海风藤对阿尔茨海默病的治疗作用及其作用机理的实验研究
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摘要
研究背景阿尔茨海默病(Alzheimer disease,AD)亦称老年性痴呆,是1907年由德国精神病学家Alois Alzheimer描述并命名的,它是以进行性认知和记忆功能下降为特征的大脑退行性变性疾病。随着社会的老龄化,其发病率呈逐年上升趋势,已成为一种严重危害人类健康的疾病。尽管在过去的二十年里,对AD致病基因的研究取得了明显的进展,但目前,AD的病因仍不十分清楚。AD的治疗也只停留在对症治疗的阶段。因此,加强对AD发病机制的研究,寻找积极有效的治疗方法,具有重要的经济和社会意义。
AD的病理变化为广泛的大脑萎缩。在受累脑区,有两个明显的病理学特征:老年斑(SP)和神经原纤维缠结(NFT)。老年斑由神经细胞外的β淀粉样蛋白(Aβ)聚集而成,而β淀粉样蛋白则是由β淀粉样前体蛋白(APP)异常剪切所产生。淀粉样蛋白级联假说认为,神经细胞外聚集的Aβ激活了炎症和氧化损伤。病理学也证实,老年斑周围常围绕着激活的小胶质细胞和星形胶质细胞。Aβ还使细胞骨架蛋白Tau蛋白磷酸化,进而缠绕形成了神经原纤维缠结。老年斑和神经原纤维缠结共同作用,促进了神经退行性变的发生。
中药海风藤是胡椒科植物风藤(Piper kadsura(Choisy)Ohwi)的干燥藤茎。具祛风湿、通经络、理气之功效,主治风寒湿痹,关节疼痛,筋脉拘挛,跌打损伤,哮喘,久咳等症。韩恩吉教授等应用水母发光蛋白的方法,发现海风藤水提物能抑制Aβ25-35诱导的神经细胞胞浆钙离子升高并随海风藤浓度增加而增强,提示海风藤对神经细胞有保护作用。韩恩吉教授等还应用细胞培养、Northernblot和逆转录-聚合酶链反应(RT-PCR)的方法观察了海风藤水提物对SK-N-SH细胞中APP mRNA的影响,证明海风藤可选择性抑制APP mRNA表达,并且随浓度的增加而增强,随时间的延长而增强。根据淀粉样蛋白级联假说,细胞外聚集的Aβ激活了炎症和氧化损伤,从而促成了神经退行性变的发生。所以我们认为,海风藤对神经细胞是有保护作用的。那么,本研究所关心的问题是,这种保护和治疗作用是否会在痴呆模型动物体内发生,海风藤抑制APP基因表达的有效活性成分是什么。
研究目的为了观察海风藤是否对痴呆模型大鼠有神经保护作用,我们首先建立了侧脑室注射Aβ大鼠痴呆模型,在海风藤水提物灌胃后,观察痴呆模型大鼠行为学和组织学的改变,大鼠脑神经元内Aβ、炎症因子TNF-α和IL-6以及突触素的表达,脑内NO、NOS的含量。然后,我们用生药学的方法,将海风藤进行分离,再将各分离组分作用于SK-N-SH细胞。通过观察各分离组分作用后,SK-N-SH细胞中APP和Aβ的表达,来确定海风藤中抑制APP和Aβ表达的有效活性成分。
方法与结果
1.痴呆大鼠模型的建立及海风藤水提物对痴呆模型大鼠行为学和组织学的影响
1.1侧脑室注射Aβ建立大鼠痴呆模型
选择通过筛选训练的大鼠60只,分为正常对照组、模型组、假手术组、阳性对照组(布洛芬)、海风藤高剂量、低剂量组,每组各10只大鼠。
正常对照组不给予处置;其余五组大鼠用水合氯醛350mg/kg bw腹腔注射麻醉,头部固定于立体定位仪。模型组、阳性对照组、海风藤高剂量、低剂量组均在定位仪定位下向侧脑室内注入10μg Aβ(25-35);假手术组钻开颅骨后,侧脑室内注入等量生理盐水。各组大鼠手术后7天开始灌胃给药,阳性对照组给予布洛芬20mg/kg bw,海风藤高剂量组给予140mg/100g bw,低剂量组给予46.1mg/100g bw。正常对照组、模型组及假手术组则给予等量蒸馏水灌胃。
1.2学习、记忆能力的测定
各组大鼠采用Morris水迷宫进行学习、记忆功能的测定,记录逃避潜伏期,逃避潜伏期越短,大鼠的学习、记忆能力越强。同时,测定大鼠跨越平台次数,跨越平台次数越多说明大鼠的学习、记忆能力越强。
Morris水迷宫检测结果:1)正常对照组、假手术组与模型组相比,逃避潜伏期明显减少,跨越平台次数明显增多,差异有统计学意义(P＜0.05),而正常对照组与假手术组相比,逃避潜伏期和跨越平台次数无明显差异,说明侧脑室注射Aβ,引起大鼠学习记忆能力下降;2)在水迷宫测试的第4、第5天,阳性对照组与模型组相比,逃避潜伏期减少,跨越平台次数增多,差异有统计学意义(P＜0.05),说明痴呆模型大鼠经抗炎药物布洛芬治疗后,其学习记忆能力明显提高;3)在水迷宫测试的第4、第5天,海风藤高、低剂量组与模型组相比,逃避潜伏期减少,跨越平台次数增多,差异有统计学意义(P＜0.05),说明痴呆模型大鼠经海风藤水提物治疗后,其学习记忆能力明显提高;4)海风藤高、低剂量组与正常对照组相比,逃避潜伏期增加,跨越平台次数减少,差异具有统计学意义(P＜0.05),说明经海风藤治疗的痴呆模型大鼠学习记忆能力尚未恢复到正常水平;5)海风藤高剂量组和低剂量组相比,逃避潜伏期减少,跨越平台次数增多,但差异无统计学意义(P＞0.05)。
1.3大鼠海马的组织学观察
大鼠海马HE染色结果:正常对照组大鼠海马CA1区细胞排列整齐、均匀,细胞结构完整;模型组大鼠海马CA1区细胞排列不整齐、细胞结构不完整、边界不清、细胞间距加大;阳性对照组、海风藤高、低剂量组大鼠CA1区细胞较模型组排列整齐、均匀,结构也较完整。
2.海风藤水提物对痴呆模型大鼠额叶、海马神经元淀粉样蛋白(Aβ)、突触素和胶质细胞中炎症因子表达的影响
本实验采用免疫荧光染色结合图像分析方法观察侧脑室注射痴呆模型大鼠额叶、海马神经元淀粉样蛋白(Aβ)、TNF-α、IL-6的表达,NOS、NO的含量和突触素的表达。
海马神经元Aβ表达结果如下:1)模型组大鼠海马神经元Aβ荧光强度明显增强,其平均阳性率高于正常对照组和假手术组,差异具有统计学意义(P＜0.05),说明脑室内注射Aβ可以造成痴呆模型大鼠海马神经元Aβ的表达升高;2)阳性对照组与模型组相比,其海马神经元Aβ荧光强度明显减弱,平均阳性率明显降低,差异有统计学意义(P＜0.05),说明抗炎药物布洛芬能够降低痴呆模型大鼠海马神经元Aβ的表达;3)海风藤水提物高、低剂量组与模型组相比,其海马神经元Aβ的荧光强度明显减弱,平均阳性率明显降低,差异具有统计学意义(P＜0.05),说明海风藤水提物能够降低痴呆模型大鼠海马神经元Aβ的表达;4)海风藤高、低剂量组之间相比,海风藤高剂量组海马神经元Aβ的荧光强度略低于低剂量组,平均阳性率也有一定程度降低,但差异无统计学意义(P＞0.05)。说明海风藤高、低剂量组之间,在降低Aβ的表达上,无明显差别。
