芒果苷对脑缺血再灌注损伤的保护作用及其机制研究
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摘要
研究背景和目的
脑血管疾病是世界最常见的疾病之一,因其发病率高、致残率高、死亡率高的特点,严重威胁人类生命与健康。缺血性脑血管病(ischemic cerebrovascular disease, ICVD)指供应大脑血流的主要动脉发生狭窄或闭塞,结果导致脑组织(特别是大脑中动脉区域)急剧缺血缺氧,最终引起局部脑组织发生缺血或坏死,出现相应的临床症状。缺血性脑卒中后可通过溶解血栓或机械再通的方式恢复缺血区的血液灌注,但一些病人在缺血后再灌注后不仅没有使组织功能恢复,反而使缺血所致的功能障碍和结构破坏进一步加重,即我们所说的脑缺血再灌注损伤(cerebral ischemia-reperfusion (IR) injury)。脑缺血再灌注损伤是一个复杂的病理生理过程,近来研究发现,脑血流中断和再灌注使脑的细胞产生损伤是一个快速的级联反应,这个级联反应包括许多环节,如能量障碍、细胞酸中毒、兴奋性氨基酸释放增加、细胞内钙失稳态、自由基生成、凋亡基因激活等。这些环节互为因果,彼此重叠,并相互联系,形成恶性循环,最终导致细胞凋亡或坏死。目前虽然对脑缺血的病理生理机制研究取得重大进展,但治疗选择仍然有限。一种成功抢救缺血组织的治疗方式是恢复再灌注,溶解血栓的重组组织型纤溶酶原激活剂型(recombinant tissue plasminogen activator, rt-PA)是唯一批准临床使用的药物,然而其时间窗有限(3h-4.5h),且造成出血并加重缺血再灌注损伤的风险。另一种方法则是以缺血级联反应中的各个关键环节为靶点,阻断其发展,达到神经保护的目的。遗憾的是虽然目前许多神经保护剂在动物实验上能缩小脑梗塞体积并改善神经功能,但没有一种被证实临床有效。
天然存在的抗氧化物质所具有的神经保护作用近来成为人们关注的焦点。芒果苷(mangineferin, MAG)是一种天然的多酚类物质,广泛存在于许多药材如百合科植物知母以及芒果叶中。芒果苷分子中的四个芳香族羟基使其具有较强的抗辐射以及抗氧化作用。大量已发表的体内及体外研究表明芒果苷在炎症、氧化应激、肿瘤生长、微生物感染、代谢调节、免疫调节以及辐射保护方面具有广泛的药理学活性。
本研究通过在整体动物水平和细胞水平上来建立神经元缺血再灌注损伤模型,探讨芒果苷对缺血性脑损伤的保护作用及其可能的机制,从而为芒果苷更安全、合理的应用于临床提供理论依据。
研究方法
在整体动物实验中,(1)采用线栓法阻塞大鼠中动脉(middle cerebral artery occlusion, MCAO),建立大鼠脑缺血再灌注损伤模型,观察MAG对脑缺血再灌注损伤大鼠的神经功能评分,评价造模是否成功。大鼠脑缺血2h,再灌注24h后,观察存活大鼠行为变化,参考Zea-longa的5分制评分标准进行神经行为学评分。在剂量研究中,将大鼠随机分成6组：模型组、假手术组、阳性对照组(edaravone, Eda)、MAG各剂量组(5mg/kg、10mg/kg,20mg/kg)。假手术组除不插线外,其余步骤同上。假手术组与模型组均给予等体积生理盐水。HE染色观察组织形态学改变,观察MAG对大鼠缺血大脑的梗塞面积及含水量的影响；(2)较长周期给药实验中,将大鼠随机分成6组：模型组、假手术组、阳性对照组、MAG的高、中、低各剂量组,造模成功后分别于第2、4、6、8d,称量体重,采用姿势反射试验、肢体不对称试验两种方法作为行为学评价指标,观察MAG对大鼠缺血再灌注损伤后运动功能康复的影响；(3)氧化应激生化指标的检测：实验大鼠分组同上。脑缺血2h,再灌注24h后,比色法检测MAG对脑组织匀浆中丙二醛(malonaldehyde, MDA),还原型谷胱甘肽(glutathione, GSH)含量和超氧化物歧化酶(superoxide dismutase, SOD)活性的影响；(4)炎症生化指标的检测：实验大鼠分组同上。RT-PCR检测MAG对脑组织匀浆中炎性因子肿瘤坏死因子α (tumor necrosis factor-α, TNF-α)、白介素-1β(Interleukin-1β, IL-1β)在转录水平表达的影响,随后实验采用ELISA法对上述两种炎性因子在蛋白质水平加以测定；(5)体外培养PC12细胞,应用化学方法建立细胞氧糖剥夺(oxygen-glucose deprivation, OGD)损伤模型,观察细胞形态学改变,并且应用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium, MTT)法观察不同浓度MAG对PC12细胞活力的影响,测定细胞外液乳酸脱氢酶(Lactate dehydrogenase, LDH)浓度。
研究结果
1.MAG对大鼠脑缺血再灌注损伤神经行为学的影响
假手术组大鼠均无异常症状,行为评分为0；模型组动物出现不能完全伸展对侧前爪,或向对侧转圈或向对侧倾倒等的神经损伤症状,其行为学评分与假手术模型组有显著性差异(P=0.000)。芒果苷中剂量组、高剂量组与模型组相比可显著降低行为学评分(P=0.023,P=0.030)。阳性对照组与模型组比较,行为学评分显著改善(P=0.020)。而芒果苷低剂量组与模型组相比无显著差异(P=0.818)。
2.MAG对大鼠脑缺血再灌注损伤大脑组织病理学的影响
组织形态学结果显示：假手术组的大鼠脑组织细胞形态规则,连接紧密。模型组的大鼠细胞周围间隙增宽,神经细胞大小不等,毛细血管萎缩,神经细胞肿胀,排列紊乱,细胞核呈深染固缩,部分胞质自溶,周围出现较大空泡,缺血脑组织形态改变严重。各给药组的大鼠均可见神经细胞缺血性改变减轻,细胞轻度肿胀,间质较致密,坏死细胞数量减少。其中MAG高剂量组可见神经细胞缺血性改变明显减轻,血管周围间隙和神经细胞周围间隙增宽明显减轻,优于模型组,坏死细胞数量明显减少。
3.MAG对大鼠脑缺血再灌注损伤脑梗塞体积及水肿的影响
图像分析行MCAO术后的大鼠脑冠切面,与假手术组相比,经TTC染色后可见明显且可重复出现的脑梗塞。假手术组的脑组织无梗塞。芒果苷低剂量处理组与模型组比较,无显著性差异(P=0.093),芒果苷中、高剂量组均可显著缩小缺血侧脑组织梗塞体积(P=0.001,P=0.000)。阳性对照组与模型组比较,脑梗塞体积显著降低(P=0.001)。与假手术组比较(62.46±0.63%),脑缺血所致的脑含水量显著增高(65.81±1.81%)说明了脑水肿的存在,模型组的脑组织含水量显著高于假手术组(P=0.000)；与模型组相比,芒果苷低、中、高剂量组(5mg/kg、10mg/kg、20mg/kg)脑组织含水量显著降低(P=0.006,P=0.000,P=0.000)。阳性对照组脑组织含水量显著降低(P=0.000),与模型组相比有统计学意义。
4.MAG对大鼠较长时间给药后体重及神经功能恢复的影响
①体重变化率：所有大鼠除假手术组外术后均体重下降。模型组由于缺血再灌注引起的损伤,造成体重呈下降趋势。比较MCAO术后2、4、6、8d各组间体重变化率的差异,结果显示与假手术组同时间点(1.49±8.70,4.61±7.55,7.54±6.83,11.65±6.99)比较,模型组MCAO术后第2d、4d、6d和8d体重变化率显著降低(-10.36±2.55,-13.33±±6.05,-12.97±±7.61,-12.39±8.23),有显著性差异(P均<0.01)。与模型组比较,MAG高剂量组(20mg/kg)在造模后4、6d时可显著改善缺血再灌注引起的体重下降(-5.34±10.30,-5.06±8.34),有显著性差异(P均<0.05)。MAG低、中剂量组(5、10mg/kg)及阳性对照组(6mg/kg)给药后可改善缺血再灌注引起的体重下降,但无统计学差异(P均>0.05)。
②姿势反射试验：假手术组大鼠肢体功能正常,评分为O。MCAO术后第2d、4d、6d和8d各时间点与假手术组(0±0)比较,模型组有显著性增加(1.90±0.13,1.71±0.32,1.74±0.22,1.41±0.43),具有显著性差异(P--0.000)。与模型组比较,芒果苷低剂量组(5mg/kg)各时间点均无显著性差异(P均>0.05)外,中、高剂量组(10.20mg/kg)各时间点大鼠姿势反射测量值均显著下降(P均<0.01)；与模型组比较,阳性对照组仅在2、6d两个测量时间点对大鼠脑缺血引起的运动障碍有显著的改善作用(P=0.000,P=0.000)。
③肢体不对称试验：与假手术组(18.08±17.50,0±23.98,5±27.76,13.46±21.25)比较,造模后第2d、4d、6d和8d各时间点模型组前肢不对称应用评分均显著升高(68.89±21.33,52.78±33.36,48.89±35.16,52.78±30.01),具有显著性差异(P=0.000)。比较MCAO术后2、4、6、8d时各组间肢体不对称评分的差异,结果显示与模型组比较,仅MAG高剂量(20mg/kg)在各个时间点均有显著下降(P均<0.01)；芒果苷低、中剂量组(5、10mg/kg)和阳性对照组(6mg/kg)虽然在MCAO术后能改善在各个时间点的肢体不对称评分,但只有第2d和第8d有显著意义(P均<0.01)。
