摘要
目的:缺血性脑血管病是常见病、多发病,病死率和致残率均高。脑缺血后继发性脑损伤是一个复杂的病理过程,其中细胞内Ca2+稳态失调在脑缺血的发病机制中起着重要作用。细胞内钙超载使受Ca2+调节的多种酶类被激活,导致膜磷脂分解和细胞骨架破坏,从而造成细胞的凋亡坏死。鞘氨醇激酶(Sphingosine kinase,SphK)是催化鞘氨醇磷酸化为1-磷酸鞘氨醇(sphingosine-1-phosphate, S1P)的关键酶,其中,SphK2由于其催化活性以及在细胞的特殊定位而影响着细胞内Ca2+的水平,进而发挥着促凋亡的作用。氧化苦参碱(Oxymatrine, OMT)是从豆科槐属植物苦参(Sophora flavescens Ait)中提取的生物碱,具有四环喹嗪啶类结构。OMT具有抗炎、抗氧化、抗病毒及调节免疫等多方面药理作用,但其作用机制尚不完全明确,近几年对于OMT的研究很多,多集中在抗肿瘤及抗心肌梗死等方面,关于OMT在脑梗死中作用的研究较少。本实验以线栓法建立大鼠大脑中动脉闭塞(middle cerebral artery occlusion, MCAO)模型,应用Western Blot, RT-PCR和免疫组织化学技术测定脑梗死后SphK2的动态表达规律;应用OMT作为干预药物,研究OMT对脑梗死后脑水肿、脑梗死体积及SphK2表达的影响,旨在探讨OMT在脑梗死中的脑保护作用及其作用机制。
方法:成年健康雄性Sprague-Dawley大鼠应用Longa等的线栓法建立永久性大脑中动脉阻塞脑缺血模型。实验一用来观察SphK2在脑缺血后的动态表达。分为两组:正常组和MCAO组。MCAO组包括6个时间点:3 h, 6 h, 12 h, 24 h, 48 h, 72 h。分别应用免疫组化、Western Blot以及逆转录多聚酶链反应(Reverse Transcription–Polymerase Chain Reaction, RT-PCR)检测脑组织中SphK2的表达变化情况。实验二用来研究OMT对SphK2及血脑屏障紧密连接蛋白claudin-5的影响。大鼠被随机分成3组:假手术组(Sham)、MCAO组、OMT组(MCAO + OMT 120 mg/kg)。脑缺血模型制成后立刻腹腔注射OMT,假手术组及MCAO组给予等容量生理盐水,手术后24 h对大鼠进行神经功能评分。评分完毕将动物断头处死,用干湿重法测定脑组织含水量,用2% 2,3,5-三苯基四唑氮红(triphenyltetrazolium chloride, TTC)染色法测算脑梗死体积,用免疫组化、Western Blot及RT-PCR来观察SphK2及claudin-5的表达情况。
结果:
1与正常对照组相比,MCAO组SphK2于脑缺血后12 h开始升高,72 h达高峰(P < 0.05)。
2 OMT可以改善神经功能的缺失,降低脑水肿以及脑梗死面积。与MCAO组相比,OMT组行为学评分下降(P < 0.05);脑水肿含量减少(MCAO vs. OMT: 83.08±0.56 vs. 81.00±0.43, P < 0.01);脑梗死面积减少(MCAO vs. OMT: 46.66±3.36 vs. 26.01±6.10, P <0.01)。
3 OMT下调SphK2的表达。脑缺血后,缺血灶周围脑组织SphK2阳性细胞数、蛋白水平以及mRNA表达水平均上升,给予OMT后,其表达下调(Western Blot: MCAO vs. OMT: 2.21±0.34 vs. 1.74±0.06, P < 0.05; RT-PCR: MCAO vs. OMT: 0.45±0.01 vs. 0.34±0.02, P < 0.05)。
4 OMT可以改善血脑屏障的通透性。与假手术组相比,脑缺血后claudin-5表达明显下降。RT-PCR以及Western Blot均显示OMT可以上调缺血脑组织内claudin-5的表达(RT-PCR: MCAO vs. OMT: 0.47±0.04 vs. 0.55±0.01, P < 0.05; Western Blot: MCAO vs. OMT: 0.95±0.04 vs. 1.25±0.14, P < 0.05)。
结论:SphK2在脑缺血的损伤过程中表达上调,给予OMT干预后可以有效减少神经系统损伤以及组织损伤。其作用可能与下调SphK2,抑制凋亡;上调claudin-5,改善血脑屏障有关。
Objective: Ischemia with high mortality and serious disability is the most common type of cerebral vascular disease. The pathological mechanism for ischemic injury was a perplexing cascade reaction, the perturbation of calcium homeostasis plays an important role in cerebral ischemic pathogenesis. The intracellular calcium overload activated kinds of enzymes regulated by Ca2+, induced membrane phospholipid resolved and brokedown cytoskeleton with subsequent cell death. Sphingosine kinase (SphK) is a critical enzyme that catalyzes the phosphorylation of sphingosine to sphingosine-1-phosphate. The catalytic activity and specific subcellular localization also contributes to the pro-apoptotic effect of SphK2, which affect the intracellular Ca2+. Oxymatrine (OMT) is an alkaloid extracted from Sophora flavescens Ait and its molecular formula is C15H24N2O2, with a tetracyclic quinolizine structure. In both preclinical and clinical studies, it has been confirmed to possess a variety of biological activities including anti-inflammatory, anti-oxidant, anti-cancer and anti-anaphylaxis properties. In recent years, there are many studies conducted on OMT, most of them are focused on anti-hepatitis and anti-carcinoma, but there are rare reports on the effects of OMT on cerebralvascular diseases. This study tested the dynamic expression of SphK2 in ischemic rats brain tissues using Western Blot, RT-PCR and immunohistochemistry technology; What’s more we studied the influence of OMT on brain water content and infarction volume to testify the neuroprotective effect of OMT on cerebral ischemia and the underlying mechanism.
