三七三醇皂苷促脑梗死大鼠血管新生改善脑灌注机制研究

发布时间：2018-08-31 19:51

【摘要】：研究背景:脑卒中是一种具有高发病率、死亡率、致残率的常见神经系统疾病,其中约87%是缺血性卒中。尽早恢复缺血区脑灌注,挽救缺血半暗带濒死神经元是脑梗死治疗的重心和目标。多项动物实验证实促进脑梗死后血管新生可减轻神经功能缺损,缩小脑梗死体积。三七三醇皂苷(Panaxatriol Saponins,PTS),是从中药三七中提取的有效成份,其中三七皂苷R1、人参皂苷Rg1、人参皂苷Re为PTS的主要组成成分,前期多项研究证实PTS对脑梗死大鼠具有保护作用,但具体机制不明。近期研究表明PTS三种主要成分均有促人脐静脉内皮细胞增殖作用,然而目前尚未有PTS在缺血脑损伤动物体内促血管新生方面的报道。目的:(1)观察PTS对大鼠脑缺血再灌注模型的保护作用;(2)明确PTS促进脑梗死后微血管新生改善脑灌注作用;(3)探讨PTS促进脑梗死后血管新生相关机制。方法:取SPF级SD雄性大鼠,随机分为(1)假手术组:腹腔注射生理盐水,每日1次;(2)MCAO(大脑中动脉梗死)模型组:MCAO+等量生理盐水,腹腔注射,每日1次;(3)PTS治疗组:MCAO+PTS,腹腔注射PTS,50mg/kg/d。缺血再灌注后不同时间点进行神经行为学评分及脑组织TTC染色评价PTS对大鼠脑梗死的治疗作用;采用Micro-PET影像技术评价PTS对脑血流灌注的影响;RT-PCR和Western blot技术检测不同时间点血管生长相关因子基因及蛋白表达的变化;免疫荧光染色法评价脑梗死缺血半暗带区微血管密度的改变;为进一步评价PTS促血管内皮细胞增殖作用,予MCAO大鼠Brdu腹腔注射,行Brdu与CD31免疫荧光共定位,评价新生血管内皮细胞数目;为深入探讨PTS促血管新生机制,采取环靶明(Shh通路特异性阻断剂)联合PTS予MCAO大腹腔注射,Western blot技术评价Shh信号通路关键蛋白的表达及其对下游血管生长因子的影响。结果:(1)PTS对MCAO大鼠的脑保护作用:TTC及Neuron染色结果表明,PTS能够明显减小缺血再灌注后3天、7天、14天时间点的大鼠脑梗死体积,与对照组比较有统计学意义;Longa神经功能评分表明,PTS给药3天开始能够改善MCAO大鼠神经行为学评分,并持续到14天,与对照组比较P0.05,有统计学意义。(2)PTS增加MCAO大鼠缺血侧脑灌注:Micro-PET/CT扫描结果显示,MCAO造模后1天18F-PDG脑摄取明显减少,术后14天复查Micro-PET可见PTS治疗组18F-PDG脑摄取量明显增加,表明脑血流量明显改善,经与对照组比较P0.05,有统计学意义。(3)PTS增加缺血侧皮层血管生长相关因子的mRNA表达水平:RT-PCR结果表明,MCAO术后VEGF的mRNA水平随时间延长,呈持续增高样趋势,并且在第3天时间点PTS治疗组与对照组之间产生差异(P0.05),治疗组优于对照组,并且这种效果可以延续到14天;Ang-1的mRNA水平表达趋势同VEGF;VEGFR2、Tie-2、CD31的mRNA表达水平在MCAO术后持续增高,并且在第7天达到高峰,14天时有所下降,治疗组与对照组从第3天开始产生差异,治疗组升高的幅度大于对照组(P0.05);α-SMA的mRNA水平在MCAO术后的表达的趋势等同于Ang-1,同样在第3天开始,治疗组与对照组间产生差异,治疗组升高的幅度明显大于对照组(P0.05)。(4)PTS上调缺血侧大脑皮层VEGF、Ang-1及VEGR2、Tie-2蛋白表达:western blot免疫印迹结果显示,MCAO术后大鼠大脑皮层VEGF蛋白水平在术后第7天达到高峰,第14天有下降趋势,PTS与对照组比较在第3天,7天,14天时间点有统计学意义(P0.05);Ang-1的蛋白表达水平随时间延长呈逐渐增加趋势,且PTS与对照组比较在第3天,7天,14天时间点有统计学意义(P0.05);在术后7天时间点,PTS治疗组能够明显上调VEGFR2、Tie-2蛋白表达水平,经统计学处理优于对照组(P0.05)。(5)PTS增加缺血侧皮层微血管密度:研究结果表明缺血缺氧诱导病灶周围大量的血管新生,而且微血管密度在脑梗死后7天达到高峰,14天时有所下降,PTS治疗组在第7天和14天时间点CD31/α-SMA阳性的微血管密度明显高于对照组(P0.05)。(6)PTS增加缺血侧皮层血管内皮细胞增殖:CD31与Brdu免疫荧光共染显示MCAO术后存在血管内皮细胞增殖,PTS治疗组CD31与Brdu共定位细胞明显多于对照组,两组比较P0.05,有统计学意义。(7)PTS激活Shh信号通路,上调血管生长因子表达:术后7天westernblot检测Shh通路主要信号蛋白(Shh、Smo、Ptch-1)表达,结果显示,术后PTS能够明显上调Shh、Smo、Ptch-1表达,环靶明靶向阻断Shh信号后明显减少PTS诱导的VEG及Ang-1表达,表明PTS可能主要通过激活Shh信号通路促进脑梗死后血管新生。结论:研究结果表明,脑梗死后PTS能够通过激活Shh信号通路,上调缺血侧脑组织VEGF/VEGFR2和Ang-1/Tie-2系统的表达,增加脑微血管密度,改善缺血区脑灌注,进而减小脑梗死体积,改善神经行为学评分。该实验结果为PTS的临床推广应用提供更多的理论依据,为中医药治疗脑梗死的研究提供新的思路和方法。
[Abstract]:BACKGROUND: Stroke is a common nervous system disease with high morbidity, mortality and disability, of which about 87% is ischemic stroke. Early recovery of cerebral perfusion in the ischemic region and rescue of dying neurons in the ischemic penumbra are the focus and goal of the treatment of cerebral infarction. Panaxatriol Saponins (PTS) is an effective component extracted from Panax notoginseng. Panaxatriol R1, ginsenoside Rg1 and ginsenoside Re are the main components of PTS. Previous studies have confirmed that PTS has protective effect on cerebral infarction rats, but the specific mechanism is unclear. Studies have shown that PTS can promote the proliferation of human umbilical vein endothelial cells, but there is no report on the role of PTS in promoting angiogenesis in ischemic brain injury. (3) To explore the mechanism of PTS promoting angiogenesis after cerebral infarction. Methods: SPF grade SD male rats were randomly divided into (1) sham operation group: intraperitoneal injection of normal saline once a day; (2) MCAO (middle cerebral artery infarction) model group: MCAO + normal saline, intraperitoneal injection once a day; (3) PTS treatment group: MCAO + PTS, intraperitoneal injection of PTS, 50mg / kg / d. Neurobehavioral score and TTC staining were performed at different time points after perfusion to evaluate the therapeutic effect of PTS on cerebral infarction in rats; Micro-PET imaging was used to evaluate the effect of PTS on cerebral blood flow perfusion; RT-PCR and Western blot were used to detect the gene and protein expression of angiogenesis-related factors at