5.MAG对缺血再灌注大鼠脑SOD、GSH和MDA水平的影响
模型组脑缺血半球SOD活力显著低于假手术组(P=0.007)；与模型组比较,MAG低、中、高剂量组(5mg/kg、10mg/kg、20mg/kg)大脑SOD水平显著高于模型组(P=0.050,P=0.011,P=0.001),表明芒果苷对脑缺血再灌注后脑组织SOD活力有一定恢复作用。阳性对照药依达拉奉组大脑SOD活力显著高于模型组(P=0.014)。
模型组大脑GSH含量显著低于假手术组(P=0.000)；与模型组比较,仅高剂量组(20mg/kg)脑组织GSH含量显著高于模型组(P=0.002),低、中剂量组(5mg/kg、10mg/kg)则无显著性差别(P=0.994,P=0.171)；阳性对照药依达拉奉组GSH含量显著高于模型组(P=0.001)。
模型组大脑MDA含量与假手术组比较有显著升高(P=0.000),表明缺血再灌注后脑组织脂质过氧化反应强烈,伴有脑组织自由基的聚积以及抗氧化酶活性的降低；与模型组比较,MAG低、中、高剂量(5mg/kg、10mg/kg、20mg/kg)脑组织MDA含量显著低于模型组(P=0.000,P=0.000,P=0.000),表明MAG对缺血再灌注后脑组织脂质过氧化反应有显著抑制作用。阳性对照药依达拉奉组MDA含量显著低于模型组(P=0.000)。
6.MAG对缺血再灌注大鼠脑炎症因子IL-1β,TNF-α的影响
定量RT-PCR分析显示在假手术组中,脑组织IL-1βmRNA与TNF-α mRNA的表达较低,在缺血再灌注后均显著上升。缺血2h,再灌注24h后,模型组缺血脑组织匀浆中IL-1β mRNA与TNF-α mRNA的表达均显著高于假手术组(P=0.004,P=0.000)。与模型组比较,芒果苷低、中、高剂量组显著降低IL-1pmRNA的表达(P=0.000,P=0.000,P=0.000),且呈一定剂量依赖性；与模型组比较,MAG低、中、高剂量组显著降低TNF-α mRNA的表达(P=0.000,P=0.000,P=0.001)；阳性对照药依达拉奉与模型组比较也显著降低IL-1βmRNA与TNF-α mRNA的表达(P=0.001, P=0.000)。ELISA法在蛋白质水平也证实了上述对炎症因子的抑制作用。
7.MAG对PC12细胞OGD损伤后细胞形态学的影响
正常对照组PC12细胞生长速度快,接种3d后,在倒置显微镜下可看到PC12细胞贴壁生长,形状呈梭形、三角形,边界清楚,胞浆丰富,突触相互交织成网状,折光性强,呈典型的神经元形态。模型组细胞折光度减弱,细胞圆缩、脱落。模型中PC12细胞的损伤及药物保护的形态学表现基本一致,MAG各组和阳性对照组的细胞损伤程度均有所减轻,呈现一定的药物形态保护。
8.MAG对PC12细胞OGD损伤后细胞存活率及LDH漏出量的的影响
MAG低、中、高剂量组(5,10,20μM)及阳性对照药依达拉奉(10μM)对氧糖剥夺模拟的缺血性损伤有明显的神经保护作用。与正常对照组比较,模型组细胞存活率显著降低(P=0.000)。MAG各组呈剂量依赖性的提高细胞存活率,与模型组比较有显著差异(P<0.01)。与正常对照组比较,模型组细胞LDH释放量显著增加(P<0.01)；而MAG各组呈剂量依赖性减少LDH释放量,与模型组比较有显著差异(P<0.01)。阳性对照组提高细胞存活率,降低LDH漏出量,与模型组相比有统计学意义(P<0.01)。
结论
MAG能够在大鼠脑缺血再灌注后显著升高脑组织中SOD活性和GSH含量,降低MDA含量,从而增强自由基清除能力,减轻脂质过氧化损伤；降低脑组织中炎症因子IL-1β mRNA与TNF-α mRNA基因的表达,从蛋白质水平也证实了对上述两个炎症因子的抑制作用,从而显示其抗炎作用；同时MAG还能增强氧糖剥夺损伤后PC12细胞的存活力,降低细胞上清中LDH含量。这些可能是MAG能改善脑缺血后大脑的组织形态学变化,缩小脑梗死面积,减轻脑水肿损伤,促进神经功能恢复,有效治疗局灶性脑缺血再灌注损伤,发挥神经保护作用的重要机制。
Background and objective
Cerebrovascular disease is one of the most common diseases worldwide. It is seriously threatening human life and health because of its high morbidity, disability rate and mortality. Ischemic cerebrovascular disease (ICVD) refers to occurrences of stenosis and occlusion of main arteries which resulted in the sudden loss of blood and oxygen to the cerebral tissue, especially in middle cerebral artery region. Local brain tissue is ischemic and necrotic ultimately, and appears corresponding clinical symptoms. Restoration of blood flow following ischemic stroke can be achieved by means of thrombolysis or mechanical recanalization. However, for some patients, reperfusion may exacerbate the injury initially caused by ischemia, producing a so-called "cerebral reperfusion injury". Cerebral ischemia-reperfusion injury is a complex pathophysiological process, there are many links in this cascade reaction, including energy shortage, cellular acidosis, increased releasing of excitatory amino acid, destabilization of intracellular Ca2+, production of free radicle, releasing of inflammatory cytokines and activation of apoptosis gene. These closely linked to each other cause and effect, overlapping each other, form a vicious cycle leading to brain cell apoptosis or necrosis. Despite great advances in the mechanism of the pathophysiology of cerebral ischemia, therapeutic options remain limited. One successful treatment strategy for salvaging ischemic tissue is reperfusion. Only recombinant tissue-plasminogen activator (rt-PA) for thrombolysis is currently approved for use in the treatment of this devastating disease. However, its use is limited by its short therapeutic window (3h-4.5h), complications derived essentially from the risk of hemorrhage, and the potential damage from reperfusion/ischemic injury. The other potential approach is to try to impede the ischemic cascade by targeting various components of the cascade that are deemed to be of importance. This latter approach is called the neuroprotection strategy. Despite many successful treatment experiments in animals regarding both infarct size reduction and improved functional outcome, no neuroprotective drug demonstrated unequivocal efficacy in clinical trials.
