表达，差异有统计学意义（P＜0.05）；L-Shi组在24h和72h可升高HO-1、Nrf2的mRNA表达，但差异无统计学意义（P＞0.05）。与Vehicle组相比，H-Shi可明显增加SOD，减低MDA的含量，差异有统计学意义（P＜0.05）；L-Shi组可增加SOD，减低MDA的含量，但差异无统计学意义（P＜0.05）。
结论：缺血小鼠脑组织中的HO-1水平出现动态表达的变化过程，紫草素可通过改善脑缺血再灌注损伤后的神经功能缺失，减轻脑水肿，减小脑梗死体积，诱导HO-1、Nrf2、p-PI3K及p-AKT的表达，使SOD含量明显增加，MDA含量明显减少，从而对局灶性缺血再灌注脑损伤小鼠发挥其神经保护作用，同时这种保护作用可被PI3K特异性拮抗剂LY294002阻断，因此我们推测紫草素的神经保护可能是通过PI3K/HO-1通路的激活实现其神经保护作用。
第二部分紫草素对局灶性脑缺血再灌注损伤小鼠的抗炎作用及其相关机制研究
目的：观察脑缺血再灌注损伤后TLR4/MAPKs/NF-κB/TNF-α的表达变化，研究紫草素对脑缺血再灌注损伤所致的炎症反应的作用，并探讨紫草素抗炎相关作用机制。
方法：选用成年雄性CD-1小鼠为研究对象，应用改良线栓法制备大脑中动脉缺血再灌注模型。试验1：将CD-1小鼠随机分为2组：假手术组(Sham)，手术组(MCAO)，根据不同的时间点又分为3h、6h、24h、48h、72h五个亚组。试验2：将CD-1小鼠随机分为4组：假手术组(Sham)、手术组(Vehicle)、低剂量紫草素干预组(L-Shi)(10mg/Kg)、高剂量紫草素干预组(H-Shi)(25mg/Kg)。Sham组：假手术前以灌胃方式给予等量PBS+1%DMSO，每日一次，连续三天，Vehicle组：缺血再灌注前以灌胃方式给予等量PBS+1%DMSO，每日一次，连续三天，L-Shi组：缺血再灌注前以灌胃方式给予紫草素10mg/Kg，每日一次，连续三天，H-Shi组：缺血再灌注前以灌胃方式给予紫草素25mg/Kg，每日一次，连续三天。各组取材前进行神经功能评分，分别用免疫组化，Western-blotting和RT-qPCR的方法检测缺血脑组织中TLR4，P38MAPK，NF-κB，TNF-αmRNA和蛋白水平的变化。
结果：在缺血再灌注早期TLR4、p-p38MAPK的表达呈逐渐增高的趋势，在24h达到高峰，可持续到48h，于72h逐渐开始下降。两种剂量紫草素干预后均可以使脑缺血再灌注后缺血脑组织炎症反应降低，TLR4/MAPKs/NF-κB/TNF-α mRNA表达下降。基因水平表达统计结果显示，H-Shi可明显减轻TLR4，p-p38MAPK，NF-κB，TNF-α表达，差异有统计学意义（P＜0.05），而L-Shi组虽可减轻缺血后上调的TLR4，p-p38MAPK，NF-κB，TNF-α表达，但结果无统计学差异（P＞0.05）。免疫组化和Western blot与基因水平表达统计结果一致。
结论：紫草素对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，减轻脑水肿，减小脑梗死体积，同时缺血再灌注损伤后的脑组织的TLR4，p-p38MAPK，NF-κB，TNF-α的表达变化与脑组织损伤程度一致，提示脑缺血再灌注后TLR4/MAPKs/NF-κB通路的参与了缺血后损伤；紫草素可下调TLR4，p-p38MAPK，TNF-α，抑制NF-κB的核转位，从而减轻缺血后的过度炎症反应，实现缺血后的神经保护作用。
第三部分紫草素对局灶性脑缺血再灌注损伤小鼠的血脑屏障保护作用及其相关机制研究
目的：观察脑缺血再灌注损伤后血脑屏障通透性变化，探讨紫草素对血脑屏障的保护作用及相关机制。