Methods: Male, healthy Sprague-Dawley rats were subjected to permanent focal cerebral ischemia by the middle cerebral artery occlusion (MCAO). Experiment 1 was used to evaluate the longitudinal expression of SphK2 in the cerebral ischemia. Two groups were studied, including normal-control group and the MCAO group. The MCAO group included 3 h, 6 h, 12 h, 24 h, 48 h and 72 h sub-groups. We tested the dynamic expression of SphK2 in ischemic rat brain tissues using Western Blot, RT-PCR and immunohistochemistry technology. Experiment 2 was used to detect OMT’s influence on SphK2 and claudin-5 expression. The rats were randomly divided into 3 groups: sham operated group that received equal volume 0.9% NaCl (Sham); MCAO group that received equal volume 0.9% NaCl after MCAO (MCAO); OMT group received OMT at 120 mg/kg after MCAO (OMT). OMT were administered immediately through intraperitoneal injection after the MCAO. For Sham and MCAO group, equal volume saline was administered in the same manner. Neurological behavior was evaluated at 24 h after operation then rats were sacrificed. Brain water content was measured by wet-dry method ; Infarct volume was analyzed with 2, 3, 5- triphenyltetrazolium chloride (TTC) staining. SphK2 and claudin-5 expression were measured by immunohistochemistry, Western Blot and RT-PCR.
Results:
1 The expression of SphK2 was upregulated compared with normal-control (P < 0.05), beginning at 12 h, and peaking at 72 h after MCAO.
2 OMT ameliorated neurologic deficits, reduced brain water content and infarct volume. Neurological deficit score in OMT group was decreased compared with MCAO group (P < 0.05); No infarction was observed in Sham group. Compared with MCAO group, the infarct volume was significantly reduced in OMT high dose group (MCAO vs. OMT: 46.66±3.36 vs. 26.01±6.10, P < 0.01); Compared with MCAO group, the water content was significantly reduced in OMT high dose group (MCAO vs. OMT: 83.08±0.56 vs. 81.00±0.43, P < 0.01).
3 OMT downregulated the expression of SphK2. The number of positive cells, the protein level and the mRNA level were increased after MCAO, and the overexpression of SphK2 was downregulated with administration of OMT (Western Blot: MCAO vs. OMT: 2.21±0.34 vs. 1.74±0.06, P < 0.05; RT-PCR: MCAO vs. OMT: 0.45±0.01 vs. 0.34±0.02, P < 0.05).
4 OMT ameliorated BBB permeability. RT-PCR and Western Blot showed that OMT upregulated the expression of claudin-5 in the cerebral ischemia (RT-PCR: MCAO vs. OMT: 0.47±0.04 vs. 0.55±0.01, P < 0.05; Western Blot: MCAO vs. OMT: 0.95±0.04 vs. 1.25±0.14, P < 0.05).
Conclusion: SphK2 increased in the delayed injury processes after cerebral ischemia. Systemic administration of OMT is effective which leads to decreased neurologic impairment and tissue injury and increased survival under cerebral ischemic conditions. Those effects may be through down-regulation of SphK2, inhibiting apoptosis; up-regulation of claudin-5, ameliorating BBB permeability.
引文
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