摘要
目的：本研究在中医络病理论指导下,对老年性痴呆(AD)脑微血管发病机制及其在脑神经元损伤中的作用进行探讨,同时阐明依据络病理论建立的复方制剂-通心络(TXL)对AD脑微血管损伤的治疗优势以及其通过保护脑微血管实现保护脑神经元的作用途径,为本药临床治疗AD提供有力的实验依据。
采用SAMP8小鼠作为研究对象,实验分为SAMR1空白对照组,SAMP8组、TXL(高、中、低)三个剂量组,石杉碱甲(SSJJ)组,共6个组,每组20只小鼠,小鼠给药量均为10ml/kg体重,灌胃给药,每日一次,连续90日,SAMR1组给予相同体积溶剂。实验结束后,观察各组小鼠一般情况、认知记忆功能、脑神经元损伤、脑皮层p淀粉样蛋白(Aβ)的沉积情况。
本研究继续采用SAMP8小鼠作为研究对象,仍以SAMR1为对照,继第一部分实验证明了小鼠脑皮层Ap沉积后,采用免疫组化方法观察各组小鼠脑微血管数目改变、内皮功能损伤情况、管周炎症因子的表达改变。
本研究采用细胞实验,分为空白组：HBMEC用10%FBS的DMEM培养基培养30h；模型组：HBMEC用10%FBS的DMEM培养基培养6h后,加入终浓度为20μmol/L Aβ1-42作用24h诱导细胞损伤；TXL组：HBMEC用10%FBS的DMEM培养基培养并分别加入终浓度为100、200、400ug/ml的TXL预处理6h后加入终浓度为20μmol/LAβ1-42作用24 h诱导细胞损伤。SSJJ组：HBMEC用10% FBS的DMEM培养基培养并加入终浓度为2μmol/L的SSJJ预处理6h后加入终浓度为20μmol/l Aβ1-42作用24h诱导细胞损伤。实验结束,倒置显微镜下观察细胞形态改变,SRB检测细胞的活性,硝酸酶还原法检测各组NO水平、ELISA检测各组细胞上清液中VEGF、IL-1β、IL-6、TNF-a水平；westemblot检测eNOS、NF-κB、HIF-1α、VEGF蛋白表达；RealtimePCR检测VEGFmRNA的表达。
本研究采用原代培养脑神经元的方法,分为正常神经元组(空白组)、正常HBMEC上清液+神经元组(正常组)、Ap损伤HBMEC上清液+神经元组(模型组)、Ap损伤神经元组(Ap损伤组)、TXL干预HBMEC上清液+Ap+神经元组(TXL组),SSJJ干预HBMEC上清液+Ap+神经元组(SSJJ组),共六个实验组,空白组加入100%无血清DMEM培养液,Ap损伤组加入终浓度为10μmol/1Aβ,其余各组均加入50%内皮细胞上清液和50%无血清DMEM培养液,将细胞置于温度37℃、5%C02、饱和湿度的培养箱中培养12h。实验结束后倒置显微镜下观察细胞的形态改变、HE染色观察细胞的形态改变、免疫荧光显微镜下观察细胞坏死和凋亡、MTT检测各组脑神经元的活性、流式细胞仪检测细胞凋亡率,westemblot检测凋亡蛋白酶Caspase-3、凋亡蛋白bax、bcl-2表达,realtimePCR检测baxmRNA、bcl-2mRNA表达。
1.1各组小鼠一般情况改变的结果显示SAMR1小鼠毛色纯白、活泼好动,反应灵敏；SAMP8小鼠背部毛色无光泽,倦怠乏力,精神萎顿,四肢蜷缩,行动迟缓,眯眼抖动、缩肩拱背老化神态。与SAMP8组比较,TXL各剂量组小鼠表征都出现不同程度的改善,且以高剂量组改善尤为显著,SSJJ组小鼠较SAMP8组小鼠有所改善,但较TXL高、中剂量组稍差。
1.2各组小鼠水迷宫实验结果显示潜伏期：在参考记忆检测中,和SAMR1对照组比较,SAMP8组空间参考记忆潜伏期逐渐延长,P<0.01,TXL组潜伏期缩短,与SAMP8组比较,P<0.05或P<0.01,尤以高剂量组为著；SSJJ组潜伏期缩短,与SAMP8组比较没有统计学意义,P>0.05。在空间工作记忆检测中,与SAMR1正常对照组比较,SAMP8组空间工作记忆潜伏期逐渐延长,P<0.05或P<0.01,在第1、2、3天TXL组潜伏期缩短,与SAMP8组比较,P<0.05或P<0.01,在第1、2天SSJJ组潜伏期缩短,与SAMP8组比较,P<0.05。
