青蒿琥酯对大鼠蛛网膜下腔出血后血脑屏障的保护及机制研究

发布时间：2018-08-30 07:41

【摘要】：背景和目的蛛网膜下腔出血(Subarachnoid Hemorrhage,SAH)主要发生于相对年轻的人群,其导致的高死残率对社会和家庭造成了巨大的损失。目前认为,SAH的病理生理机制主要涉及脑神经血管网络,该网络包含脑内大血管下游所有的小血管以及与这些血管相关的所有细胞。血脑屏障(Blood-brain barrier,BBB)是神经血管网络中最为重要的结构之一,它的损伤与SAH后发生的血管性脑水肿有着极为紧密的联系。因此,保护BBB免受损伤将有助于SAH病人的康复。青蒿琥酯,作为半合成水溶性的青蒿素衍生物,已经成为脑型疟疾和其他各种重型疟疾的标准治疗药物。青蒿琥酯的抗疟作用高效、安全且耐受良好。而且,大量的研究也指出,其具有广泛的药理学活性,例如抗寄生虫、抗肿瘤、抗炎和抗微生物等。我们知道,神经系统疾病的发生涉及多种病因和复杂的病理生理机制。近来的研究也显示单一作用机制和具有严重副作用的药物不可能用于治疗神经系统疾病。因此,具有多种作用效应的青蒿琥酯可能在预防和治疗神经系统疾病方面具有重要的应用潜力。更为重要的是,青蒿琥酯能特异性地在脑组织中保持高浓度。新近研究发现,青蒿琥酯能够缓解小鼠实验性脑型疟疾导致的BBB损伤,而且这种缓解与疟原虫的杀灭无关。另外,FTY720(S1P1的激动剂)联合青蒿琥酯治疗小鼠脑型疟疾可以明显减轻BBB的损伤并提高其存活率。因此,我们有理由相信青蒿琥酯对SAH具有治疗作用。1-磷酸鞘氨醇受体-1(Sphingosine-1-phosphate receptor-1,S1P1)主要表达于血管内皮细胞上。S1P1特异性地与Gi/o家族结合,在维持血流依赖性血管网络方面具有重要的作用。在小鼠体内全部或仅内皮细胞上敲除S1P1基因都会对血管丛产生极为严重的后果。以往的研究指出,激活S1P1可保护多种卒中模型导致的脑损伤,如:增加S1P的表达可以提高S1P1的活性,进而阻止SAH后小鼠早期脑损伤的发展。但是,对S1P1在保护受损脑组织中涉及的下游信号通路并没有完全弄清。研究显示,S1P1既可通过激活PI3K/Akt信号通路抑制凋亡、阻止Fox O3a活化并最终促进PC12细胞的存活;也可使心肌细胞在缺氧条件下存活;还可促进脂肪干细胞分化成为表达内皮型NO合酶的内皮样细胞等。因此,pi3k是s1p1分子一个极为重要的下游效应器。而且,taddei等人发现激活pi3k/akt分子可以灭活gsk-3β并阻止β-catenin的核转位,这样有利于解除其对claudin-5表达的抑制并最终增加claudin-5的表达。类似研究也指出,小鼠脑出血后激活pi3k/akt信号通路可以减少gsk-3β的激活并稳定β-catenin,最终增加紧密连接蛋白claudin-3和claudin-5的表达。众所周知,紧密连接蛋白claudin-3和claudin-5对维护bbb的完整性极为重要。高效地促进claudin-3和claudin-5的表达,可以有效地缓解各种中枢神经系统疾病导致的bbb损伤。基于上述研究成果,我们推测sah后给予青蒿琥酯可以激活s1p1进而保护bbb的完整性。我们利用脑血管内穿法来构建sah大鼠模型,进而研究sah后青蒿琥酯对bbb的保护效应和潜在机制。材料与方法整个课题研究包括在体和离体实验两部分。脑血管内穿法被用于构建大鼠sah模型。1、18分sah分级评分法被用于评价大鼠sah后24和72小时的出血量,以此来判断模型构建成功与否。2、改良garcia神经功能评分被用于检测大鼠sah后24和72小时的神经系统功能。3、脑水肿程度则通过检测大鼠sah后24和72小时的脑水含量来进行评价。4、血脑屏障完整性则通过检测大鼠sah后24小时脑组织的伊文斯蓝渗出量、伊文斯蓝的自发荧光以及透射电镜来进行评估。5、大鼠sah后24小时s1p1表达量则通过免疫组化染色来进行检测。6、westernblot被用于检测大鼠sah后24或72小时,s1p1、sphk1、sphk2、p-akt/akt、p-gsk3β/gsk3β、p-β-catenin/β-catenin、claudin-3和claudin-5蛋白的表达情况。此外,离体实验中,s1p1的表达量也使用westernblot来检测。7、cck-8被用于检测大鼠脑血管内皮细胞在给予青蒿琥酯24小时后的活性。8、在体实验中,sirna的抑制效果则使用rt-pcr来进行评估。9、自发性高血压大鼠血压的测定,则使用mrbp血压测量仪在2%高盐溶液给予后,每周检测一次。10、sah后24或72小时,青蒿琥酯的治疗作用通过神经功能评分、脑水含量、伊文斯蓝渗出量、伊文斯蓝自发荧光和透射电镜进行评价。11、青蒿琥酯对sah后bbb保护涉及的具体分子机制,则通过分别给予不同拮抗剂(如DMS,VPC23019和Wortmannin)和si RNA(如S1P1 si RNA和Scr siRNA)进行确认。12、青蒿琥酯的预防作用则通过检测SAH后24小时,大鼠的神经功能、脑水含量、以及伊文斯蓝渗出量来评价。结果1、我们发现,相对高浓度的青蒿琥酯(100mg/kg,200mg/kg)可以有效地缓解SAH后模型大鼠的神经功能缺损、抑制脑水肿并减轻BBB的损伤。2、青蒿琥酯在在体和离体实验中,均能明显增加S1P1的表达。但对神经鞘氨醇激酶Sph K1和Sph K2的表达则没有影响,而且给予神经鞘氨醇激酶抑制剂N,N-dimethylsphingosine,并不能逆转青蒿琥酯的作用。3、200mg/kg青蒿琥酯可以提高SAH 24h后蛋白分子p-Akt、Claudin-3和Claudin-5的表达,并降低p-GSK-3/GSK-3β和p-β-catenin/β-catenin的比率。青蒿琥酯的这些作用可以被VPC23019、wortmannin和S1P1 si RNA所逆转。4、预先给予200mg/kg青蒿琥酯,并不能提高SAH后24h模型大鼠的神经功能评分,而且脑水含量和伊文斯蓝的渗出量也未明显减少。但预防性给予青蒿琥酯,可以明显减轻自发性高血压大鼠(SHR)慢性高血压导致的BBB损伤。结论1、青蒿琥酯可以通过维护血脑屏障完整性进而减少脑水肿,来缓解SAH导致的脑损伤。因其可显著提高S1P1表达量,而对神经鞘氨醇激酶Sph K1和Sph K2表达的影响则不大,所以,此作用可能与提高S1P的浓度无关。2、S1P1与PI3K信号通路关系密切,S1P1激活能够增加PI3K/Akt的活性,进而灭活GSK-3β并稳定β-catenin,最终提高SAH模型大鼠紧密连接蛋白Claudin-3和Claudin-5的表达,保护了血脑屏障。3、高血压可以提高正常SHR大鼠血脑屏障的通透性,预先给予青蒿琥酯能够显著减轻其在高血压诱导下的血脑屏障损伤。