The neuroprotective role of naturally occurring antioxidants has recently been a focus of attention. Mangiferin (MAG) is one such naturally occurring polyphenol that is widely found in many herbs such as Mangifera indica L and Anemarrhena asphodeloides Bunge. In the mangiferin molecule, four aromatic hydroxyl groups determine its strong antiradical and antioxidant properties. Numerous published in vitro and in vivo studies about mangiferin, demonstrated many pharmacological activities on inflammation, oxidative stress, tumor growth, microorganism infections, metabolic regulations, immune regulations, and radioprotection.
The aim of this research is to set up the model of cerebral ischemia-reperfusion injury in vivo and in vitro and investigate the protective effects and mechanisms of mangiferin on the ischemic cerebral injury. It can provide a theoretical basis for safer and more reasonable uses of this therapy in clinic.
Methods
In vivo,(1) We set up the cerebral ischemia-reperfusion injury model by blocking middle cerebral artery with filament and observed effects of MAG on the neurological deficit score. Neurological deficits in the animals were assessed of the groups after2h of ischemia and24h of reperfusion. At24h after reperfusion, the behavioral characteristics were examined using a standard scale for a five-point neurological assessment (Longa et al.,1989). Briefly, rats were scored as follows:0=no deficit;1=failure to extend contralateral paw fully;2=circling to the contralateral side;3=falling to the contralateral side; and4=no spontaneous walking with a depressed level of consciousness. In the dose-dependence study, rats were randomly divided into6groups:model group, sham-operated group, positive control (edaravone) group, and MAG-treated groups (5mg/kg,10mg/kg and20mg/kg). In the sham-operated rats, the external carotid artery was surgically prepared for insertion of the filament but the filament was not inserted. All groups were injected, besides sham group and model group were gave isotonic Na chloride. HE staining was used to detect the histomorphological changes of brain tissue. The size of cerebral infarction and the water content were measured in the rats.(2) In the long-term experiment, the rats were randomly divided into6groups:model group, sham-operated group, positive control group, and MAG-treated groups of high, medium and low doses. Postural reflex test and limb use asymmetry test were performed as behavioral indexes at2th,4th,6th and8th day after the rats were subjected to focal cerebral ischemia-reperfusion injury for evaluating the recovery of motor function.(3) Biochemical evaluation of oxidative stress:The experiment designs the groups as above. The content of malonaldehyde (MDA), glutathione(GSH) and activity of superoxide dismutase(SOD) in brain tissue after cerebral ischemia for2h followed reperfusion for24h was determined by colorimetry.(4) Biochemical evaluation of inflammation:The experiment designs the groups as above. mRNA expression of cytokines TNF-a and IL-1β in brain tissue was measured by the RT-PCR method. ELISA experiments were next performed at protein levels. In vitro,(5) By establishing oxygen-glucose deprivation (OGD) model of PC12cells, we observed the morphological change of the cells. At the same time, MTT assay was employed to detect the cellular survival rate and biochemical method was used to determine LDH activity.
Result
1. Effect of MAG on neurological deficits in rats after focal cerebral ischemia-reperfusion injury
The rats in sham-operated group have no deficit and were scored0; The rats in model group fail to extend contralateral paw fully and circle or fall to the contralateral side. The neurological score of ischemic model group was significantly high as compared with sham-operated group (P=0.000), indicating induction of ischemia. Treatment with MAG (10,20mg/kg) significantly reduced the neurological deficit (,P=0.023,P=0.030). Edaravone-treated animals exhibited a significantly improved neurological function (P=0.005) compared with the ischemic model group. However, we did not find a significant difference in neurobehavioral improvement in the5mg/kg MAG-treated groups (P=0.818).
2. Effect of MAG on brain histopathology in rats after focal cerebral ischemia-reperfusion injury
Histomorphological changes:In model group, the ischemic cortex showed necrosis of neuron, swelling of cell, cytoplasm rarefaction, light dyeing and vacuolization, while in the sham group it showed no such pathological changes:the nucleolus was clear, round with intact karyolemma.Treatment groups to a certain extent were ameliorated. MAG of high-dose significantly ameliorated the above-mentioned pathological changes, compared with the model group, and necrosis cells obviously eliminated.
3. Effect of MAG on cerebral infarct size, edema in rats after cerebral ischemia-reperfusion injury
Image analysis of TTC-stained coronal sections showed reproducible and readily detectable volume of infarction (31.51±9.78%) in MCAO group as compared to sham. In sham operation group, the infarct areas were unconspicuous. No significant difference was found in5mg/kg MAG-treated groups (P=0.093) in comparison to model group. The cerebral infarct size was significantly smaller in both10mg/kg and20mg/kg MAG-treated group than in MCAO model group(P=0.001,P=0.000). Edaravone-treated group has reduced the infarct volume significantly as compared with the MCAO ischemia group. Ischemia-reperfusion injury led to a significant increase of the brain water content as compared to sham operation group, suggesting the presence of brain edema. Treatment with MAG (5mg/kg,10mg/kg and20mg/kg) significantly limited the increase of brain water content after MCAO (P=0.006, P=0.000, P=0.000). Positive control (edaravone) group reduced the brain water content, the differences was statistically significant compared with the modle group (P=0.000).