1.3各组小鼠脑皮层脑神经元损伤的结果显示与SAMR1正常对照组比较,SAMP8组神经元损伤严重,P<0.01,与SAMP8组比较,TXL剂量组神经元损伤减轻,且呈明显量效关系,P<0.05或P<0.01,SSJJ组神经元损伤减轻,与SAMP8组比较,P<0.01。
1.4各组小鼠脑皮层Aβ沉积的结果显示刚果红着色的淀粉样沉积呈砖红色或樱桃色,弱阳性则成紫红色。与SAMR1正常对照组比较,SAMP8组Aβ的沉积较多,P<0.05,TXL高、低剂量组和SSJJ组皮层细胞着色多为紫红色,较SAMR1组皮层染色较重,与SAMP8组比较,Aβ的沉积显著减少,P<0.01,两药比较无统计学意义,P>0.05。
2.1各组小鼠血浆中NO的改变结果显示与SAMR1正常组比较,SAMP8组血浆中NO含量明显降低,P<0.01,TXL各剂量组NO含量升高,与SAMP8组比较,P<0.05或P<0.01,且呈明显的量效关系,SSJJ组改善不明显,与SAMP8组比较,P>0.05。
2.2各组小鼠脑皮层微血管数目的改变结果显示与SAMR1正常组比较,SAMP8组脑皮层微血管数目明显减少,P<0.01,TXL各剂量组脑皮层中血管数均明显增多,与SAMP8组比较,P<0.05或P<0.01,且呈明显的量效关系,SSJJ组脑微血管数目改善不明显,与SAMP8组比较,P>0.05,与TXL组比较,P<0.05或P<0.01。
2.3各组小鼠脑皮层ET-1表达的改变结果显示与SAMR1正常组比较,SAMP8组脑皮层ET-1表达明显增强,P<0.01,TXL高、中剂量组表达减弱,与SAMP8组比较,P<0.05或P<0.01,呈明显量效关系,SSJJ组脑皮层ET-1表达改善不明显,与SAMP8组比较,P>0.05,与TXL高剂量组比较,P<0.05。
2.4各组小鼠脑皮层IL-6表达的改变结果显示与SAMR1正常组比较,SAMP8组脑皮层微血管周围IL-6表达增强,P<0.01,TXL各剂量组脑皮层微血管周围IL-6表达减弱,与SAMP8组比较,P<0.01或P<0.05,SSJJ组IL-6表达无明显改变,与SAMP8组比较,P>0.05。
2.5各组小鼠脑皮层IL-1β表达的改变结果显示与SAMR1正常组比较,SAMP8组脑皮层中微血管周围IL-1β表达增强,P<0.01,与SAMP8组比较,TXL脑皮层中微血管周围IL-1β表达明显减弱,P<0.01,SSJJ组微血管周围IL-1β表达减弱,P<0.05,与TXL组比较,P<0.05。
2.6各组小鼠脑皮层TNF-α表达的改变结果显示与SAMR1正常组比较,SAMP8组脑皮层中微血管周围TNF-α表达明显增强,P<0.01,与SAMP8组比较,TXL高剂量组皮层中微血管周围TNF-α表达明显减弱,P<0.05,SSJJ组无明显改变,P>0.05。
3.1各组细胞形态改变结果显示镜下可见,空白组HBMEC胞体呈梭形,典型的“铺路石”形状,细胞数量多,而模型组细胞数量显著减少,大量细胞受损,呈圆形,细胞之间连接断裂,还可见细胞死亡漂浮于培养液中。TXL组细胞数量与模型组比较数量较多,细胞贴壁良好,形态正常,少见损伤和死亡细胞,SSJJ组细胞形态较模型组改善不明显。
3.2各组HBMEC活性改变结果显示与空白组比较,模型组HBMEC细胞活性下降,P<0.05,TXL组和SSJJ组HBMEC活性较模型组有所改善,P<0.05或P<0.01,且TXL组优于SSJJ组,P<0.05。
3.3各组HBMEC中NO的改变结果显示与空白组比较,模型组HBMEC中NO水平下降,P<0.01,而TXL组NO水平较模型组有所改善,P<0.05或P<0.01,SSJJ组NO水平改善不明显,与模型组比较,P>0.05。
3.4各组HBMEC中eNOS的改变结果显示与空白组比较,模型组HBMEC中eNOS表达减弱,P<0.01,而TXL组eNOS水平较模型组有所改善,P<0.05或P<0.01,SSJJ组eNOS表达改善不明显,与模型组比较,P>0.05,与TXL组比较,P<0.05。