[Abstract]:BACKGROUND AND OBJECTIVE Subarachnoid hemorrhage (SAH) mainly occurs in relatively young people, resulting in high mortality and disability rate which has caused enormous losses to society and families. Blood-brain barrier (BBB) is one of the most important structures in the neural-vascular network. Its damage is closely related to vascular brain edema after SAH. Therefore, protecting BBB from injury will help the rehabilitation of SAH patients. Sexual artemisinin derivatives have become standard treatments for cerebral malaria and other serious forms of malaria. Artesunate is highly effective, safe and well tolerated. Moreover, numerous studies have shown that it has a wide range of pharmacological activities, such as antiparasite, antitumor, anti-inflammatory and antimicrobial activities. Recent studies have also shown that drugs with single mechanism of action and serious side effects are unlikely to be used to treat nervous system diseases. Therefore, artesunate with multiple effects may have important potential applications in the prevention and treatment of nervous system diseases. More importantly, artesunate can specifically maintain high concentrations in brain tissue. Recent studies have shown that artesunate can alleviate BBB damage caused by experimental cerebral malaria in mice, and this remission has nothing to do with the eradication of malaria parasites. In addition, FTY720 (S1P1 agonist) combined with artesunate in the treatment of cerebral malaria in mice can be demonstrated. Therefore, we have reason to believe that artesunate has therapeutic effect on SAH. Sphingosine-1-phosphate receptor-1 (S1P1) is mainly expressed in vascular endothelial cells. S1P1 is specifically bound to the Gi/o family and plays an important role in maintaining blood flow-dependent vascular network. Knocking out the S1P1 gene on all or only endothelial cells in mice can have extremely serious consequences for the vascular plexus. Previous studies have shown that activating S1P1 protects against brain damage caused by various stroke models, such as increasing the expression of S1P can increase the activity of S1P1, thereby preventing the development of early brain damage in mice after SAH. The downstream signaling pathways involved in the protection of damaged brain tissues by S1P1 have not been fully understood. Studies have shown that S1P1 can inhibit apoptosis, prevent Fox O3a activation and ultimately promote the survival of PC12 cells by activating PI3K/Akt signaling pathway; it can also promote the survival of cardiomyocytes under hypoxia conditions; and it can also promote the differentiation of adipose-derived stem cells into expressed cells. Therefore, PI3K is a very important downstream effector of s1p1. Moreover, Taddei et al. found that activation of PI3K / Akt can inactivate GSK-3 beta and prevent the nuclear translocation of beta-catenin, which is beneficial to relieve its inhibition of claudin-5 expression and eventually increase claudin-5 expression. Activation of pi3k/akt signaling pathway after intracerebral hemorrhage in mice reduces GSK-3 beta activation and stabilizes beta-catenin, and ultimately increases the expression of tight junction proteins Claudin-3 and claudin-5. It is well known that tight junction proteins Claudin-3 and claudin-5 are very important for maintaining the integrity of bbb. Based on the above results, we hypothesized that artesunate could activate S1P1 and protect the integrity of BBB after sah. We constructed SAH rat model by cerebrovascular endopuncture, and then studied the protective effect and potential mechanism of artesunate on BBB after sah. Methods The whole subject was divided into two parts: in vitro and in vivo. Cerebrovascular endoscopy was used to construct rat SAH model. 