额叶胶质细胞TNF-α、IL-6表达结果如下:1)模型组大鼠的额叶胶质细胞TNF-α、IL-6荧光强度增强,其平均阳性率高于正常对照组和假手术组,差异具有统计学意义(P＜0.05),说明脑室内注射Aβ可以造成痴呆模型大鼠额叶胶质细胞TNF-α和IL-6的表达升高;2)阳性对照组与模型组相比,其额叶胶质细胞TNF-α和IL-6荧光强度明显减弱,平均阳性率明显降低,差异有统计学意义(P＜0.05),说明抗炎药物布洛芬能够降低痴呆模型大鼠额叶胶质细胞TNF-α和IL-6的表达;3)海风藤水提物高剂量组与模型组相比,其额叶胶质细胞TNF-α和IL-6荧光强度明显减弱,平均阳性率明显降低,差异有统计学意义(P＜0.05);4)而海风藤水提物低剂量组虽然降低了痴呆模型大鼠额叶胶质细胞TNF-α和IL-6的表达,但差异并无统计学意义(P＞0.05),说明海风藤水提物能够降低痴呆模型大鼠额叶胶质细胞TNF-α和IL-6的表达,海风藤高剂量组的疗效优于低剂量组。
大鼠脑组织NO、NOS含量检测结果如下:1)模型组大鼠脑组织中NO和NOS的含量增高,其含量高于正常对照组和假手术组,差异具有统计学意义(P＜0.05),说明脑室内注射Aβ可以造成痴呆模型大鼠脑组织中NO和NOS的表达升高;2)阳性对照组与模型组相比,其脑组织中NO和NOS含量明显降低,差异具有统计学意义(P＜0.05),说明抗炎药物布洛芬能够降低痴呆模型大鼠脑组织中NO和NOS的表达;3)海风藤水提物高剂量组与模型组相比,脑组织中NO和NOS含量明显降低,差异具有统计学意义(P＜0.05);4)海风藤水提物低剂量组与模型组相比,其脑组织NO和NOS的含量降低,但差异不具有统计学意义(P＞0.05),说明海风藤水提物能够降低痴呆模型大鼠脑组织中NO和NOS的表达,海风藤高剂量组的疗效优于低剂量组。
额叶、海马突触素表达结果如下:1)模型组大鼠额叶、海马神经元突触素荧光强度明显减弱,其平均阳性率低于正常对照组和假手术组,差异具有统计学意义(P＜0.05),说明脑室内注射Aβ可以造成痴呆模型大鼠额叶、海马神经元突触素的表达降低;2)阳性对照组与模型组相比,其额叶、海马神经元突触素荧光强度明显增强,平均阳性率明显升高,差异具有统计学意义(P＜0.05),说明抗炎药物布洛芬能够增加痴呆模型大鼠额叶、海马神经元突触素的含量;3)海风藤水提物高剂量组与模型组相比,其额叶、海马神经元突触素荧光强度明显增强,平均阳性率明显升高,差异具有统计学意义(P＜0.05),说明海风藤水提物能够增加痴呆模型大鼠额叶、海马突触素的表达;4)海风藤水提物低剂量组与模型组相比,其海马神经元突触素荧光强度明显增强,平均阳性率明显升高,差异具有统计学意义(P＜0.05),5)海风藤低剂量组与模型组相比,其额叶神经元突触素荧光强度未见增强,平均阳性率差异无统计学意义(P＞0.05),说明低剂量海风藤水提物仅有增高痴呆模型大鼠海马,没有增高其额叶神经元突触素表达的作用。
3.海风藤单体复合物毕拨明宁碱/二氢毕拨明宁碱对SK-N-SH细胞内APP基因转录和蛋白表达的抑制作用
3.1海风藤单体的制备
福建产海风藤药材用水提取后,提取液浓缩得到浸膏。浸膏加水溶解后,依次用石油醚,乙酸乙酯,正丁醇萃取,分别得到石油醚萃取相,乙酸乙酯萃取相和正丁醇萃取相。乙酸乙酯萃取相用95%乙醇溶解后,加硅胶拌样,经硅胶柱色谱,氯仿-丙酮梯度洗脱,收集洗脱流份。其中氯仿-丙酮(9:1)洗脱流份Fr.31用石油醚-丙酮反复结晶后,得到化合物1(HFT-1)。
HFT-1根据氢谱、碳谱的数据,并与文献对照,确定该结晶由化合物毕拨明宁碱(piperlonguminine,A)和二氢毕拨明宁碱(dihydropiperlonguminine,B)组成,由其氢谱积分确定在此结晶中A与B的比例为1:0.8。化合物A、B分子式如下:A:piperlonguminine B:dihydropiperlonguminine
3.2海风藤单体复合物毕拨明宁碱和二氢毕拨明宁碱的药理作用
将SK-N-SH细胞分为正常对照组、DMSO组(1‰DMSO)、海风藤水提物组(15g/l)、海风藤单体高剂量组(13.13μg/ml)、中剂量组(6.56μg/ml)、低剂量组(3.28μg/ml)。在细胞被药物处理22小时后,进行各项检测。共进行六次独立的重复性实验。
使用MTT的技术,检测SK-N-SH细胞在各种药物处理后的细胞增殖活性。结果显示,SK-N-SH细胞在各种药物处理24小时后,细胞活性未发生明显变化。
使用逆转录聚合酶链式反应(RT-PCR)的方法,检测SK-N-SH细胞中APP mRNA的表达。电泳结束后,用APP与β-Actin荧光强度的比率代表APP mRNA的表达量。结果发现,海风藤水提物组及海风藤单体高剂量组的荧光强度比率比正常对照组均明显降低,差异有统计学意义(P＜0.05)。而DMSO组、海风藤单体低、中剂量组的荧光强度比率与正常对照组相比,无明显差异(P＞0.05)。表明海风藤水提物(15g/l)与海风藤的毕拨明宁碱和二氢毕拨明宁碱单体复合物(13.13μg/ml)可以降低APP mRNA的表达。
使用蛋白免疫印迹(Western blot)的方法,检测SK-N-SH细胞中APP的表达。使用APP与β-Actin荧光强度的比率代表APP的表达量。结果发现,海风藤水提物组(5g/l、15g/l)与海风藤单体中、高剂量组(6.56μg/ml、13.13μg/ml)的荧光强度比率比正常对照组均明显降低,差异有统计学意义(P＜0.05)。而DMSO组、海风藤单体低剂量组(3.28μg/ml)的荧光强度比率与正常对照组相比,无明显差异(P＞0.05)。表明海风藤水提物(5g/l、15g/l)与海风藤的单体复合物毕拨明宁碱和二氢毕拨明宁碱(6.56μg/ml、13.13μg/ml)可以降低APP蛋白的表达。
采用免疫荧光染色结合图像分析的方法,检测SK-N-SH细胞中Aβ的表达。结果发现,海风藤水提物组(15g/l)与海风藤单体中、高剂量组(6.56μg/ml、13.13μg/ml)Aβ的荧光强度比对照组均明显降低,平均阳性率均明显下降,差异有统计学意义(P＜0.05)。而DMSO组、海风藤单体低剂量组(3.28μg/ml)的平均阳性率与正常对照组相比,无明显差异(P＞0.05)。表明海风藤水提物(15g/l)与海风藤的单体复合物毕拨明宁碱和二氢毕拨明宁碱(6.56μg/ml、13.13μg/ml)可以降低细胞内Aβ的表达。
结论
一向大鼠侧脑室内注射Aβ可明显降低大鼠的学习记忆能力,损害海马神经元,表明Aβ侧脑室注射模型是AD较理想的动物模型。海风藤水提物能够改善Aβ侧脑室注射AD模型大鼠的学习记忆能力,对受损的海马神经元有保护作用。
二海风藤水提物能够降低Aβ侧脑室注射AD模型大鼠神经元内Aβ的表达,减少胶质细胞内TNF-α、IL-6的表达及脑组织内NO、NOS的含量,改善脑内的慢性炎症反应。海风藤水提物能够增加Aβ侧脑室注射AD模型大鼠神经元内突触素的表达,改善因脑内的慢性炎症反应引起的突触损伤。
三海风藤单体复合物毕拨明宁碱/二氢毕拨明宁碱能够抑制SK-N-SH细胞中APP基因的转录和蛋白的表达。