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。试验1：随机将CD-1小鼠分为2组：假手术组(Sham)(n=30），手术组(MCAO)，每组又根据不同的时间点分为3h、6h、24h、48h、72h五个亚组（n=90）。试验2：随机将CD-1小鼠分为4组：假手术组(Sham)、手术组(Vehicle)、小剂量紫草素组(L-Shi)(10mg/Kg)、大剂量紫草素组(H-Shi)(25mg/Kg)。Sham组：假手术前以灌胃方式给予等量PBS+1%DMSO，每日一次，连续三天，Vehicle组：缺血再灌注前以灌胃方式给予等量PBS+1%DMSO，每日一次，连续三天，L-Shi组：缺血再灌注前以灌胃方式给予紫草素10mg/Kg，每日一次，连续三天，H-Shi组：缺血再灌注前以灌胃方式给予紫草素25mg/Kg，每日一次，连续三天。各组取材前进行神经功能评分，血脑屏障通透性通过检测血管内皮细胞紧密连接蛋白成分Claudin-5进行评价。将各组小鼠分别于相应时间点进行神经功能评分后断头处死，采用免疫组化、western blot和qR-TPCR来观察脑缺血后Claudin-5以及MMP-9的mRNA和蛋白水平动态变化，通过Evans Blue的渗出情况评估血脑屏障的完整性。
结果：在缺血再灌注早期MMP-9表达升高（P＜0.05），至24h达到高峰，可持续至72h，与正常组比较，差异有统计学意义；两种剂量紫草素干预后均可以上调脑缺血再灌注后小鼠的Claudin-5表达，同时使MMP-9表达减少，H-Shi可明显诱导Claudin-5表达增加，减少MMP-9表达，差异有统计学意义，L-Shi组可上调诱导Claudin-5表达增加，减少MMP-9，但差异无统计学意义（P＞0.05）。Claudin-5、MMP-9mRNA表达以CT值表示，Sham组、Vehicle组、L-Shi组、H-Shi组Claudin-5的CT值分别为：Claudin-51.75±0.03，0.19±0.10，0.63±0.28，1.63±0.38；MMP-90.25±0.06，1.87±0.23，1.62±0.10，1.16±0.15。Claudin-5、MMP-9蛋白的表达变化与基因水平改变类似。再灌注24h后，Evans Blue染色的结果显示，与Vehicle组相比，H-Shi能够明显减少Evans Blue的渗出，维持血脑屏障的完整性。
结论：血脑屏障通透性显著增加是导致明显的神经功能缺失、脑水肿形成的直接原因。局灶性脑缺血再灌注后，缺血脑组织中Claudin-5、MMP-9表达的变化过程与脑组织损伤程度一致；紫草素通过抑制MMP-9、上调Claudin-5的表达，保护了血脑屏障的完整性，因此改善了脑缺血的神经功能缺失、脑水肿，减小了梗死体积。
Ischemic cerebrovascular disease is the most common type of disease,which is the first leading cause of death and the most frequent cause ofpermanent disability in adults. Ischemic stroke is one of the main diseasesendangering people’s health in our state, have a high incidence, mortality,morbidity, high recurrence rate, and the characteristics of the slow recovery.The worldwide average of one in every400people suffer a stroke in anischemic cerebrovascular disease according to the World Health Organisation.There are250million patients with cerebrovascular disease each year in China,equating to more than75%with ischemic brain disease. Stroke is aslo aleading cause of mortality after heart disease, equating to9%of total deathseach year. Ischaemic stroke accounts for approximately80–85%of all cases,with approximately30%of the former undergoing haemorrhagictransformation.