3.5各组]HBME上清液中VEGF水平改变结果显示与空白组比较,模型组VEGF水平降低,P<0.01,而TXL组VEGF表达较模型组有所改善,P<0.05或P<0.01,SSJJ甲组VEGF水平升高,与模型组比较,P<0.05。
3.6各组HBMEC中VEGF表达改变的结果显示与空白组比较,模型组VEGF表达减弱,P<0.01,而TXL组VEGF表达较模型组有所改善,P<0.05或P<0.01。SSJJ甲组VEGF改善不明显,与模型组比较,P>0.05。TXL组优于SSJJ甲组,P<0.05。
3.7各组HBMEC中VEGFmRNA表达结果显示与空白组比较,模型组VEGFmRNA表达减弱,P<0.01,而TXL组VEGFmRNA表达增强,与模型组比较,P<0.05或P<0.01,SSJJ组VEGFmRNA表达改善不明显,与模型组比较,P>0.05。
3.8各组HBMEC细胞中HIF-1α改变结果显示与空白组比较,模型组HIF-1α蛋白表达显著,P<0.01,TXL高剂量组可增强HBMEC中HIF-1α蛋白的表达,P<0.05,SSJJ组HIF-1α蛋白的表达改变不明显,与模型组比较,P>0.05。两药比较,TXL优于SSJJ组,P<0.05。
3.9各组HBMEC中IL-1β、IL-6、TNF-α的表达结果显示与空白组比较,模型组IL-1p、IL-6、TNF-α均升高,P<0.05或P<0.01,TXL各剂量组IL-1β、IL-6、TNF-α表达均降低,P<0.05或P<0.01,SSJJ组IL-1β较模型组下降,P<0.05,IL-6、TNF-α表达改善不明显,与模型组比较,P>0.05。
3.10各组HBMEC中NF-κB的表达结果显示与空白组比较,模型组NF-κB蛋白表达增强,P<0.01,TXL三个剂量组NF-κB蛋白表达降低,与模型组比较,P<0.01,SSJJ组NF-κB蛋白表达减弱不明显,与模型组比较,P>0.05。
4.1神经元培养形态变化结果显示本实验选取胎鼠大脑皮层部分进行原代培养,种植于孔板中的皮层神经元细胞呈圆形,透亮,分布均匀,细胞膜光滑,折光好,光晕明显。24h后细胞完全贴壁,部分细胞可见短而小的突起。第2-3天,带有突起的细胞明显增多,突起有变长的趋势,胞体较大,多呈梭形、球形和三角形,细胞具有立体感。加入阿糖胞苷作用后,扁平多边形的神经胶质细胞几乎消除,神经元细胞表现出特征形态。第4-5天,神经元细胞迅速分化,细胞形态非常典型,细胞立体感很强,胞体饱满,胞浆丰富,核仁清晰,双极和多极神经元细胞随处可见,突起多而粗,末端出现分支,神经突起延伸形成网络结构。
4.2.1倒置显微镜下观察：空白组和正常组皮层神经元胞体呈梭形或三角形,聚堆生长,相互交织成网状,折光性好。Ap损伤组和模型组细胞损伤严重,细胞形态改变,细胞突起变短或变少,折光性差,且有坏死细胞漂浮,且以模型组表现最为明显,TXL组和SSJJ组较模型组均有不同程度改善而以TXL组为著。
4.2.2 HE染色显示：各组神经元形态的改变结果中空白组、正常组神经元细胞胞体大而透亮,胞核和核仁清晰可见,神经元突起长、多,末端分支形成典型的神经网络；模型组和Ap损伤组神经元细胞数量减少,胞体模糊,神经元突起明显稀疏,变短变少,甚至消失,且以模型组损伤为重。TXL和SSJJ组细胞状态与模型组相比均有好转,且以TXL组改善明显。
4.3各组神经元细胞坏死和凋亡改变结果显示：PI染色下,空白组和正常组皮层神经元呈少个红染细胞,表明坏死细胞较少,而Ap损伤组和模型组红染细胞较多,坏死细胞数量增加,且以模型组的细胞改变明显,TXL和SSJJ组均有改善,以TXL组为著。Hoechst 33342染色下空白组和正常组皮层神经元呈低蓝染色胞核和胞膜完整,少有凋亡细胞,Aβ1-42损伤组和模型组细胞损伤严重,细胞呈亮蓝染色,有些细胞呈典型的凋亡形态,在细胞周边形成凋亡小体,且以模型组的改变明显,TXL和SSJJ组均有不同程度改善,以TXL组为著。
4.4各组神经元活性的改变结果显示：空白组与正常组比较无统计学差异,P>0.05,与两组相比,Ap损伤组及模型组神经元活性下降,且模型组最为显著,P<0.05,TXL组神经元的活性较模型组升高,P<0.05。SSJJ组的神经元活性与模型组比较,没有统计学意义,P>0.05。