4. Long-term motor functional assessment and body weight of MAG after cerebral ischemia-reperfusion injury
①the weight variation ratio:Due to the surgical procedures, all the rats showed a loss of body weight after their operation, except rats in the sham-operated group. The weight of rats in the model group was significantly decreased. Compared with sham-operated group at each tested time point (2th、4th、6th and8th day), the weight variation ratio after MCAO in the model group was significantly decreased (-10.3612.55,-13.33±6.05,-12.97±7.61,-12.39±8.23) and the data presented highly significantly difference(all P (0.01). Compared with model group at each tested time point, the weight variation ratio of MAG in high dose (20mg/kg) group were just significantly increased (-5.34±10.30,-5.06±8.34) at4th and6th day (all P (0.05). Although statistically not significant, there was a general uptrend to body weight in the low and middle groups(5-.1Omg/kg), and the positive control group (6mg/kg) at each tested time point(2th、4th、6th and8th day) after surgery.
②Postural reflex test:The limb function of rats in the sham-operated group was normal and scores0. Compared with sham-operated group(0±0) at each tested time point (2th,4th、6th and8th day), the model group were significantly increased (1.90+0.13,1.71±0.32,1.74±0.22,1.41±0.43) on the postural reflex test neurological score, and the data presented highly significantly difference (all P<0.01). Compared with model group, the rats treated with MAG (5mg/kg) showed no improvement on the limb abnormality after MCAO (all P>0.05). The rats treated with MAG (10、20mg/kg) showed significant improvement on the limb abnormality after MCAO (all P<0.01) and positive group treated with edaravone (6mg/kg) improved only at2th and6th day.
③limb use asymmetry test:On limb use asymmetry test score, compared with sham-operated group(18.08±17.50,0±23.98,5±27.76,13.46+21.25) at each tested time point (2th、4th、6th and8th day), the model group were significantly increased (66.43±32.78,69.29±23.69,50.36±17.81,51.79±40.03) on the asymmetry test score, and the data presented highly significantly difference(P=0.000). Compared with the model group at each tested time point (2th、4tth、6th and8th day), only MAG high dose (20mg/kg) group were significantly decreased(37.22±10.34,25130.52,8.33±35.27,14.44±29.84)? and the data presented highly significantly difference(all P<0.01). The rats treated with MAG (5、10mg/kg) and edaravone (6mg/kg) showed improvement on the asymmetry test score after MCAO, but still significant difference at2th and8th day (all P<0.01).
5. Effect of MAG on brain SOD, GSH and MDA levels after cerebral ischemia-reperfusion
The SOD activities in the ischemic hemisphere were significantly decreased (P=0.007) in ischemic model group as compared with the sham-operated group. Treatment with MAG (5mg/kg,10mg/kg and20mg/kg) significantly increased the SOD levels (P=0.044, P=0.009, f=0.001) compared with ischemic control group. Treatment with edaravone (6mg/kg/i.v.) significantly reversed the decreased SOD levels (P=0.012) as compared with the ischemic group.
The GSH levels in the ischemic hemisphere were significantly decreased (P=0.000) in ischemic model group as compared with the sham-operated group. Treatment with MAG (20mg/kg) significantly increased the GSH content (P=0.002) compared with ischemic control group, but middle and low group (5mg/kg and10mg/kg)show no significant difference (P=0.994, P=0.171). Treatment with edaravone (6mg/kg) significantly reversed the decreased GSH levels (P=0.001) as compared with the ischemic group.
Treatment with MAG (5mg/kg,10mg/kg and20mg/kg) significantly increased the GSH content (P=0.000, p=0.000) compared with ischemic control group. Treatment with edaravone (6mg/kg/i.v.) significantly reversed the decreased GSH levels (P=0.000) as compared with the ischemic group.
The content of brain MDA in the ischemic hemisphere was significantly increased (P=0.000) in ischemic model group as compared with the sham-operated group. Treatment with MAG (5mg/kg, lOmg/kg and20mg/kg) significantly decrease the MDA content(P=0.000, P=0.000, P=0.000) compared with ischemic control group, indicating inhibition of lipid peroxidation after cerebral ischemia reperfusion. Treatment with edaravone (6mg/kg/i.v.) significantly decreased MDA content (P-0.000) as compared with the ischemic group.
6. Effects of MAG on expression of TNF-a mRNA, IL-1β mRNA in cerebral ischemia/reperfusion injury rat brain tissue
RT-PCR analysis showed that the expressions of IL-1β, TNF-a mRNA were seldom in brain of sham groups and all increased in brain of cerebral ischemia/reperfusion rat. Compared to the sham operation group, expression of TNF-a and IL-1β mRNA in ischemic brain tissue homogenate all increased after2h cerebral ischemia followed by24h reperfusion(P=0.004, P=0.000). Significantly, treatment with MGA (5mg/kg,10mg/kg and20mg/kg) dose-dependently decreased expression of IL-1β mRNA in tissue homogenate in comparison to the ischemic model grouptP=0.000, P=0.000, P=0.000). Meanwhile, treatment with MGA (5mg/kg,10mg/kg and20mg/kg) significantly decreased the mRNA expressions of TNF-a. Treatment with edaravone (6mg/kg/i.v.) significantly decreased the expressions of IL-1β and TNF-a mRNA as compared with the ischemic group (P=0.001, P=0.000). ELISA experiments were next conformed the results at protein levels.
7. Effect of MAG on the morphology of PC12cell exposed to OGD injury
When cultured after3days, rat pheochromocytoma (PC12) cells in normal medium group, showed fine growing, well wall-adherented with lucent endochylema. The hallmarks of differentiation into neuronal-like cells are triangular or spindle-shaped cell body followed by outgrowth of multiple neurites from the cell body with multiple branches. The photographs of rat PC12cell that were exposed to oxygen-glucose deprivation showed obvious cell body damage, shrinkage, and cellular debris. In contrast, the cells treated with MAG (5μM,10μM and20μM) or edaravone (10μM) were much better preserved and did not show damage like that of model group. Thus, MAG appears to have a protective effect against oxygen-glucose deprivation-induced injury as well as edaravone.
8. Effect of MAG on the Cellular survival rate and LDH efflux of PC12cell exposed to OGD injury
The three dosage groups of MAG (5μM,10μM,20μM) and positive control group (edaravone,10μM) showed a clear neuroprotective effect against oxygen-glucose deprivation induced ischemic injury. Compared with the normal control group, cellular survival rate in the model group significantly decreased (P=0.000). Moreover, we observed that, compared with model group, The three dosage groups of MAG increased cell survival rate (P<0.01), and more the MAG concentration, higher the cell survival rate. Compared with the normal control group, the level of LDH in the model group significantly increased (P<0.01). MAG (5μM,10μM,20μM) significantly decreased the LDH efflux (P<0.01), and more the MAG concentration, lower the level of LDH. Edaravone-treated group significantly significantly enhanced the cell viability (P<0.01) and reduced the level of LDH (P<0.01) as compared with the model group.
Conclusion
In conclusion, MAG could increase the level of SOD and the content of GSH, decrease the content of MDA in brain tissue, which demonstrated its activity of scavenging free-radical and ability of attenuating lipid peroxidation damage; MAG could decrease the expression of IL-1β mRNA and TNF-a mRNA in brain tissue, which suppressed cerebral ischemia-reperfusion-induced inflammation.These results were next confirmed by ELISA at protein levels; meanwhile, MAG treatment significantly enhanced the cell viability and reduced the levels of LDH. Thus, MAG could ameliorate morphological changes, decrease the cerebral infarction size, attenuate tissue edema and improve neurological recovery, which could be of the important mechanisms of effective therapy effect after focal cerebral ischemia.