Brain tissue secondary injuries to ischemia often leads to an aggravatedillness after cerebral ischemia/reperfusion. Cerebral ischemia/reperfusioninjury is aslo a complex pathophysiologic process which is not been clarifiednow. With the rapid development of modern immunology and molecularbiology, recent studies found that the injuries caused by cerebral blood flowcessation and reperfusion are a rapid cascade reaction which includesinflammatory damage, oxidative stress, excitatory amino acid toxicity,apoptosis, intracellular calcium overload, free radical damage and so on.These pathophysiologic processes overlap and intercommunicate then form avicious cycle which direct/indirect resulting in blood-brain barrier disruptionand brain edema formation, neurological deficit, as well as cell apoptosis ornecrosis.
Nowadays, beyond only a restricted number of hospitalized patients profiting from thrombolytic therapy, the vast majority of patients sufferingfrom stroke resulted in disability even death owing to the limited therapies.Delaying the development of brain damage and protecting neuron areremained. There a large number of agents have been proved to displayanti-inflammation, anti-oxidation properties but never employed in treatingcerebral ischemia. It is a focus in neuroscience that looking for the idealneuroprotective agents that can block ischemic cascade reaction.
Shikonin is one of the major naphthoquinone pigment extracted from atraditional herbal medicine Lithospermum erythrorhizon. Evidence has shownthat shikonin possesses a wide range of biological effects, such asantimicrobial, anticancer, antithrombotic, and anti-inflammatory activities totreat thermal edema, arthritis, atherosclerosis. However, the molecular targetsand mechanisms underlying shikonin are not completely characterized, andthe effect of the shikonin in acute stroke is still unknown. In the present study,we demonstrated the unexplored potential of shikonin for the treatment ofcerebral ischemic damage and its potential mechanism.
ICR mice were induced into focal cerebral ischemiaby transient middlecerebral artery occlusion (MCAO), and received shikonin treatmentimmediately after MCAO. The present study examined the ability of shikonin,the major constituents of Chinese herb Lithospermum erythrorhizon, to induceexpression of HO-1, p85(PI3-kinase) and AKT phospholation, NF-E2-relatedfactor-2(Nrf2) and analyzed its signaling mechanism in mice brain. Theneurological deficits, brain water content, infarct volume and the expression ofTLR4, p-p38MAPK, NF-κB, TNF-α, MMP-9and claudin-5were measured at24h and72h after cerebral ischemia/reperfusion. The activities of superoxidedismutase (SOD) and malondialdehyde (MDA) content in ischemic corticaltissue were aslo detected to examine the oxidative response at24h and72hafter ischemia.
The study was divided into three part list as below.
PartⅠAnti-oxidative effect of shikonin on focal cerebralischemia/reperfusion injury in mice and its mechanism of signal transduction pathway
Objective: This study is to evaluate the time course expression regularityof HO-1, Nrf2, p85(PI3-kinase) and p-AKT, and estimate the anti-oxidativeeffects of shikonin on focal cerebral ischemia/reperfusion injury in mice andexplore its possible mechanisms and related signal transduction pathways.
Methods: CD-1mice were subjected to transient focal cerebralischemia/reperfusion model was established by middle cerebral arteryocclusion using modified suture occlusion technique. Experiment1: The micewere randomly divided into Sham group(n=30), MCAO group(n=90), Thetime course expression of HO-1in the brain tissue after MCAO. Two groupswere studied, including Sham group, MCAO group. The last two groupincluded3h,6h,24h,48h,72h sub-groups. Experiment2: Shikonin’sneuroprotection against damage from cerebral ischemia/reperfusion. Shikoninwas injected intraperitoneally after MCAO. Mice were reanesthetized andkilled at24h and72h after MCAO. In this part, mice were individed into4groups randomly. Shikonin was injected intraperitoneally immediately afterMCAO. Group1: Sham-operated group (Sham): animals received shamoperation; group2: Vehicle controls (Vehicle): animals received transientMCAO and equal volume PBS including1%DMSO respectively once a dayfor three times before surgery; group3: Shikonin low dose group (L-Shi):animals received transient MCAO and10mg/kg of Shikonin respectively oncea day for three times before surgery; group4: MCAO-Shikonin high dosegroup (H-Shi): animals received transient MCAO and25mg/kg of Shikoninrespectively once a day for three times before surgery. Drug or solvent wasadministered before MCAO, then once daily thereafter. Experiment3:Shikonin was administered before MCAO. Sham-operated group (Sham):animals received sham operation; vehicle controls (Vehicle): animals receivedtransient MCAO and equal volume PBS including1%DMSO; Shikonin lowdose group (L-Shi): animals received transient MCAO and10mg/kg ofShikonin respectively once a day for three times before surgery; MCAO-Shikonin high dose group (H-Shi): animals received transient MCAO and25 mg/kg of Shikonin respectively once a day for three times before surgery;Shikonin+LY-294002group: animals received transient MCAO and25mg/kg of Shikonin respectively once a day for three times before surgery andLY-294002(10μL10mM dissolved in3%DMSO). Infarct volume wasmeasured by TTC staining and morphologic changes were observed by H.E.we measured superoxide dismutase (SOD) and malondialdehyde (MDA) ofcerebral tissue to evaluate antioxidation activitis of shikonin.Immunohistochemistry, RT-qPCR and Western blot were used to analyse theexpression of HO-1, p85(PI3-kinase) and p-AKT and Nrf2.