4.5各组神经元凋亡率的改变结果显示：空白组与正常组比较无统计学差异,P>0.05,与两组相比,Ap损伤组及模型组神经元细胞凋亡率升高,P<0.01,模型组升高显著,P<0.05,TXL与SSJJ组的凋亡率较模型组均出现下降,与模型组比较,P<0.05,两组之间比较,P<0.05。
4.6各组神经元凋亡相关蛋白酶caspase-3的改变结果显示：空白组与正常组比较无统计学差异,P>0.05,与两组相比,Ap损伤组及模型组神经元细胞凋亡有关蛋白酶caspase-3表达增强,且模型组最为显著,P<0.05,TXL组caspase-3表达减弱,与模型组比较,P<0.05,SSJJ组caspase-3表达减弱不明显,与模型组比较,P>0.05。
4.7各组神经元凋亡蛋白bax、bcl-2的改变结果显示：空白组与正常组比较无统计学差异,P>0.05,与两组相比,Aβ损伤组及模型组神经元细胞凋亡蛋白bax表达增强,bcl-2表达减弱,且模型组最为明显,P<0.05,TXL与SSJJ组的bcl-2表达较模型组改善,P<0.05。
4.8各组神经元凋亡基因bax、bcl-2mRNA的改变结果显示：空白组与正常组比较无统计学差异,P>0.05,与两组相比,Aβ损伤组及模型组神经元细胞中baxmRNA表达增强,bcl-2mRNA表达减弱,且模型组最为明显,P<0.05或p<0.01,与模型组比较,TXL组baxmRNA表达降低,P<0.05,bcl-2mRNA表达增强,p<0.01,而SSJJ组baxmRNA降低,P<0.05,bcl-2mRNA改善不明显,P>0.05。
1.首先运用中医络病理论探讨AD病机及治疗,脑之络脉分为运行经气为主的气络与运行血液为主的脉络,共同成为维持脑神活动的物质基础,基于气血相关的络病理论特色,AD虽病在气络,但亦不能忽视脉络结构与功能异常对其影响,基于精、气、神之间相互转化关系,指出元气亏虚、气络损伤是AD重要病机,气虚运血无力,脉络瘀阻,脉络末端供血供气、津血互换、营养代谢失常,亦可加重气络损伤,引起脑神失用,成为AD发病的病理基础,益气活血通络是AD的有效治法。
2.基于中医脉络与西医中、小血管及微循环解剖形态相关性,吸取西医学关于AD微血管发病机制的最新进展,突破以往把AD单纯理解为神经变性性疾病的观念,从微血管损伤探讨AD的发病机制,结果显示快速老化型模型小鼠脑部微血管出现结构与功能损伤：脑皮层ET-1表达升高、NO分泌降低,微血管数目明显减少,管周炎性因子显著增加；离体细胞研究结果显示Aβ不仅可以引起HBMEC结构与功能损伤,Aβ与内皮细胞共孵育的上清液还可明显诱导脑神经元调亡,这不仅为西医学AD微血管发病机制假说提供了实验依据,也丰富了气血相关脉络学说的科学内涵。
3.本研究结果显示,TXL可明显改善快速老化型小鼠记忆与认知功能,显著保护其脑微血管结构与功能；通过减轻Aβ所致脑微血管内皮细胞损伤有效减少脑神经元凋亡,初步揭示了TXL治疗AD微血管保护机制,为其在临床治疗AD提供了实验依据,也佐证了络病理论在AD中的指导价值。
Objective:Under the guidance of collateral disease theory,we study medicine senile dementia (AD) pathogenesis of cerebral microvascular injury in Neuronal damage, which will provide reliable experimental data to clarify the compound-Cardio Contact (TXL) basised of collateral disease theory on brain microvascular injury and its protection of Neuron by the way of protecting cerebral microvascular.