Background Alzheimer disease(AD)is named after German psychiatrist and neural anatomist Alois Alzheimer,who described it in 1907.AD is a neurodegenerative disease characterized by progressive cognitive and memory decline.With the aging of the society,its incidence is increasing year by year.Alzheimer disease has already become a disease which seriously damages people's health.Although apparent improvements for the virulence genes of AD have been achieved in the past 20 years,so far,the reason of AD is still not clear.And the treatment of AD still stays in the period of symptomatic treatment.Therefore,enhancing the research for AD pathogenesis,looking for positive and effective method of AD treatment is of vital economic and social significance.
The pathological change of AD is comprehensive atrophy of brain.At the afflicted brain area,there are two apparent pahthological hallmarks:senile plaques(SP)and neurofibrillary tangles(NFT).SP is extracellular accumulations ofβ-Amyloid(Aβ)peptides that are derived from the abnormal proteolytic processing of the amyloid precursor protein(APP).According to amyloid protein hypothesis,extracellular accumulated Aβactivated inflammation and oxidative damage.It is also demenstrated by pathology that senile plaques are often surrounded by activated microglia and astrocytes. NFT is intraneural accumulation of hyperphosphorlated tau,a cellular skeletin protein.SP and NFT together promote the neural degeneration.
Futokadsura stem is the petiol of piper plant Kadsura.It is used to treat inflammatory diseases.The traditional function of the plant is to dispel wind-damp obstruction symdrome manifested as painful and stiff joints,tendon and muscle spasms,lower back pain,painful knees and pain from external injury.Using the method of aequorin,Enji Han found aqueous extract of futokadsura stem can inhibit the increase of intracellular Ca2+ induced by Aβ25-35.The inhibition effect increases with futokadsura stem concentration. Using the method of cell culture and RT-PCR,Enji Han also demonstrated that aqueous extract of futokadsura stem could selectively inhibit the APP gene expression in SK-N-SH cells.The inhibition effect increases with futokadsura stem concentration and the time of incubation.According to the hypothesis of amyloid protein,extracellular accumulated Aβactivates inflammation and oxidative damage,and these can cause neural degeneration.Based on these findings,we firstly want to observe if futokadsura stem has neural protective effect on dementia model rats.Then,we separated futokadsura stem,and tried to find the effective components which inhibit the expression of APP. Therefore,we assume that futokadsura stem has neural protective effect on SK-N-SH cells.What we are concerning about in this research is,whether this protective effect could happen in dementia models,and what the effective components in futokadsura stem which can inhibit APP expression are?