Results: Compared with Sham group, HO-1were upregulated at geneand protein level in ischemia/reperfusion brain, beginning at6h and peakingat24h after MCAO (P＜0.05). Treatment of shikonin for indicated timeperiods increased up-regulation of HO-1in ischemic brain after cerebralischemia. In addition, treatment of various concentrations of shikonin for24halso increased HO-1expression in a concentration-dependent manner.Treatment with shikonin for indicated time periods, or with variousconcentrations of shikonin also increased HO-1mRNA expression in atime-dependent or a concentration dependent manners. Taken together, thesefindings demonstrate that shikonin increases HO-1protein and mRNAexpression. Shikonin high dose (25mg/kg) upregulated Nrf2and HO-1inMCAO-affected brain tissue, The Nrf2CT of Vehicle group, MCAO group,L-Shi group, H-Shi group were (0.22±0.27,0.85±0.10,0.70±0.05,0.53±0.06). Shikonin reduced infarct volume (P＜0.05), and behavioral deficitscaused by MCAO. And also, shikonin significantly increased the activities ofSOD and decreased the production of MDA at24h and72h after ischemia inH-Shi group. Phospho-PI3K and phospho-Akt were examined withimmunohistochemistry, Western blot and RT-qPCR. Few cells were stainedwith phospho-PI3K and phospho-Akt in the cortex in sham group byimmunohistochemistry. In vehicle group, the number of positive cells ofphospho-PI3K and phospho-Akt significantly decreased in the ischemic cortex.In H-Shi group, the number of positive cells of phospho-PI3K and phospho-Akt was significantly increased compared with vehicle group (P <0.05). However, there were no significant differences about the positive cellsbetween vehicle group and L-Shi group. In agreement with the results ofwestern blotting, the mRNA and protein expression of phospho-PI3K andphospho-Akt were down-regulated in Vehicle group compared with Shamgroup (P <0.05). The expression of those factors was significantly increasedin H-Shi group (P<0.05). Whereas L-Shi group did not display changes ofphospho-PI3K and phospho-Akt expression compared with Vehicle group. Tofurther investigate whether the PI3K/Akt pathway mediates theneuroprotection of shikonin in cerebral I/R injury, we treated the mice with aPI3K inhibitor LY-294002(i.c.v.,10μL10mM LY-294002dissolved in3%DMSO) at15min before ischemia. Western blot analysis showed that thePI3K inhibitor LY-294002inhibited the increase of p-Akt level induced byshikonin treatment and almost restored p-Akt level to basal level. In addition,shikonin-induced HO-1expression was antagonized by treatment with PI-3kinase inhibitors LY294002.