1.The cognitive and memory dysfunction of AD model and the intervention of TXL
In this study, mice were divided into six groups, SAMR1 control group, SAMP8 group, TXL three dose groups (high, medium, low), Huperzine A (SSJJ) group, Each group included 20 mice which were Fedded of 10m1/kg basis of body weight, once a day for 90 days, the same volume of solvent of SAMR1. in the End of the experiment, mice were characterized by changes observed in each group, cognitive and memory dysfunction,neuronal loss, cortical amyloid (Aβ) deposition conditions.
2.The cerebral microvascular injury of AD model and and the intervention of TXL
In this study, SAMP8 mice continue to be used as the research object, SAMR1 still as control, following the first part of the experiment proved that Aβdeposition in brain cortex after immunohistochemical method to observe the changes in the number of mice brain microvascular endothelial function damage, the expression in perivascular inflammatory changes.
3. The injury of brain microvascular endothelial cells (HBMEC) induced by Aβ, and the intervention of TXL
In this study, cell experiment, divided into six groups,control group: HBMEC with 10% FBS in DMEM medium for 30h after the test; model group: HBMEC with 10% FBS in DMEM culture medium after 6h, adding a final concentration of 20μmol/L the role of Aβ1-42 induced cell damage 24 h; TXL Group:HBMEC DMEM with 10% FBS in the culture medium and the final concentration was added 100,200,400 ug/ml of the TXL preconditioning 6h, adding a final concentration of 20μmol/L Aβ-42,24h, induced cell damage. SSJJ Group:HBMEC with 10% FBS in DMEM culture.And add a final concentration of 2μmol/L of SSJJ pretreatment 6h, adding a final concentration of 20μmol/l Aβ1-42 effect 24 h induced cell damage. In the End of the experiment, cells were observed under the inverted microscope, morphological changes, SRB detection of cell activity, nitric acid were detected by enzyme NO reduction levels, various groups in the supernatant VEGF, IL-lβ, IL-6, TNF-a expression by ELISA detection; eNOS, HIF-la, VEGF protein expression by westernblot detect; the expression of VEGFmRNA by RealtimePCR detect.
4. The effect of cerebral microvascular endothelial cells injuried by Aβto neurons and the intervention of TXL
In this study, the method of primary cultured neurons were used, neurons were divided into normal group (control group), normal HBMEC supernatants+neurons group (normal group), Aβinjury HBMEC supernatants+neurons (model group), Aβgroup of damaged neurons (Aβinjury group), TXL intervention HBMEC supernatants+Aβ+neurons group (TXL group), SSJJ intervention HBMEC supernatants+Aβ+neurons group (SSJJ group), a total of six experimental group, control group adding 100% serum-free DMEM with nutrient solution, and the remaining 50% of each group were on 50% of endothelial cells in serum-free supernatant and DMEM medium, cells were placed in temperature of 37℃,5%CO2, saturated humidity The incubator 12h. After the end of the experiment the cells were observed under inverted microscope morphological changes, HE staining cells morphological changes, immune cell necrosis was observed under fluorescence microscope, the activity of neurons in each group by MTT test, the apoptosis rate by flow cytometry, apoptotic protease Caspase-3, apoptotic protein bax, bcl-2 expression by Western Blot detection, the bax mRNA, bcl-2 mRNA expression realtime PCR detection.
1.1 The characterization of SAMR1 mice showed changes in white coat color, lively, responsive; SAMP8 mouse pelage is dull, lassitude, mental Weidun, limbs curled up, move slowly, squinting jitter, reduced shoulder Arch aging demeanor. And SAMP8 compared, TXL each dose group are characterized by different degrees of improvement, and high-dose group improved significantly, SSJJ mice compared with SAMP8 mice had improved, compared with TXL, middle dose group little worse.