脑血管疾病是世界最常见的疾病之一,因其发病率高、致残率高、死亡率高的特点,严重威胁人类生命与健康。缺血性脑血管病(ischemic cerebrovascular disease, ICVD)指供应大脑血流的主要动脉发生狭窄或闭塞,结果导致脑组织(特别是大脑中动脉区域)急剧缺血缺氧,最终引起局部脑组织发生缺血或坏死,出现相应的临床症状。缺血性脑卒中后可通过溶解血栓或机械再通的方式恢复缺血区的血液灌注,但一些病人在缺血后再灌注后不仅没有使组织功能恢复,反而使缺血所致的功能障碍和结构破坏进一步加重,即我们所说的脑缺血再灌注损伤(cerebral ischemia-reperfusion (IR) injury)。脑缺血再灌注损伤是一个复杂的病理生理过程,近来研究发现,脑血流中断和再灌注使脑的细胞产生损伤是一个快速的级联反应,这个级联反应包括许多环节,如能量障碍、细胞酸中毒、兴奋性氨基酸释放增加、细胞内钙失稳态、自由基生成、凋亡基因激活等。这些环节互为因果,彼此重叠,并相互联系,形成恶性循环,最终导致细胞凋亡或坏死。目前虽然对脑缺血的病理生理机制研究取得重大进展,但治疗选择仍然有限。一种成功抢救缺血组织的治疗方式是恢复再灌注,溶解血栓的重组组织型纤溶酶原激活剂型(recombinant tissue plasminogen activator, rt-PA)是唯一批准临床使用的药物,然而其时间窗有限(3h-4.5h),且造成出血并加重缺血再灌注损伤的风险。另一种方法则是以缺血级联反应中的各个关键环节为靶点,阻断其发展,达到神经保护的目的。遗憾的是虽然目前许多神经保护剂在动物实验上能缩小脑梗塞体积并改善神经功能,但没有一种被证实临床有效。
天然存在的抗氧化物质所具有的神经保护作用近来成为人们关注的焦点。芒果苷(mangineferin, MAG)是一种天然的多酚类物质,广泛存在于许多药材如百合科植物知母以及芒果叶中。芒果苷分子中的四个芳香族羟基使其具有较强的抗辐射以及抗氧化作用。大量已发表的体内及体外研究表明芒果苷在炎症、氧化应激、肿瘤生长、微生物感染、代谢调节、免疫调节以及辐射保护方面具有广泛的药理学活性。
本研究通过在整体动物水平和细胞水平上来建立神经元缺血再灌注损伤模型,探讨芒果苷对缺血性脑损伤的保护作用及其可能的机制,从而为芒果苷更安全、合理的应用于临床提供理论依据。
研究方法
在整体动物实验中,(1)采用线栓法阻塞大鼠中动脉(middle cerebral artery occlusion, MCAO),建立大鼠脑缺血再灌注损伤模型,观察MAG对脑缺血再灌注损伤大鼠的神经功能评分,评价造模是否成功。大鼠脑缺血2h,再灌注24h后,观察存活大鼠行为变化,参考Zea-longa的5分制评分标准进行神经行为学评分。在剂量研究中,将大鼠随机分成6组：模型组、假手术组、阳性对照组(edaravone, Eda)、MAG各剂量组(5mg/kg、10mg/kg,20mg/kg)。假手术组除不插线外,其余步骤同上。假手术组与模型组均给予等体积生理盐水。HE染色观察组织形态学改变,观察MAG对大鼠缺血大脑的梗塞面积及含水量的影响；(2)较长周期给药实验中,将大鼠随机分成6组：模型组、假手术组、阳性对照组、MAG的高、中、低各剂量组,造模成功后分别于第2、4、6、8d,称量体重,采用姿势反射试验、肢体不对称试验两种方法作为行为学评价指标,观察MAG对大鼠缺血再灌注损伤后运动功能康复的影响；(3)氧化应激生化指标的检测：实验大鼠分组同上。脑缺血2h,再灌注24h后,比色法检测MAG对脑组织匀浆中丙二醛(malonaldehyde, MDA),还原型谷胱甘肽(glutathione, GSH)含量和超氧化物歧化酶(superoxide dismutase, SOD)活性的影响；(4)炎症生化指标的检测：实验大鼠分组同上。RT-PCR检测MAG对脑组织匀浆中炎性因子肿瘤坏死因子α (tumor necrosis factor-α, TNF-α)、白介素-1β(Interleukin-1β, IL-1β)在转录水平表达的影响,随后实验采用ELISA法对上述两种炎性因子在蛋白质水平加以测定；(5)体外培养PC12细胞,应用化学方法建立细胞氧糖剥夺(oxygen-glucose deprivation, OGD)损伤模型,观察细胞形态学改变,并且应用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium, MTT)法观察不同浓度MAG对PC12细胞活力的影响,测定细胞外液乳酸脱氢酶(Lactate dehydrogenase, LDH)浓度。
研究结果
1.MAG对大鼠脑缺血再灌注损伤神经行为学的影响
假手术组大鼠均无异常症状,行为评分为0；模型组动物出现不能完全伸展对侧前爪,或向对侧转圈或向对侧倾倒等的神经损伤症状,其行为学评分与假手术模型组有显著性差异(P=0.000)。芒果苷中剂量组、高剂量组与模型组相比可显著降低行为学评分(P=0.023,P=0.030)。阳性对照组与模型组比较,行为学评分显著改善(P=0.020)。而芒果苷低剂量组与模型组相比无显著差异(P=0.818)。
2.MAG对大鼠脑缺血再灌注损伤大脑组织病理学的影响
组织形态学结果显示：假手术组的大鼠脑组织细胞形态规则,连接紧密。模型组的大鼠细胞周围间隙增宽,神经细胞大小不等,毛细血管萎缩,神经细胞肿胀,排列紊乱,细胞核呈深染固缩,部分胞质自溶,周围出现较大空泡,缺血脑组织形态改变严重。各给药组的大鼠均可见神经细胞缺血性改变减轻,细胞轻度肿胀,间质较致密,坏死细胞数量减少。其中MAG高剂量组可见神经细胞缺血性改变明显减轻,血管周围间隙和神经细胞周围间隙增宽明显减轻,优于模型组,坏死细胞数量明显减少。
3.MAG对大鼠脑缺血再灌注损伤脑梗塞体积及水肿的影响
图像分析行MCAO术后的大鼠脑冠切面,与假手术组相比,经TTC染色后可见明显且可重复出现的脑梗塞。假手术组的脑组织无梗塞。芒果苷低剂量处理组与模型组比较,无显著性差异(P=0.093),芒果苷中、高剂量组均可显著缩小缺血侧脑组织梗塞体积(P=0.001,P=0.000)。阳性对照组与模型组比较,脑梗塞体积显著降低(P=0.001)。与假手术组比较(62.46±0.63%),脑缺血所致的脑含水量显著增高(65.81±1.81%)说明了脑水肿的存在,模型组的脑组织含水量显著高于假手术组(P=0.000)；与模型组相比,芒果苷低、中、高剂量组(5mg/kg、10mg/kg、20mg/kg)脑组织含水量显著降低(P=0.006,P=0.000,P=0.000)。阳性对照组脑组织含水量显著降低(P=0.000),与模型组相比有统计学意义。
4.MAG对大鼠较长时间给药后体重及神经功能恢复的影响
①体重变化率：所有大鼠除假手术组外术后均体重下降。模型组由于缺血再灌注引起的损伤,造成体重呈下降趋势。比较MCAO术后2、4、6、8d各组间体重变化率的差异,结果显示与假手术组同时间点(1.49±8.70,4.61±7.55,7.54±6.83,11.65±6.99)比较,模型组MCAO术后第2d、4d、6d和8d体重变化率显著降低(-10.36±2.55,-13.33±±6.05,-12.97±±7.61,-12.39±8.23),有显著性差异(P均<0.01)。与模型组比较,MAG高剂量组(20mg/kg)在造模后4、6d时可显著改善缺血再灌注引起的体重下降(-5.34±10.30,-5.06±8.34),有显著性差异(P均<0.05)。MAG低、中剂量组(5、10mg/kg)及阳性对照组(6mg/kg)给药后可改善缺血再灌注引起的体重下降,但无统计学差异(P均>0.05)。
②姿势反射试验：假手术组大鼠肢体功能正常,评分为O。MCAO术后第2d、4d、6d和8d各时间点与假手术组(0±0)比较,模型组有显著性增加(1.90±0.13,1.71±0.32,1.74±0.22,1.41±0.43),具有显著性差异(P--0.000)。与模型组比较,芒果苷低剂量组(5mg/kg)各时间点均无显著性差异(P均>0.05)外,中、高剂量组(10.20mg/kg)各时间点大鼠姿势反射测量值均显著下降(P均<0.01)；与模型组比较,阳性对照组仅在2、6d两个测量时间点对大鼠脑缺血引起的运动障碍有显著的改善作用(P=0.000,P=0.000)。
③肢体不对称试验：与假手术组(18.08±17.50,0±23.98,5±27.76,13.46±21.25)比较,造模后第2d、4d、6d和8d各时间点模型组前肢不对称应用评分均显著升高(68.89±21.33,52.78±33.36,48.89±35.16,52.78±30.01),具有显著性差异(P=0.000)。比较MCAO术后2、4、6、8d时各组间肢体不对称评分的差异,结果显示与模型组比较,仅MAG高剂量(20mg/kg)在各个时间点均有显著下降(P均<0.01)；芒果苷低、中剂量组(5、10mg/kg)和阳性对照组(6mg/kg)虽然在MCAO术后能改善在各个时间点的肢体不对称评分,但只有第2d和第8d有显著意义(P均<0.01)。
5.MAG对缺血再灌注大鼠脑SOD、GSH和MDA水平的影响