Conclusions: Nrf2, HO-1were induced at the early stage after MCAO.Shikonin reduce cerebral infarct volume and improve neurologic impairmentand protected the brain from damage caused by MCAO, we found thatshikonin increased HO-1mRNA and protein expression time-dependently. Inaddition, shikonin-induced HO-1expression was attenuated by PI3-kinase(phosphatidylinositol3-kinase) inhibitors LY294002. Treatment of mice withshikonin also induced p85(PI3-kinase) and AKT phospholation. Shikoninalso increased NF-E2-related factor-2(Nrf2) accumulation in the nucleus.Moreover, shikonin-induced increase of Nrf2was reduced by PI3-kinaseinhibitors. Thus, shikonin may be useful as a therapeutic agent for thetreatment of cerebral ischemic-associated disorders. The neuroprotection ofshikonin was accomplished by antioxidation. These findings suggest thatshikonin-increased HO-1expression is mediated by Nrf2activation throughthe PI3-kinase/AKT pathway.
PartⅡ Anti-inflammatory effect of shikonin on focal cerebral ischemia/reperfusion injury in mice and its mechanism of signaltransduction pathway
Objective: This study is to evaluate the expression regularity of TLR4,MAPKs, NF-κB, TNF-α and estimate the anti-inflammatory effects ofshikonin on focal cerebral ischemia/reperfusion injury in mice and explore itspossible mechanisms and related signal transduction pathways.
Methods: CD-1mice were subjected to transient focal cerebralischemia/reperfusion model in rats was established by middle cerebral arteryocclusion using modified suture occlusion technique. Experiment1: The micewere randomly divided into Sham group(n=30), MCAO group(n=90), Thetime course expression of TLR4, MAPKs, NF-κB, TNF-α in the brain tissueafter MCAO. Two groups were studied, including Sham group, MCAO group.The last two group included3h,6h,24h,48h and72h sub-groups.Experiment2: Shikonin’s neuroprotection against damage from cerebralischemia/reperfusion. In this part, mice were individed into4groups randomly.Shikonin (98%) was purchased from Santa Cruz Biotechnology; it wasdissolved in dimethyl sulfoxide (DMSO) as a10mmol/L stock solution andstored at–20℃. For all experiments the final concentration of the testedcompound was prepared by diluting the stock with PBS including1%DMSO.Shikonin was administered before MCAO. Mice were reanesthetized andkilled at24h and72h after MCAO. In this part, mice were individed into4groups randomly. Group1: Sham-operated group (Sham): animals receivedsham operation; group2: Vehicle controls (Vehicle): animals receivedtransient MCAO and equal volume PBS including1%DMSO respectivelyonce a day for three times before surgery; group3: Shikonin low dose group(L-Shi): animals received transient MCAO and10mg/kg of Shikoninrespectively once a day for three times before surgery; group4: MCAO-Shikonin high dose group (H-Shi): animals received transient MCAO and25mg/kg of Shikonin respectively once a day for three times before surgery.Drug or solvent was administered before MCAO, then once daily thereafter.Immunohistochemistry, RT-qPCR and Western blot were used to analyse the expression of TLR4, MAPKs, NF-κB, TNF-α.
Results: TLR4, p-p38MAPK, NF-κB and TNF-α were examined withimmunohistochemistry, Western blot and RT-qPCR. Few cells were stainedwith TLR4, p-p38MAPK, NF-κB and TNF-α in the cortex in sham group byimmunohistochemistry. In vehicle group, the number of positive cells of TLR4,p-p38MAPK, NF-κB and TNF-α significantly increased in the ischemic cortex,among which the location of NF-κB is in nucleus. In H-Shi group, the numberof positive cells of TLR4, p-p38MAPK, NF-κB and TNF-α was significantlydecreased compared with vehicle group (P <0.05). Moreover, positive nucleiof NF-κB were also reduced and lots of cells labeled by NF-κB were stainedonly in cytoplasm (P <0.05). However, there were no significant differencesabout the positive cells between vehicle group and L-Shi group.We found the protein and mRNA levels of TLR4, p-p38MAPK, NF-κB andTNF-α in ischemic tissue were upregulated at24h and72h after MCAO. Wefirst analyzed the protein levels of nuclear NF-κB p65and total TLR4,p-p38MAPK, TNF-α and MMP-9by western blot. The NF-κB p65was rich incytosolic fractions but poor in nuclear extracts in brain tissue of Sham group.In contrast, the protein level of NF-κB in Vehicle group was significantlyenhanced in nuclear fraction and concurrently decreased in cytosol at24h and72h after ischemia, indicating the translocation of these NF-κB subunits fromthe cytosol to the nucleus. High dose of shikonin significantly decreased theexpressions TLR4, p-p38MAPK, NF-κB and TNF-α at24h and72h afterMCAO. However, there were no significant differences in the protein levels ofTLR4, p-p38MAPK, NF-κB and TNF-α between Vehicle group and L-Shigroup. Another parallel set of samples treated with L-Shi and H-Shi werealso evaluated for mRNA expression of TLR4, p-p38MAPK, NF-κB andTNF-α by RT-qPCR. In agreement with the results of western blotting, themRNA expression of TLR4, p-p38MAPK, NF-κB and TNF-α wereup-regulated in Vehicle group compared with Sham group (P <0.05). Theover-expression of those factors was significantly decreased in H-Shi group(P<0.05). Whereas L-Shi group did not display changes of TLR4, p-p38MAPK, NF-κB and TNF-α expression compared with Vehicle group.