Objective To observe whether futokadsura stem has neural protective effect on dementia model rats,firstly,Aβwas injected to lateral ventricle of rats to establish dementia rats model.After intragastric administration with aqueous extract of futokadsura stem,the expression of Aβ,inflammatory factors TNF-αand IL-6,synaptophysin and the content of NO,NOS were detected.Secondly, using chemical method,futokadsura stem was separated and the separated components were added to SK-N-SH cells.After the action of these components,the expression of APP and Aβin SK-N-SH cells,and the content of Aβin culture medium were detected.Then we tried to determine the effective components in futokadsura stem which could inhibit the expression of APP and Aβ.
Methods and Results
1.Establishment of dementia model rats and the effect of aqueous extract of futokadsura stem for ethology and histology of dementia model rats
1.1 Aβwas injected to lateral ventricle of rats to establish dementia model rats
60 trained rats were selected,and were divided into 6 groups:normal control group,model group,sham group,positive control group(ibuprofen), high dose futokadsura stem group and low dose futokadsura stem group.Each group has 10 rats.
Normal control group received no treatment;other 5 groups were anesthetized with chloral hydrate(350mg/kg bw)by intraperitoneal injection. The heads of rats were set on the stereotaxis instrument.For the model group, positive control group,high and low dose futokadsura stern group,10μg Aβ(25-35)was injected to the lateral ventricle;for sham group,the same dose of sodium chloride were injected when the skull was opened.Each group began intragastric administration after 7 days,positive group was administered ibuprofen by 20mg/kg bw,high dose futokadsura stem group was administered aqueous extract of futokadsura stem by 140mg/100g bw,low dose group was administered aqueous extract of futokadsura stem by 46.1mg/100g bw. Normal control group,model group and sham group were administered the same dose of distilled water.
1.2 Determination of learning and memory ability of rats
Learning and memory ability of rats from each group were determined by Morris water maze experiment.The escape latencies were recorded.The shorter the escape latencies are,the better their learning and memory ability is. At the same time,we detected the spanning platform times of rats.The more times rats spanned the platform,the better their learning and memory ability is.
Morris water maze experiment results showed that,1)Compared with escape latencies of rats in model group,those in normal control group and sham group decreased obviously,but the spanning platform times increased apparently,the difference is significant(P＜0.05).There were no diffence between escape latencies of rats in normal control group and sham group.It meant that learning ability of rats decreased when Aβwas injected to their lateral ventricles.2)In the 4~(th)and 5~(th)day of Morris water maze experiment, rats in positive control group have lower escape latencies and higher spanning platform times compared with rats in model group.The difference is also significant(P＜0.05).This demonstrated that when treated with anti-inflammatory medicine ibuprofen,dementia model rats could have improved learning and memory ability.3)In the 4~(th)and 5~(th)day of Morris water maze experiment,rats in high or low dose futokadsura stem group have lower escape latencies and higher spanning platform times compared with rats in model group.The difference is significant(P＜0.05).This demonstrated that when treated with aqueous extract of futokadsura stem,dementia model rats could have improved learning and memory ability.4)However,rats in high or low dose futokadsura stem had higher escape latency and lower spanning platform times,compared with rats in normal control group.The diffence is significant(P＜0.05).This meant that even if treated with futokadsura stem, the learning and memory ability of dementia model rats could not restore to normal state.5)When rats in high and low dose futokadsura stem groups were compared,rats in high dose group had lower escape latencies and higher spanning platform times.However,the difference is not significant(P＞0.05).
1.3 Histology of rats hippocampus
HE staining of rats hippocampus showed that,in normal control group, cells in CA1 region of rats hippocampus were arranged in order,the structure of cells was integrated;in model group,cells in CA1 region of rats hippocampus were not arranged in order,the structure of cells was not integrated,the boundary of cells was not clear,the gap between cells was enlarged;in positive control group,high and low dose of futokadsura stem group,cells in CA1 region of rats hippocampus were arranged in order,the structure of cells was fairly integrated.
2.The effect of aqueous extract of futokadsura stem on the expression of Aβ,synaptophysin in frontal lobe and hippocampus neurons and on the expression of inflammatory factors in frontal lobe gliocytes of dementia model rats.
Using immuno-fluorescence staining combined with image analysis,we observed the expression of Aβ,TNF-α,IL-6 and synaptophysin in frontal lobe and hippocampus of dementia model rats which was established by lateral ventricle Aβinjection.
The results of Aβexpression in hippocampus:1)Rats in model group had higher fluorescence intensity of Aβin the hippocampus than rats in normal control group and sham group had.The mean positive ratios were of significant difference(P＜0.05).This demonstrated that dementia model rats established by lateral ventricle Aβinjection could increase the expression of Aβin the hippocampus;2)Rats in positive control group had lower fluorescence intensity of Aβin the hippocampus compared with rats in model group.The mean positive ratios were of significant difference(P＜0.05).This meant that anti-inflammatory medicine ibuprofen could decrease the expresson of Aβin the hippocampus of dementia model rats;3)Compared with rats in model group,rats in high or low dose futokadsura stem group had lower fluorescence intensity of Aβin hippocampus,The mean positive ratios were of significant difference(P＜0.05),This meant that aqueous extract of futokadsura stern could decrease the expression of Aβin the hippocampus of dementia model rats;4)When rats in high and low dose futokadsura stem groups were compared,rats in high dose group had lower fluorescence intensity of Aβin hippocampus,but the mean positive ratios were of no significant difference.It showed that high dose futokadsura stern had similar treatment effect than low dose in decreasing the expression of Aβ.