1.2 The water maze latency of mice experiments showing:in the reference memory detection, and SAMR1 control group, SAMP8 group gradually extend the spatial reference memory latency, P<0.01, TXL group latency, and SAMP8 group, P<0.05 or P<0.01, especially with high-dose group; SSJJ group latency, and SAMP8 group was not statistically significant, P>0.05. Detection in spatial working memory, and SAMR1 normal control group, SAMP8 group of spatial working memory latency gradually extended, P<0.05 or P<0.01, TXL group in 1,2,3 days latency, and SAMP8 control group, P<0.05 or P<0.01, set in 1,2 days SSJJ latency, and SAMP8 control group, P<0.05.
1.3 The cerebral cortex of mice neuronal damage showing that compared with SAMR1 normal control group, the neurons serious of SAMP8 group, P<0.01, TXL dose of neurons to reduce, and SAMP8 group, P<0.05 or P<0.01, SSJJ group of neurons to reduce, and SAMP8 group, P<0.01, two-drug treatment showed no statistical significance, P>0.05.
1.4 The cortex of mice showing that Aβdeposition in Congo red amyloid deposition was painted brick red or cherry, purple weak positive is into. And SAMR1 normal control group, SAMP8 group of more Aβdeposition, P<0.05, TXL of three dose groups and SSJJ cortical cells mostly colored purple, heavier than the SAMR1 group of cortical staining, and SAMP8 group, the deposition Aβdecreased significantly, P<0.01, no significant difference between the two drugs, P>0.05.
2.1 The changes of NO in plasma among groups showed that:compared with SAMR1 mice, the levels of NO in SAMP8 group plasma significantly decreased, P<0.01, the levels of NO in each dose TXL group was significantly higher, and SAMP8 group, P<0.05 or P<0.01. TXL higher dose group which is most obvious, SSJJ group had no obvious improvement, compared with SAMP8 group, P>0.05.
2.2 The number of microvascular changes in mice brain cortex showed that:compared with SAMR1 control group, the number of cortical microvessels reduced significantly in SAMP8 group, P<0.01, each TXL dose group were increased significantly, compared with SAMP8 group, P<0.05 or P<0.01, SSJJ number of obvious improvement in brain microvessels, compared with SAMP8 group, P> 0.05.
2.3 The expression of ET-1 in mice cerebral cortex showed that: compared with SAMR1 group, the expression of ET-1 in SAMP8 group was increased significantly, P<0.01. Each TXL dose group was reduced, compared with the SAMP8 group, P<0.05 or P<0.01, the expression of ET-1 in SSJJ group was not improved obviously, compared with SAMP8 group, P>0.05.
2.4 The expression of IL-6 in mice cerebral cortex showed that: compared with SAMR1 group, the expression of IL-6 in SAMP8 around microvessels was increased, P<0.01, TXL dose groups decreased, compared with SAMP8, P<0.01 or P<0.05, the expression of IL-6 in SSJJ group was not changed significantly, compared with SAMP8 group, P>0.05.
2.5 The expression changes of IL-1βin brain cortex showed that: compared with SAMR1 control group, that in SAMP8 group increased, P<0.01, the expression of IL-lβin TXL group was significant lower, compared with SAMP8 group, P<0.01, the IL-1βaround capillaries decreased, compared with SAMP8 group, P<0.05.
2.6 The expression of TNF-a in mice cerebral cortex showed that: compared with SAMR1 control group, the TNF-a around the capillaries in SAMP8 group brain was increased significantly, P<0.01, the microvascular expression in TXL dose group was decreased significantly, compared with the model group P<0.05, SSJJ group of capillaries around the TNF-a expression was not significantly changed, compared with SAMP8 group, P>0.05.
3.1 The morphological changes in each group showed visible microscope, normal HBMEC fusiform cell bodies, the typical "cobblestone" shape, cell quantity, while the number of cells in model group decreased significantly, a large number of cell damage, but also cell death can be seen floating in the culture medium. TXL cells compared with the model number of larger number of good adherent cells, morphologically normal, a rare injury and death of cells, compared with model group, SSJJ cells form was little improvement.
3.2 The change of cells in each group showed that:compared with the control group, model group HBMEC cytotoxicity, P<0.05, the activity TXL group and SSJJ group HBMEC improved than the model group, P<0.05 or P<0.01, compared with the TXL and SSJJ group, P<0.05.
3.3 The change of NO in HBMEC in each group showed that:compared with control group, model group, NO levels in HBMEC decreased, P<0.01, while the TXL Group NO improvement compared with model group, P<0.05 or P<0.01, SSJJ Group NO levels had no obvious improvement, compared with model group, P>0.05.