Conclusions: The expression of TLR4, p-p38MAPK, NF-κB andTNF-α were up-regulated after ischemia. Systemic administration of shikoninis effective which can decrease the expression of TLR4, p-p38MAPK, NF-κBand TNF-α. Therefore, the excessive inflammation of the brain ischemia wasalleviated.
Part Ⅲ The permeability of blood-brain barrier after focal cerebralischemia in rats and the protection of shikonin
Objective: This study is to evaluate the time course expression ofClaudin-5and MMP-9and explore the underling regulation mechanisms ofshikonin.
Methods: CD-1mice were subjected to transient focal cerebralischemia/reperfusion model in rats was established by middle cerebral arteryocclusion using modified suture occlusion technique. Experiment1: The micewere randomly divided into Sham group(n=30), MCAO group(n=90), Thetime course expression of Claudin-5and MMP-9in the brain tissue afterMCAO. Two groups were studied, including Sham group, MCAO group. Thelast two group included3h,6h,24h,48h and72h sub-groups. Experiment2:shikonin’s neuroprotection against damage from cerebral ischemia/reperfusion.shikonin was administered before MCAO. Mice were reanesthetized andkilled at24h and72h after MCAO. In this part, mice were individed into4groups randomly. Group1: Sham-operated group (Sham): animals receivedsham operation; group2: Vehicle controls (Vehicle): animals receivedtransient MCAO and equal volume PBS including1%DMSO respectivelyonce a day for three times before surgery; group3: Shikonin low dose group(L-Shi): animals received transient MCAO and10mg/kg of Shikoninrespectively once a day for three times before surgery; group4: MCAO-Shikonin high dose group (H-Shi): animals received transient MCAO and25mg/kg of Shikonin respectively once a day for three times before surgery.Drug or solvent was administered before MCAO, then once daily thereafter.RT-qPCR and Western blot were used to analyse the expression of Claudin-5 and MMP-9. The loss of BBB integrity was assessed by leakage of Evans bluefrom microvessels after intravenous injection.
Results: There was considerable leak of Evans blue into the ischemicbrain after MCAO, showing that the BBB had been disrupted in the ischemichemisphere. Compared with Vehicle group, Evans blue evasion of braintissues was significantly decreased in H-Shi group (P<0.05). Whileclaudin-5's expression was increased in both L-Shi and H-Shi groups (P<0.01) at gene and protein levels by western blotting and RT-qPCR at24h and72h, but low dose of shikonin did not display significant level. At the sametime, the MMP-9expression was obviously decreased in H-Shi group (P<0.05), relative to that in normal group.
Conclusions: The blood-brain barrier was dramatically disrupted at theearly stage of focal cerebral ischemia, which was responsible for the brainedema. Shikonin normalizing Claudin-5by inhibiting MMP-9couldcontribute to the alleviated infarct volume, neurological deficits and brainedema in the study.
引文
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