The results of TNF-α、IL-6 expression in frontal lobe:1)Rats in model group had higher fluorescence intensity of TNF-α、IL-6 in the frontal lobe gliocytes than rats in normal control group and sham group had.The mean positive ratios were of significant difference(P＜0.05).This demonstrated that dementia model rats established by lateral ventricle Aβinjection could increase the expression of TNF-α、IL-6 in the frontal lobe gliocytes;2)Rats in positive control group had lower fluorescence intensity of TNF-α、IL-6 in the frontal lobe gliocytes compared with rats in model group.The mean positive ratios were of significant difference(P＜0.05).This meant that anti-inflammatory medicine ibuprofen could decrease the expresson of TNF-α、IL-6 in the frontal lobe gliocytes of dementia model rats;3)Compared with rats in model group,rats in high dose futokadsura stem group had lower fluorescence intensity of TNF-α、IL-6 in frontal lobe gliocytes,The mean positive ratios were of significant difference(P＜0.05);4)Although rats in low dose futokadsura stem groups had lower fluorescence intensity of TNF-α、IL-6 in frontal lobe gliocytes,the mean positive ratios were of no significant difference(P＞0.05).It showed that aqueous extract of futokadsura stem could decrease the expression of TNF-α、IL-6 in frontal lobe gliocytes,the high dose group had better treatment effect than low dose group in decreasing the expression of TNF-α、IL-6.
The detection of NO,NOS content in rats brain:1)Rats in model group had higher content of NO,NOS in the brain than rats in normal control group and sham group had.It was of significant difference(P＜0.05).This demonstrated that dementia model rats established by lateral ventricle Aβinjection could increase the expression of NO,NOS in the brain of dementia model rats;2) Rats in positive control group had lower content of NO,NOS in the brain compared with rats in model group.It was of significant difference(P＜0.05). This meant that anti-inflammatory medicine ibuprofen could decrease the expresson of in the brain of dementia model rats;3)Compared with rats in model group,rats in high dose futokadsura stem group had lower content of NO,NOS in the brain,It was of significant difference(P＜0.05);4)Although rats in low dose futokadsura stem groups had lower content of NO,NOS in the brain,It was of no significant difference(P＞0.05).It showed that aqueous extract of futokadsura stem could decrease the expression of NO,NOS in the brain,the high dose group had better treatment effect than low dose group in decreasing the expression of NO,NOS.
The results of synaptophysin(SYP)expression in rats frontal lobe and hippocampus:1)Rats in model group had lower fluorescence intensity of SYP in the frontal lobe and hippocampus than rats in normal control group and sham group had.The mean positive ratios were of significant difference(P＜0.05).This demonstrated that dementia model rats established by lateral ventricle Aβinjection could decrease the expression of SYP in the frontal lobe and hippocampus;2)Rats in positive control group had higher fluorescence intensity of SYP in the frontal lobe and hippocampus compared with rats in model group.The mean positive ratios were of significant difference(P＜0.05). This meant that anti-inflammatory medicine ibuprofen could increase the expresson of SYP in the frontal lobe of dementia model rats;3)Compared with rats in model group,rats in high dose futokadsura stem group had higher fluorescence intensity of SYP in frontal lobe and hippocampus,The mean positive ratios were of significant difference(P＜0.05),It showed that aqueous extract of futokadsura stem could increase the expression of SYP in frontal lobe and hippocampus,4)Compared with rats in model group,rats in low dose futokadsura stem group had higher fluorescence intensity of SYP in hippocampus,The mean positive ratios were of significant difference(P＜0.05),5)Rats in low dose futokadsura stem groups did not have higher fluorescence intensity of SYP in frontal lobe,the mean positive ratios were of no significant difference(P＞0.05).It meant that low dose aqueous extract of futokadsura stem could only increase the expression of SYP in the hippocampus,not the frontal lobe of dementia model rats.
3.Inhibition of APP gene transcription and protein expression in SK-N-SH cells by piperlonguminineldihydropiperlonguminine components separated from futokadsura stem
3.1 Preparation of futokadsura stem monomers components
Futokadsura stem from Fujian province was first extracted with water,after concentration,we got the extract.Then,it was extracted with petroleum ether, acetic ether and normal butyl alcohol in turn.The acetic ether extract phase was dissolved by 95%ethanol,then subject to silica gel column chromatography,chloroform-acetone gradient elution.Chloroform-acetone(9:1) elution phase Ft.1 was repeatedly crystallized by petroleum ether-acetone, and HFT-1 was obtained.
According to the data of ~1HNMR and ~(13)CNMR spectra,referring to related literature,we determined the crystal was composed of piperlonguminine(A) and dihydropiperlonguminine(B),the ratio of A to B is 1:0.8.The structures of piperlonguminine and dihydropiperlonguminine are showed below:
A:piperlonguminine
B:dihydropiperlonguminine
3.2 Pharmacological effect of piperlonguminine/ dihydropiperlonguminine components separated from futokadsura stem
SK-N-SH cells were divided into normal control group,DMSO group(1‰DMSO),aqueous extract group(15g/I),high dose of monomers components group(13.13μg/ml),middle dose of monomers components group(6.56μg/ml), low dose of monomers components group(3.28μg/ml).After 22 hours of treatment,different indexes were detected.6 reproducible experiments were performed.
Using the method of MTT,SK-N-SH cells proliferation assay was performed.The results showed that,after 22 hours different treatments,no significant difference was found for SK-N-SH cells proliferation among different groups.
Using the method of RT-PCR,APP gene expression was detected in SK-N-SH cells.After electrophoresis,the fluorescence intensity ratio between APP andβ-Actin was used to represent the expression of APP mRNA.The results showed that the fluorescence intensity ratios in aqueous extract group and high dose of monomers components group were apparently lower than the ratios in normal control group.The difference was significant.However,the ratios of DMSO group,middle dose and low dose of monomers components group had no significant difference compared to the ratios of normal control group.This demonstrated that aqueous extract of futokadsura stem(15g/I)and piperlonguminine/dihydropiperlonguminine components separated from futokadsura stem(13.13μg/ml)could reduce the expression of APP mRNA.
Using the method of Western blot,amyloid precursor protein(APP) expression was detected in SK-N-SH cells.The fluorescence intensity ratio between APP andβ-Actin was used to represent the expression of amyloid precursor protein.The results showed that the fluorescence intensity ratios in aqueous extract group and high dose of monomers components group were apparently lower than the ratios in normal control group.The difference was significant.However,the ratios of DMSO group,middle dose and low dose of monomers components group had no significant difference compared to the ratios of normal control group.This demonstrated that aqueous extract of futokadsura stem(15g/I)and piperlonguminine/dihydropiperlonguminine components separated from futokadsura stem(13.13μg/ml)could reduce the expression of amyloid precursor protein.