模型组脑缺血半球SOD活力显著低于假手术组(P=0.007)；与模型组比较,MAG低、中、高剂量组(5mg/kg、10mg/kg、20mg/kg)大脑SOD水平显著高于模型组(P=0.050,P=0.011,P=0.001),表明芒果苷对脑缺血再灌注后脑组织SOD活力有一定恢复作用。阳性对照药依达拉奉组大脑SOD活力显著高于模型组(P=0.014)。
模型组大脑GSH含量显著低于假手术组(P=0.000)；与模型组比较,仅高剂量组(20mg/kg)脑组织GSH含量显著高于模型组(P=0.002),低、中剂量组(5mg/kg、10mg/kg)则无显著性差别(P=0.994,P=0.171)；阳性对照药依达拉奉组GSH含量显著高于模型组(P=0.001)。
模型组大脑MDA含量与假手术组比较有显著升高(P=0.000),表明缺血再灌注后脑组织脂质过氧化反应强烈,伴有脑组织自由基的聚积以及抗氧化酶活性的降低；与模型组比较,MAG低、中、高剂量(5mg/kg、10mg/kg、20mg/kg)脑组织MDA含量显著低于模型组(P=0.000,P=0.000,P=0.000),表明MAG对缺血再灌注后脑组织脂质过氧化反应有显著抑制作用。阳性对照药依达拉奉组MDA含量显著低于模型组(P=0.000)。
6.MAG对缺血再灌注大鼠脑炎症因子IL-1β,TNF-α的影响
定量RT-PCR分析显示在假手术组中,脑组织IL-1βmRNA与TNF-α mRNA的表达较低,在缺血再灌注后均显著上升。缺血2h,再灌注24h后,模型组缺血脑组织匀浆中IL-1β mRNA与TNF-α mRNA的表达均显著高于假手术组(P=0.004,P=0.000)。与模型组比较,芒果苷低、中、高剂量组显著降低IL-1pmRNA的表达(P=0.000,P=0.000,P=0.000),且呈一定剂量依赖性；与模型组比较,MAG低、中、高剂量组显著降低TNF-α mRNA的表达(P=0.000,P=0.000,P=0.001)；阳性对照药依达拉奉与模型组比较也显著降低IL-1βmRNA与TNF-α mRNA的表达(P=0.001, P=0.000)。ELISA法在蛋白质水平也证实了上述对炎症因子的抑制作用。
7.MAG对PC12细胞OGD损伤后细胞形态学的影响
正常对照组PC12细胞生长速度快,接种3d后,在倒置显微镜下可看到PC12细胞贴壁生长,形状呈梭形、三角形,边界清楚,胞浆丰富,突触相互交织成网状,折光性强,呈典型的神经元形态。模型组细胞折光度减弱,细胞圆缩、脱落。模型中PC12细胞的损伤及药物保护的形态学表现基本一致,MAG各组和阳性对照组的细胞损伤程度均有所减轻,呈现一定的药物形态保护。
8.MAG对PC12细胞OGD损伤后细胞存活率及LDH漏出量的的影响
MAG低、中、高剂量组(5,10,20μM)及阳性对照药依达拉奉(10μM)对氧糖剥夺模拟的缺血性损伤有明显的神经保护作用。与正常对照组比较,模型组细胞存活率显著降低(P=0.000)。MAG各组呈剂量依赖性的提高细胞存活率,与模型组比较有显著差异(P<0.01)。与正常对照组比较,模型组细胞LDH释放量显著增加(P<0.01)；而MAG各组呈剂量依赖性减少LDH释放量,与模型组比较有显著差异(P<0.01)。阳性对照组提高细胞存活率,降低LDH漏出量,与模型组相比有统计学意义(P<0.01)。
结论
MAG能够在大鼠脑缺血再灌注后显著升高脑组织中SOD活性和GSH含量,降低MDA含量,从而增强自由基清除能力,减轻脂质过氧化损伤；降低脑组织中炎症因子IL-1β mRNA与TNF-α mRNA基因的表达,从蛋白质水平也证实了对上述两个炎症因子的抑制作用,从而显示其抗炎作用；同时MAG还能增强氧糖剥夺损伤后PC12细胞的存活力,降低细胞上清中LDH含量。这些可能是MAG能改善脑缺血后大脑的组织形态学变化,缩小脑梗死面积,减轻脑水肿损伤,促进神经功能恢复,有效治疗局灶性脑缺血再灌注损伤,发挥神经保护作用的重要机制。
Background and objective
Cerebrovascular disease is one of the most common diseases worldwide. It is seriously threatening human life and health because of its high morbidity, disability rate and mortality. Ischemic cerebrovascular disease (ICVD) refers to occurrences of stenosis and occlusion of main arteries which resulted in the sudden loss of blood and oxygen to the cerebral tissue, especially in middle cerebral artery region. Local brain tissue is ischemic and necrotic ultimately, and appears corresponding clinical symptoms. Restoration of blood flow following ischemic stroke can be achieved by means of thrombolysis or mechanical recanalization. However, for some patients, reperfusion may exacerbate the injury initially caused by ischemia, producing a so-called "cerebral reperfusion injury". Cerebral ischemia-reperfusion injury is a complex pathophysiological process, there are many links in this cascade reaction, including energy shortage, cellular acidosis, increased releasing of excitatory amino acid, destabilization of intracellular Ca2+, production of free radicle, releasing of inflammatory cytokines and activation of apoptosis gene. These closely linked to each other cause and effect, overlapping each other, form a vicious cycle leading to brain cell apoptosis or necrosis. Despite great advances in the mechanism of the pathophysiology of cerebral ischemia, therapeutic options remain limited. One successful treatment strategy for salvaging ischemic tissue is reperfusion. Only recombinant tissue-plasminogen activator (rt-PA) for thrombolysis is currently approved for use in the treatment of this devastating disease. However, its use is limited by its short therapeutic window (3h-4.5h), complications derived essentially from the risk of hemorrhage, and the potential damage from reperfusion/ischemic injury. The other potential approach is to try to impede the ischemic cascade by targeting various components of the cascade that are deemed to be of importance. This latter approach is called the neuroprotection strategy. Despite many successful treatment experiments in animals regarding both infarct size reduction and improved functional outcome, no neuroprotective drug demonstrated unequivocal efficacy in clinical trials.