3.4 The changes of eNOS in the HBMEC group showed that:compared with the control group, the expression of eNOS in model group HBMEC decreased, P<0.01, compared with model group, the eNOS in TXL group improved, P<0.05 or P<0.01, compared with the model group, the eNOS level of in SSJJ group was not obvious, P>0.05, compared with the TXL group, P<0.05.
3.5 The expression of VEGF change in cells supernatant showed that: compared with the control group, VEGF levels decreased in model group, P<0.01, compared with model group, VEGF expression TXL group improved P<0.05 or P<0.01. compared with model group, the VEGF level SSJJ Group increased, P<0.05.
3.6 The expression of VEGF change in HBMEC showed that:compared with the control group, the VEGF expression in model group was decreased, P<0.01, compared with model group, the VEGF expression in TXL group improved, P<0.01 the VEGF expression in SSJJ Group improved, compared with the model group, P<0.05.
3.7 The expression of VEGFMRNA in each HBMEC group showed that: compared with the control group, the VEGFMRNA expression model group reduced, P<0.01, compared with model the model group, the VEGFMRNA expression in TXL group, P<0.05 or P<0.01. SSJJ group had no obvious improvement, P>0.05.
3.8 The changes of HIF-1a in HBMEC group cells showed that: compared with control group, the HIF-1a protein expression in model group was increased, P<0.01, compared with the model group, the expression of TXL high dose group was enhanced, P<0.05, the expression changes of HIF-la protein in SSJJ group was not obvious, P>0.05, compared with two drugs, TXL group was better than SSJJ group, P<0.05.
3.9 The expression of IL-1β, IL-6, TNF-a in HBMEC group showed that: compared with the control group, the IL-1β, IL-6, TNF-a in model group were higher, P<0.05 or P<0.01, the IL-1β, IL-6, TNF-a expression in TXL group was reduced, P<0.05 or P<0.01, the IL-1βin SSJJ group decreased, P<0.05, the expression of IL-6, TNF-a was no obvious improvement, compared with model group, P>0.05.
3.10 The results of the expression of NF-κB signaling pathway in HBMEC:compared with the control group, the expression of NF-κB protein in model group increased, P<0.01, the expression in TXL three doses was reduced, Compared with model group, P<0.01, the NF-kB protein expression of SSJJ group decreased was not obvious, compared with the model group, P>0.05.
4.1 Cultured neurons showed morphological changes in cerebral cortex of the experimental portion of selected primary culture, grown on plates in the cortical neurons were round, transparent, uniform, smooth membrane, refractive good, clear halo. After 24h cells fully adherent, some short and small cells can be seen protruding.2-3 days, with significantly increased cell processes, the processes have become the trend of long, large cell body bes spindle, spherical and triangular cells with three-dimensional. The role of adding cytosine arabinoside, the flat polygons almost eliminate glial cells, neuronal cells showed morphological characteristics.4-5 days, the rapid differentiation of neuronal cells, cell shape is very typical cell dimension a strong, full cell body, cytoplasm-rich, clear nucleolus, bipolar and multipolar neurons everywhere, protruding over the rough, at end of branches, the formation of neurites extended network.
4.2 The showing of morphological changes of neurons in each group
4.2.1 Under inverted microscope:the control group and normal group of spindle cell bodies of cortical neurons, or triangle, poly heap growth, interwoven into the mesh, good refraction. Aβinjury group and model group cell injury seriously, cell morphology, cell processes became shorter, or fewer, refraction difference, and there are dead cells floating, and Model group, the most significant performance, compared with the model group, TXL group and SSJJ group improved, and TXL group showed the most significant.
4.2.2 HE staining:the group of neurons showed morphological changes in the control group, normal control group, neuronal cell bodies large and bright, clearly visible nucleus and nucleolus, neurite length, and more, forming a typical nerve terminal branch network; model group and Aβinjury group reduction in the number of neurons, cell bodies fuzzy, sparse neuronal processes significantly, fewer shorter, or even disappear, and Model group, the most serious injury. compared with the model, TXL and SSJJ group was improved, and the TXL group improved significantly.