Using the method of immuno-fluorescence staining combined with image analysis,Aβexpression was detected in SK-N-SH cells.The results showed that the fluorescence intensity of Aβin aqueous extract group and high dose of monomers components group was apparently lower than that in normal control group.The mean positive ratios were of significant difference.However,the mean positive ratios in DMSO group,middle dose and low dose of monomers components group had no significant difference compared to the ratios of normal control group.This demonstrated that aqueous extract of futokadsura stem(15g/I)and piperlonguminine/dihydropiperlonguminine components separated from futokadsura stem(13.13μg/ml)could reduce the expression of Aβ.
Conclusion
1.Aβlateral ventricle injection to rats could decrease the their learning and memory ability,damage the neurons in hippocampus.It is a fairly ideal AD animal model.Aqueous extract of futokadsura stem could ameliorate the learning and memory ability of dementia model rats,protect the damaged neurons in hippocampus.
2.Aqueous extract of futokadsura stem could decrease the expression of Aβin neurons,reduce the expression of inflammatory factors TNF-α,IL-6 and NO, NOS in gliocytes,ameliorate the chronic inflammatory response in the brain of Aβlateral ventricle injection AD model rats.Aqueous extract of futokadsura stem could also increase the expression of synaptophysin in the neurons of AD model rats,improve the synapse damage caused by chronic inflammatory response.
3.Piperlonguminine/dihydropiperlonguminine components separated from Futokadsura stem could inhibit the expression of APP mRNA and APP in SK-N-SH.
AD的病理变化为广泛的大脑萎缩。在受累脑区,有两个明显的病理学特征:老年斑(SP)和神经原纤维缠结(NFT)。老年斑由神经细胞外的β淀粉样蛋白(Aβ)聚集而成,而β淀粉样蛋白则是由β淀粉样前体蛋白(APP)异常剪切所产生。淀粉样蛋白级联假说认为,神经细胞外聚集的Aβ激活了炎症和氧化损伤。病理学也证实,老年斑周围常围绕着激活的小胶质细胞和星形胶质细胞。Aβ还使细胞骨架蛋白Tau蛋白磷酸化,进而缠绕形成了神经原纤维缠结。老年斑和神经原纤维缠结共同作用,促进了神经退行性变的发生。
中药海风藤是胡椒科植物风藤(Piper kadsura(Choisy)Ohwi)的干燥藤茎。具祛风湿、通经络、理气之功效,主治风寒湿痹,关节疼痛,筋脉拘挛,跌打损伤,哮喘,久咳等症。韩恩吉教授等应用水母发光蛋白的方法,发现海风藤水提物能抑制Aβ25-35诱导的神经细胞胞浆钙离子升高并随海风藤浓度增加而增强,提示海风藤对神经细胞有保护作用。韩恩吉教授等还应用细胞培养、Northernblot和逆转录-聚合酶链反应(RT-PCR)的方法观察了海风藤水提物对SK-N-SH细胞中APP mRNA的影响,证明海风藤可选择性抑制APP mRNA表达,并且随浓度的增加而增强,随时间的延长而增强。根据淀粉样蛋白级联假说,细胞外聚集的Aβ激活了炎症和氧化损伤,从而促成了神经退行性变的发生。所以我们认为,海风藤对神经细胞是有保护作用的。那么,本研究所关心的问题是,这种保护和治疗作用是否会在痴呆模型动物体内发生,海风藤抑制APP基因表达的有效活性成分是什么。
研究目的为了观察海风藤是否对痴呆模型大鼠有神经保护作用,我们首先建立了侧脑室注射Aβ大鼠痴呆模型,在海风藤水提物灌胃后,观察痴呆模型大鼠行为学和组织学的改变,大鼠脑神经元内Aβ、炎症因子TNF-α和IL-6以及突触素的表达,脑内NO、NOS的含量。然后,我们用生药学的方法,将海风藤进行分离,再将各分离组分作用于SK-N-SH细胞。通过观察各分离组分作用后,SK-N-SH细胞中APP和Aβ的表达,来确定海风藤中抑制APP和Aβ表达的有效活性成分。
方法与结果
1.痴呆大鼠模型的建立及海风藤水提物对痴呆模型大鼠行为学和组织学的影响
1.1侧脑室注射Aβ建立大鼠痴呆模型
选择通过筛选训练的大鼠60只,分为正常对照组、模型组、假手术组、阳性对照组(布洛芬)、海风藤高剂量、低剂量组,每组各10只大鼠。
正常对照组不给予处置;其余五组大鼠用水合氯醛350mg/kg bw腹腔注射麻醉,头部固定于立体定位仪。模型组、阳性对照组、海风藤高剂量、低剂量组均在定位仪定位下向侧脑室内注入10μg Aβ(25-35);假手术组钻开颅骨后,侧脑室内注入等量生理盐水。各组大鼠手术后7天开始灌胃给药,阳性对照组给予布洛芬20mg/kg bw,海风藤高剂量组给予140mg/100g bw,低剂量组给予46.1mg/100g bw。正常对照组、模型组及假手术组则给予等量蒸馏水灌胃。
1.2学习、记忆能力的测定
各组大鼠采用Morris水迷宫进行学习、记忆功能的测定,记录逃避潜伏期,逃避潜伏期越短,大鼠的学习、记忆能力越强。同时,测定大鼠跨越平台次数,跨越平台次数越多说明大鼠的学习、记忆能力越强。