The neuroprotective role of naturally occurring antioxidants has recently been a focus of attention. Mangiferin (MAG) is one such naturally occurring polyphenol that is widely found in many herbs such as Mangifera indica L and Anemarrhena asphodeloides Bunge. In the mangiferin molecule, four aromatic hydroxyl groups determine its strong antiradical and antioxidant properties. Numerous published in vitro and in vivo studies about mangiferin, demonstrated many pharmacological activities on inflammation, oxidative stress, tumor growth, microorganism infections, metabolic regulations, immune regulations, and radioprotection.
The aim of this research is to set up the model of cerebral ischemia-reperfusion injury in vivo and in vitro and investigate the protective effects and mechanisms of mangiferin on the ischemic cerebral injury. It can provide a theoretical basis for safer and more reasonable uses of this therapy in clinic.
Methods
In vivo,(1) We set up the cerebral ischemia-reperfusion injury model by blocking middle cerebral artery with filament and observed effects of MAG on the neurological deficit score. Neurological deficits in the animals were assessed of the groups after2h of ischemia and24h of reperfusion. At24h after reperfusion, the behavioral characteristics were examined using a standard scale for a five-point neurological assessment (Longa et al.,1989). Briefly, rats were scored as follows:0=no deficit;1=failure to extend contralateral paw fully;2=circling to the contralateral side;3=falling to the contralateral side; and4=no spontaneous walking with a depressed level of consciousness. In the dose-dependence study, rats were randomly divided into6groups:model group, sham-operated group, positive control (edaravone) group, and MAG-treated groups (5mg/kg,10mg/kg and20mg/kg). In the sham-operated rats, the external carotid artery was surgically prepared for insertion of the filament but the filament was not inserted. All groups were injected, besides sham group and model group were gave isotonic Na chloride. HE staining was used to detect the histomorphological changes of brain tissue. The size of cerebral infarction and the water content were measured in the rats.(2) In the long-term experiment, the rats were randomly divided into6groups:model group, sham-operated group, positive control group, and MAG-treated groups of high, medium and low doses. Postural reflex test and limb use asymmetry test were performed as behavioral indexes at2th,4th,6th and8th day after the rats were subjected to focal cerebral ischemia-reperfusion injury for evaluating the recovery of motor function.(3) Biochemical evaluation of oxidative stress:The experiment designs the groups as above. The content of malonaldehyde (MDA), glutathione(GSH) and activity of superoxide dismutase(SOD) in brain tissue after cerebral ischemia for2h followed reperfusion for24h was determined by colorimetry.(4) Biochemical evaluation of inflammation:The experiment designs the groups as above. mRNA expression of cytokines TNF-a and IL-1β in brain tissue was measured by the RT-PCR method. ELISA experiments were next performed at protein levels. In vitro,(5) By establishing oxygen-glucose deprivation (OGD) model of PC12cells, we observed the morphological change of the cells. At the same time, MTT assay was employed to detect the cellular survival rate and biochemical method was used to determine LDH activity.
Result
1. Effect of MAG on neurological deficits in rats after focal cerebral ischemia-reperfusion injury
The rats in sham-operated group have no deficit and were scored0; The rats in model group fail to extend contralateral paw fully and circle or fall to the contralateral side. The neurological score of ischemic model group was significantly high as compared with sham-operated group (P=0.000), indicating induction of ischemia. Treatment with MAG (10,20mg/kg) significantly reduced the neurological deficit (,P=0.023,P=0.030). Edaravone-treated animals exhibited a significantly improved neurological function (P=0.005) compared with the ischemic model group. However, we did not find a significant difference in neurobehavioral improvement in the5mg/kg MAG-treated groups (P=0.818).
2. Effect of MAG on brain histopathology in rats after focal cerebral ischemia-reperfusion injury
Histomorphological changes:In model group, the ischemic cortex showed necrosis of neuron, swelling of cell, cytoplasm rarefaction, light dyeing and vacuolization, while in the sham group it showed no such pathological changes:the nucleolus was clear, round with intact karyolemma.Treatment groups to a certain extent were ameliorated. MAG of high-dose significantly ameliorated the above-mentioned pathological changes, compared with the model group, and necrosis cells obviously eliminated.
3. Effect of MAG on cerebral infarct size, edema in rats after cerebral ischemia-reperfusion injury
Image analysis of TTC-stained coronal sections showed reproducible and readily detectable volume of infarction (31.51±9.78%) in MCAO group as compared to sham. In sham operation group, the infarct areas were unconspicuous. No significant difference was found in5mg/kg MAG-treated groups (P=0.093) in comparison to model group. The cerebral infarct size was significantly smaller in both10mg/kg and20mg/kg MAG-treated group than in MCAO model group(P=0.001,P=0.000). Edaravone-treated group has reduced the infarct volume significantly as compared with the MCAO ischemia group. Ischemia-reperfusion injury led to a significant increase of the brain water content as compared to sham operation group, suggesting the presence of brain edema. Treatment with MAG (5mg/kg,10mg/kg and20mg/kg) significantly limited the increase of brain water content after MCAO (P=0.006, P=0.000, P=0.000). Positive control (edaravone) group reduced the brain water content, the differences was statistically significant compared with the modle group (P=0.000).