1,18 points SAH grading method was used to evaluate the amount of bleeding 24 and 72 hours after SAH in order to judge whether the model was successfully constructed. 2. Modified Garcia neurological function score was used to detect 24 and 72 hours after SAH in rats. Neurological function was assessed at 24 and 72 hours after sah. Blood-brain barrier integrity was assessed at 24 hours after SAH by Evans blue exudation, Evans blue autofluorescence and transmission electron microscopy. 5. S1P1 expression at 24 hours after SAH in rats Western blot was used to detect the expression of s1p1, sphk1, sphk2, p-Akt / akt, p-gsk3 beta, p-beta-catenin / beta-catenin, Claudin-3 and claudin-5 proteins 24 or 72 hours after SAH in rats. In addition, in vitro, the expression of S1P1 was also detected by Western blot, and CCK-8 was also detected by Western blot. In vivo, siRNA inhibitory effect was evaluated by rt-pcr. In spontaneously hypertensive rats, blood pressure was measured by mrbp blood pressure meter after 2% high salt solution was given, once a week, once a week, 24 or 72 hours after sah, artesunate treatment. The therapeutic effect was evaluated by neurological function score, brain water content, Evans blue exudation, Evans blue autofluorescence and transmission electron microscopy. 11. Specific molecular mechanisms involved in the protection of BBB after SAH by artesunate were confirmed by different antagonists (such as DMS, VPC23019 and Wortmannin) and siRNA (such as S1P1 siRNA and SCr siRNA). Results 1. Relatively high concentration of artesunate (100mg/kg, 200mg/kg) could effectively alleviate the neurological impairment, inhibit brain edema and reduce the damage of BBB. 2. Artesunate significantly increased the expression of S1P1 in vivo and in vitro, but had no effect on the expression of Sph K1 and Sph K 2, and the effect of Artesunate could not be reversed by N, N-dimethylsphingosine, an inhibitor of sphingosine kinase. The expression of daughter p-Akt, Claudin-3 and Claudin-5 and the ratio of p-GSK-3/GSK-3 beta and p-beta-catenin/beta-catenin were decreased. These effects of artesunate could be reversed by VPC23019, wortmannin and S1P1si RNA. Pre-administration of 200 mg/kg artesunate did not increase the neurological function score of SAH-induced 24 h rats, and the brain water content and Evans content were also decreased. However, preventive administration of artesunate can significantly alleviate BBB injury induced by chronic hypertension in spontaneously hypertensive rats (SHR). Conclusion 1. Artesunate can alleviate brain injury induced by SAH by maintaining the integrity of blood-brain barrier and thereby reducing brain edema. S1P1 is closely related to PI3K signaling pathway. Activation of S1P1 can increase PI3K/Akt activity, inactivate GSK-3 beta and stabilize beta-catenin, and ultimately increase the tight junction proteins Claudin-3 and Claudin-5 in SAH model rats. Artesunate can significantly reduce the blood-brain barrier damage induced by hypertension.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.35

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