Morris水迷宫检测结果:1)正常对照组、假手术组与模型组相比,逃避潜伏期明显减少,跨越平台次数明显增多,差异有统计学意义(P＜0.05),而正常对照组与假手术组相比,逃避潜伏期和跨越平台次数无明显差异,说明侧脑室注射Aβ,引起大鼠学习记忆能力下降;2)在水迷宫测试的第4、第5天,阳性对照组与模型组相比,逃避潜伏期减少,跨越平台次数增多,差异有统计学意义(P＜0.05),说明痴呆模型大鼠经抗炎药物布洛芬治疗后,其学习记忆能力明显提高;3)在水迷宫测试的第4、第5天,海风藤高、低剂量组与模型组相比,逃避潜伏期减少,跨越平台次数增多,差异有统计学意义(P＜0.05),说明痴呆模型大鼠经海风藤水提物治疗后,其学习记忆能力明显提高;4)海风藤高、低剂量组与正常对照组相比,逃避潜伏期增加,跨越平台次数减少,差异具有统计学意义(P＜0.05),说明经海风藤治疗的痴呆模型大鼠学习记忆能力尚未恢复到正常水平;5)海风藤高剂量组和低剂量组相比,逃避潜伏期减少,跨越平台次数增多,但差异无统计学意义(P＞0.05)。
1.3大鼠海马的组织学观察
大鼠海马HE染色结果:正常对照组大鼠海马CA1区细胞排列整齐、均匀,细胞结构完整;模型组大鼠海马CA1区细胞排列不整齐、细胞结构不完整、边界不清、细胞间距加大;阳性对照组、海风藤高、低剂量组大鼠CA1区细胞较模型组排列整齐、均匀,结构也较完整。
2.海风藤水提物对痴呆模型大鼠额叶、海马神经元淀粉样蛋白(Aβ)、突触素和胶质细胞中炎症因子表达的影响
本实验采用免疫荧光染色结合图像分析方法观察侧脑室注射痴呆模型大鼠额叶、海马神经元淀粉样蛋白(Aβ)、TNF-α、IL-6的表达,NOS、NO的含量和突触素的表达。
海马神经元Aβ表达结果如下:1)模型组大鼠海马神经元Aβ荧光强度明显增强,其平均阳性率高于正常对照组和假手术组,差异具有统计学意义(P＜0.05),说明脑室内注射Aβ可以造成痴呆模型大鼠海马神经元Aβ的表达升高;2)阳性对照组与模型组相比,其海马神经元Aβ荧光强度明显减弱,平均阳性率明显降低,差异有统计学意义(P＜0.05),说明抗炎药物布洛芬能够降低痴呆模型大鼠海马神经元Aβ的表达;3)海风藤水提物高、低剂量组与模型组相比,其海马神经元Aβ的荧光强度明显减弱,平均阳性率明显降低,差异具有统计学意义(P＜0.05),说明海风藤水提物能够降低痴呆模型大鼠海马神经元Aβ的表达;4)海风藤高、低剂量组之间相比,海风藤高剂量组海马神经元Aβ的荧光强度略低于低剂量组,平均阳性率也有一定程度降低,但差异无统计学意义(P＞0.05)。说明海风藤高、低剂量组之间,在降低Aβ的表达上,无明显差别。
额叶胶质细胞TNF-α、IL-6表达结果如下:1)模型组大鼠的额叶胶质细胞TNF-α、IL-6荧光强度增强,其平均阳性率高于正常对照组和假手术组,差异具有统计学意义(P＜0.05),说明脑室内注射Aβ可以造成痴呆模型大鼠额叶胶质细胞TNF-α和IL-6的表达升高;2)阳性对照组与模型组相比,其额叶胶质细胞TNF-α和IL-6荧光强度明显减弱,平均阳性率明显降低,差异有统计学意义(P＜0.05),说明抗炎药物布洛芬能够降低痴呆模型大鼠额叶胶质细胞TNF-α和IL-6的表达;3)海风藤水提物高剂量组与模型组相比,其额叶胶质细胞TNF-α和IL-6荧光强度明显减弱,平均阳性率明显降低,差异有统计学意义(P＜0.05);4)而海风藤水提物低剂量组虽然降低了痴呆模型大鼠额叶胶质细胞TNF-α和IL-6的表达,但差异并无统计学意义(P＞0.05),说明海风藤水提物能够降低痴呆模型大鼠额叶胶质细胞TNF-α和IL-6的表达,海风藤高剂量组的疗效优于低剂量组。
大鼠脑组织NO、NOS含量检测结果如下:1)模型组大鼠脑组织中NO和NOS的含量增高,其含量高于正常对照组和假手术组,差异具有统计学意义(P＜0.05),说明脑室内注射Aβ可以造成痴呆模型大鼠脑组织中NO和NOS的表达升高;2)阳性对照组与模型组相比,其脑组织中NO和NOS含量明显降低,差异具有统计学意义(P＜0.05),说明抗炎药物布洛芬能够降低痴呆模型大鼠脑组织中NO和NOS的表达;3)海风藤水提物高剂量组与模型组相比,脑组织中NO和NOS含量明显降低,差异具有统计学意义(P＜0.05);4)海风藤水提物低剂量组与模型组相比,其脑组织NO和NOS的含量降低,但差异不具有统计学意义(P＞0.05),说明海风藤水提物能够降低痴呆模型大鼠脑组织中NO和NOS的表达,海风藤高剂量组的疗效优于低剂量组。
额叶、海马突触素表达结果如下:1)模型组大鼠额叶、海马神经元突触素荧光强度明显减弱,其平均阳性率低于正常对照组和假手术组,差异具有统计学意义(P＜0.05),说明脑室内注射Aβ可以造成痴呆模型大鼠额叶、海马神经元突触素的表达降低;2)阳性对照组与模型组相比,其额叶、海马神经元突触素荧光强度明显增强,平均阳性率明显升高,差异具有统计学意义(P＜0.05),说明抗炎药物布洛芬能够增加痴呆模型大鼠额叶、海马神经元突触素的含量;3)海风藤水提物高剂量组与模型组相比,其额叶、海马神经元突触素荧光强度明显增强,平均阳性率明显升高,差异具有统计学意义(P＜0.05),说明海风藤水提物能够增加痴呆模型大鼠额叶、海马突触素的表达;4)海风藤水提物低剂量组与模型组相比,其海马神经元突触素荧光强度明显增强,平均阳性率明显升高,差异具有统计学意义(P＜0.05),5)海风藤低剂量组与模型组相比,其额叶神经元突触素荧光强度未见增强,平均阳性率差异无统计学意义(P＞0.05),说明低剂量海风藤水提物仅有增高痴呆模型大鼠海马,没有增高其额叶神经元突触素表达的作用。
3.海风藤单体复合物毕拨明宁碱/二氢毕拨明宁碱对SK-N-SH细胞内APP基因转录和蛋白表达的抑制作用
3.1海风藤单体的制备
福建产海风藤药材用水提取后,提取液浓缩得到浸膏。浸膏加水溶解后,依次用石油醚,乙酸乙酯,正丁醇萃取,分别得到石油醚萃取相,乙酸乙酯萃取相和正丁醇萃取相。乙酸乙酯萃取相用95%乙醇溶解后,加硅胶拌样,经硅胶柱色谱,氯仿-丙酮梯度洗脱,收集洗脱流份。其中氯仿-丙酮(9:1)洗脱流份Fr.31用石油醚-丙酮反复结晶后,得到化合物1(HFT-1)。
HFT-1根据氢谱、碳谱的数据,并与文献对照,确定该结晶由化合物毕拨明宁碱(piperlonguminine,A)和二氢毕拨明宁碱(dihydropiperlonguminine,B)组成,由其氢谱积分确定在此结晶中A与B的比例为1:0.8。化合物A、B分子式如下:A:piperlonguminine B:dihydropiperlonguminine
3.2海风藤单体复合物毕拨明宁碱和二氢毕拨明宁碱的药理作用
将SK-N-SH细胞分为正常对照组、DMSO组(1‰DMSO)、海风藤水提物组(15g/l)、海风藤单体高剂量组(13.13μg/ml)、中剂量组(6.56μg/ml)、低剂量组(3.28μg/ml)。在细胞被药物处理22小时后,进行各项检测。共进行六次独立的重复性实验。
使用MTT的技术,检测SK-N-SH细胞在各种药物处理后的细胞增殖活性。结果显示,SK-N-SH细胞在各种药物处理24小时后,细胞活性未发生明显变化。