4.3 In each group cell, necrosis and apoptosis of neurons to change PI staining showed the blank group and control group showed less cortical neurons in the red-stained cells, indicating that very few necrotic cells, while Aβinjury group and model group of Red dye cells more, the number of dead cells increased and the model group of cells changed significantly, TXL and SSJJ group had improved to the TXL group. Hoechst33342 staining the blank group and control group showed low cortical neurons stained blue nucleus and membrane integrity, and few apoptotic cells, Aβgroup and model group injury serious cell damage, cells were stained bright blue, some cells were typical morphology of apoptosis, in cells surrounding the formation of apoptotic bodies. And changes to the model group significantly, TXL and SSJJ group were improved, especially TXL group.
4.4 The group of neuronal activity changes in results with the control group and normal group, Aβinjury group and model group neuron activity decreased, especially the model group, P<0.05, TXL group of neurons in the energy than the model group, P<0.05. SSJJ group activity of neurons compared with model group, but not statistically significant, P>0.05.
4.5 Apoptosis rate of neurons in each group showed a change with the control group and normal group, Aβinjury group and model group increased rate of neuronal apoptosis, P<0.01, between two groups, the model group increased significantly, P<0.05, compared with model group, the apoptotic rate of TXL and SSJJ group were decline, especially the model group, P<0.05, between two groups, P<0.05.
4.6 The group of neuronal apoptosis protease caspase-3 showed no change with the control group and normal group, Aβinjury group and model group, neuronal apoptosis protease caspase-3 expression, especially the model group, P<0.05, the caspase-3 expression of TXL group was decreased, compared with the model group, P<0.05, SSJJ group reducing was not obvious, compared with the model group, P>0.05.
4.7 The group of neuronal apoptosis gene bax, bcl-2 showed changes in Aβinjury group and model group, neuronal expression of apoptosis protein bax, bcl-2 expression was decreased, and the model group was most significant, P<0.05, compared with the model group,TXL and SSJJ group was improved, P<0.05,.
4.8 The group of neuronal apoptosis gene bax, bcl-2mRNA showed changes in Aβinjury group and model group, the expression of neuronal cells in baxmRNA increased, bcl-2mRNA expression decreased, and the model group was most obvious, P<0.05 or p<0.01, compared with the model group, TXL Group baxmRNA lower, P<0.05, bcl-2mRNA increase, p<0.01. The SSJJ group baxmRNA not changed significantly, P<0.05, bcl-2mRNA expression was not obvious improvement, P>0.05.
1. Firstly, using the theory of Traditional Chinese Medicine collateral disease to investigate pathogenesis and treatment of AD. Cerebral collaterals divide into gas collaterals that transport gas and blood collaterals that transport blood, which is the material basis to maintain the brain activity of God, based on blood-related Collateral Theory characteristics. Although AD is the disease on the gas collaterals, but the abnormal structure and function of blood-related Collateral can not be ignored. Based on the transforming relationship among god, gas, precision air, we find that deficiency of vital energy, the injury of gas collaterals is an important pathogenesis of AD,because that the weakness of gas and blood, Vein stasis, the disorders about the blood supply, Tianjin blood exchange, nutrition, metabolic, in the end of vein can also increase the air contact injury, which will cause loss of brain with god, as the pathological basis of AD pathogenesis, yiqihuoxuetongluo is an effective therapies to AD.
2. Based on the Correlation between Traditional Chinese Medicine context and Western medicine about the small blood vessels and microcirculation anatomy relevant, Learning Western medicine on the pathogenesis of AD microvessels latest break through in the last year on AD simply understood as the concept of neurodegenerative diseases, by the microvascular injury mechanism, we explore the pathogenesis of AD. The results show that brain structure and function of microvascular damage occurs on rapid aging model mice:cortical ET-1 increased expression, NO secretion was decreased, the number of capillaries reduced significantly, perivascular inflammatory factors increased significantly; somatic cell studies Show that Aβcan not only damage the structure and function of HBMEC, but also Aβincubated with the supernatant of endothelial cells also induced significant neuronal apoptosis, which can not only provide an experimental basis for Western medicine microvascular hypothesis of AD pathogenesis, but also Enrich the scientific content of the Context theory of gas-blood related.
3. The results show that TXL could improve the rapid aging of memory and cognitive function in type mice and protect the brain microvascular structure and function significantly; by reducing the microvascular endothelial cell injury Aβ-induced to reduce brain neuronal apoptosis, which reveale Initially that the clinical treatment of TXL is by the protection of AD microvascular mechanisms, which will provide the experimental basis for Clinical study of AD, but also prove the guiding value to AD of the collateral disease theory.
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