different time points; immunofluorescence staining was used to evaluate the effect of PTS on cerebral infarction in rats. To further evaluate the effect of PTS on proliferation of vascular endothelial cells, Brdu was injected intraperitoneally into MCAO rats and co-localized with CD31 immunofluorescence to evaluate the number of neovascular endothelial cells. Results: (1) Protective effect of PTS on the brain of MCAO rats: TTC and Neuron staining showed that PTS could significantly reduce cerebral infarction at 3, 7 and 14 days after reperfusion. Volume, compared with the control group had statistical significance; Longa neurological function score showed that PTS administration could improve the neurobehavioral score of MCAO rats from 3 days to 14 days, and lasted for 14 days, compared with the control group, P 0.05, with statistical significance. (2) PTS increased ischemic cerebral perfusion of MCAO rats: Micro-PET/CT scan showed that 1 day after MCAO modeling 18F-PDG brain. Micro PET showed that the brain uptake of 18F-PDG in PTS treatment group was significantly increased, indicating that cerebral blood flow was significantly improved, compared with the control group, P 0.05, with statistical significance. (3) PTS increased the expression of angiogenesis-related factors mRNA in ischemic cortex: RT-PCR results showed that the level of VEGF mRNA after MCAO with time The expression of Ang-1 mRNA was similar to that of VEGF, and the expression of VEGF R2, Tie-2, CD31 mRNA increased continuously after MCAO, and reached a peak on the 7th day. The expression of alpha-SMA mRNA was similar to that of Ang-1 after MCAO, and was significantly higher in the treatment group than in the control group (P 0.05). PTS up-regulated the expression of VEGF, Ang-1, VEGR2, Tie-2 protein in ischemic cerebral cortex: Western blot immunoblot showed that the level of VEGF protein in the cerebral cortex of rats after MCAO reached a peak on the 7th day after operation, and decreased on the 14th day. Compared with the control group, the expression of Ang-1 protein was statistically significant on the 3rd, 7th and 14th day (P 0.05). Compared with the control group, PTS increased the expression of VEGF R2 and Tie-2 on the 3rd day, 7th day and 14th day (P 0.05). At the 7th day after operation, PTS treatment group significantly increased the expression of VEGF R2 and Tie-2 protein, which was superior to the control group (P 0.05). (5) PTS increased the microvessel density of ischemic cortex: A study. The results showed that hypoxia-ischemia induced a large number of angiogenesis around the lesion, and the microvessel density reached a peak at 7 days after cerebral infarction, and decreased at 14 days. The CD31/alpha-SMA positive microvessel density in PTS treatment group was significantly higher than that in control group at 7 days and 14 days (P 0.05). (6) PTS increased the proliferation of vascular endothelial cells in ischemic cortex: CD31. The co-localization of CD31 and Brdu in PTS group was significantly higher than that in control group (P 0.05). (7) PTS activated Shh signaling pathway and up-regulated the expression of vascular growth factor. The main signal proteins of Shh signaling pathway (Shh, Smo, Ptch-1) were detected by Western blot 7 days after MCAO. The results showed that PTS could significantly up-regulate the expression of Shh, Smo and Ptch-1 after operation. Cyclotargeting could significantly reduce the expression of VEG and Ang-1 induced by PTS after blocking Shh signal, suggesting that PTS could promote angiogenesis after cerebral infarction mainly by activating Shh signal pathway. The results provide more theoretical basis for the clinical application of PTS and provide new ideas and methods for the study of TCM treatment of cerebral infarction. Method.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.3

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