4. Long-term motor functional assessment and body weight of MAG after cerebral ischemia-reperfusion injury
①the weight variation ratio:Due to the surgical procedures, all the rats showed a loss of body weight after their operation, except rats in the sham-operated group. The weight of rats in the model group was significantly decreased. Compared with sham-operated group at each tested time point (2th、4th、6th and8th day), the weight variation ratio after MCAO in the model group was significantly decreased (-10.3612.55,-13.33±6.05,-12.97±7.61,-12.39±8.23) and the data presented highly significantly difference(all P (0.01). Compared with model group at each tested time point, the weight variation ratio of MAG in high dose (20mg/kg) group were just significantly increased (-5.34±10.30,-5.06±8.34) at4th and6th day (all P (0.05). Although statistically not significant, there was a general uptrend to body weight in the low and middle groups(5-.1Omg/kg), and the positive control group (6mg/kg) at each tested time point(2th、4th、6th and8th day) after surgery.
②Postural reflex test:The limb function of rats in the sham-operated group was normal and scores0. Compared with sham-operated group(0±0) at each tested time point (2th,4th、6th and8th day), the model group were significantly increased (1.90+0.13,1.71±0.32,1.74±0.22,1.41±0.43) on the postural reflex test neurological score, and the data presented highly significantly difference (all P<0.01). Compared with model group, the rats treated with MAG (5mg/kg) showed no improvement on the limb abnormality after MCAO (all P>0.05). The rats treated with MAG (10、20mg/kg) showed significant improvement on the limb abnormality after MCAO (all P<0.01) and positive group treated with edaravone (6mg/kg) improved only at2th and6th day.
③limb use asymmetry test:On limb use asymmetry test score, compared with sham-operated group(18.08±17.50,0±23.98,5±27.76,13.46+21.25) at each tested time point (2th、4th、6th and8th day), the model group were significantly increased (66.43±32.78,69.29±23.69,50.36±17.81,51.79±40.03) on the asymmetry test score, and the data presented highly significantly difference(P=0.000). Compared with the model group at each tested time point (2th、4tth、6th and8th day), only MAG high dose (20mg/kg) group were significantly decreased(37.22±10.34,25130.52,8.33±35.27,14.44±29.84)? and the data presented highly significantly difference(all P<0.01). The rats treated with MAG (5、10mg/kg) and edaravone (6mg/kg) showed improvement on the asymmetry test score after MCAO, but still significant difference at2th and8th day (all P<0.01).
5. Effect of MAG on brain SOD, GSH and MDA levels after cerebral ischemia-reperfusion
The SOD activities in the ischemic hemisphere were significantly decreased (P=0.007) in ischemic model group as compared with the sham-operated group. Treatment with MAG (5mg/kg,10mg/kg and20mg/kg) significantly increased the SOD levels (P=0.044, P=0.009, f=0.001) compared with ischemic control group. Treatment with edaravone (6mg/kg/i.v.) significantly reversed the decreased SOD levels (P=0.012) as compared with the ischemic group.
The GSH levels in the ischemic hemisphere were significantly decreased (P=0.000) in ischemic model group as compared with the sham-operated group. Treatment with MAG (20mg/kg) significantly increased the GSH content (P=0.002) compared with ischemic control group, but middle and low group (5mg/kg and10mg/kg)show no significant difference (P=0.994, P=0.171). Treatment with edaravone (6mg/kg) significantly reversed the decreased GSH levels (P=0.001) as compared with the ischemic group.
Treatment with MAG (5mg/kg,10mg/kg and20mg/kg) significantly increased the GSH content (P=0.000, p=0.000) compared with ischemic control group. Treatment with edaravone (6mg/kg/i.v.) significantly reversed the decreased GSH levels (P=0.000) as compared with the ischemic group.
The content of brain MDA in the ischemic hemisphere was significantly increased (P=0.000) in ischemic model group as compared with the sham-operated group. Treatment with MAG (5mg/kg, lOmg/kg and20mg/kg) significantly decrease the MDA content(P=0.000, P=0.000, P=0.000) compared with ischemic control group, indicating inhibition of lipid peroxidation after cerebral ischemia reperfusion. Treatment with edaravone (6mg/kg/i.v.) significantly decreased MDA content (P-0.000) as compared with the ischemic group.
6. Effects of MAG on expression of TNF-a mRNA, IL-1β mRNA in cerebral ischemia/reperfusion injury rat brain tissue
RT-PCR analysis showed that the expressions of IL-1β, TNF-a mRNA were seldom in brain of sham groups and all increased in brain of cerebral ischemia/reperfusion rat. Compared to the sham operation group, expression of TNF-a and IL-1β mRNA in ischemic brain tissue homogenate all increased after2h cerebral ischemia followed by24h reperfusion(P=0.004, P=0.000). Significantly, treatment with MGA (5mg/kg,10mg/kg and20mg/kg) dose-dependently decreased expression of IL-1β mRNA in tissue homogenate in comparison to the ischemic model grouptP=0.000, P=0.000, P=0.000). Meanwhile, treatment with MGA (5mg/kg,10mg/kg and20mg/kg) significantly decreased the mRNA expressions of TNF-a. Treatment with edaravone (6mg/kg/i.v.) significantly decreased the expressions of IL-1β and TNF-a mRNA as compared with the ischemic group (P=0.001, P=0.000). ELISA experiments were next conformed the results at protein levels.
7. Effect of MAG on the morphology of PC12cell exposed to OGD injury
When cultured after3days, rat pheochromocytoma (PC12) cells in normal medium group, showed fine growing, well wall-adherented with lucent endochylema. The hallmarks of differentiation into neuronal-like cells are triangular or spindle-shaped cell body followed by outgrowth of multiple neurites from the cell body with multiple branches. The photographs of rat PC12cell that were exposed to oxygen-glucose deprivation showed obvious cell body damage, shrinkage, and cellular debris. In contrast, the cells treated with MAG (5μM,10μM and20μM) or edaravone (10μM) were much better preserved and did not show damage like that of model group. Thus, MAG appears to have a protective effect against oxygen-glucose deprivation-induced injury as well as edaravone.
8. Effect of MAG on the Cellular survival rate and LDH efflux of PC12cell exposed to OGD injury
The three dosage groups of MAG (5μM,10μM,20μM) and positive control group (edaravone,10μM) showed a clear neuroprotective effect against oxygen-glucose deprivation induced ischemic injury. Compared with the normal control group, cellular survival rate in the model group significantly decreased (P=0.000). Moreover, we observed that, compared with model group, The three dosage groups of MAG increased cell survival rate (P<0.01), and more the MAG concentration, higher the cell survival rate. Compared with the normal control group, the level of LDH in the model group significantly increased (P<0.01). MAG (5μM,10μM,20μM) significantly decreased the LDH efflux (P<0.01), and more the MAG concentration, lower the level of LDH. Edaravone-treated group significantly significantly enhanced the cell viability (P<0.01) and reduced the level of LDH (P<0.01) as compared with the model group.
Conclusion
In conclusion, MAG could increase the level of SOD and the content of GSH, decrease the content of MDA in brain tissue, which demonstrated its activity of scavenging free-radical and ability of attenuating lipid peroxidation damage; MAG could decrease the expression of IL-1β mRNA and TNF-a mRNA in brain tissue, which suppressed cerebral ischemia-reperfusion-induced inflammation.These results were next confirmed by ELISA at protein levels; meanwhile, MAG treatment significantly enhanced the cell viability and reduced the levels of LDH. Thus, MAG could ameliorate morphological changes, decrease the cerebral infarction size, attenuate tissue edema and improve neurological recovery, which could be of the important mechanisms of effective therapy effect after focal cerebral ischemia.
引文
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