使用逆转录聚合酶链式反应(RT-PCR)的方法,检测SK-N-SH细胞中APP mRNA的表达。电泳结束后,用APP与β-Actin荧光强度的比率代表APP mRNA的表达量。结果发现,海风藤水提物组及海风藤单体高剂量组的荧光强度比率比正常对照组均明显降低,差异有统计学意义(P＜0.05)。而DMSO组、海风藤单体低、中剂量组的荧光强度比率与正常对照组相比,无明显差异(P＞0.05)。表明海风藤水提物(15g/l)与海风藤的毕拨明宁碱和二氢毕拨明宁碱单体复合物(13.13μg/ml)可以降低APP mRNA的表达。
使用蛋白免疫印迹(Western blot)的方法,检测SK-N-SH细胞中APP的表达。使用APP与β-Actin荧光强度的比率代表APP的表达量。结果发现,海风藤水提物组(5g/l、15g/l)与海风藤单体中、高剂量组(6.56μg/ml、13.13μg/ml)的荧光强度比率比正常对照组均明显降低,差异有统计学意义(P＜0.05)。而DMSO组、海风藤单体低剂量组(3.28μg/ml)的荧光强度比率与正常对照组相比,无明显差异(P＞0.05)。表明海风藤水提物(5g/l、15g/l)与海风藤的单体复合物毕拨明宁碱和二氢毕拨明宁碱(6.56μg/ml、13.13μg/ml)可以降低APP蛋白的表达。
采用免疫荧光染色结合图像分析的方法,检测SK-N-SH细胞中Aβ的表达。结果发现,海风藤水提物组(15g/l)与海风藤单体中、高剂量组(6.56μg/ml、13.13μg/ml)Aβ的荧光强度比对照组均明显降低,平均阳性率均明显下降,差异有统计学意义(P＜0.05)。而DMSO组、海风藤单体低剂量组(3.28μg/ml)的平均阳性率与正常对照组相比,无明显差异(P＞0.05)。表明海风藤水提物(15g/l)与海风藤的单体复合物毕拨明宁碱和二氢毕拨明宁碱(6.56μg/ml、13.13μg/ml)可以降低细胞内Aβ的表达。
结论
一向大鼠侧脑室内注射Aβ可明显降低大鼠的学习记忆能力,损害海马神经元,表明Aβ侧脑室注射模型是AD较理想的动物模型。海风藤水提物能够改善Aβ侧脑室注射AD模型大鼠的学习记忆能力,对受损的海马神经元有保护作用。
二海风藤水提物能够降低Aβ侧脑室注射AD模型大鼠神经元内Aβ的表达,减少胶质细胞内TNF-α、IL-6的表达及脑组织内NO、NOS的含量,改善脑内的慢性炎症反应。海风藤水提物能够增加Aβ侧脑室注射AD模型大鼠神经元内突触素的表达,改善因脑内的慢性炎症反应引起的突触损伤。
三海风藤单体复合物毕拨明宁碱/二氢毕拨明宁碱能够抑制SK-N-SH细胞中APP基因的转录和蛋白的表达。
Background Alzheimer disease(AD)is named after German psychiatrist and neural anatomist Alois Alzheimer,who described it in 1907.AD is a neurodegenerative disease characterized by progressive cognitive and memory decline.With the aging of the society,its incidence is increasing year by year.Alzheimer disease has already become a disease which seriously damages people's health.Although apparent improvements for the virulence genes of AD have been achieved in the past 20 years,so far,the reason of AD is still not clear.And the treatment of AD still stays in the period of symptomatic treatment.Therefore,enhancing the research for AD pathogenesis,looking for positive and effective method of AD treatment is of vital economic and social significance.
The pathological change of AD is comprehensive atrophy of brain.At the afflicted brain area,there are two apparent pahthological hallmarks:senile plaques(SP)and neurofibrillary tangles(NFT).SP is extracellular accumulations ofβ-Amyloid(Aβ)peptides that are derived from the abnormal proteolytic processing of the amyloid precursor protein(APP).According to amyloid protein hypothesis,extracellular accumulated Aβactivated inflammation and oxidative damage.It is also demenstrated by pathology that senile plaques are often surrounded by activated microglia and astrocytes. NFT is intraneural accumulation of hyperphosphorlated tau,a cellular skeletin protein.SP and NFT together promote the neural degeneration.
Futokadsura stem is the petiol of piper plant Kadsura.It is used to treat inflammatory diseases.The traditional function of the plant is to dispel wind-damp obstruction symdrome manifested as painful and stiff joints,tendon and muscle spasms,lower back pain,painful knees and pain from external injury.Using the method of aequorin,Enji Han found aqueous extract of futokadsura stem can inhibit the increase of intracellular Ca2+ induced by Aβ25-35.The inhibition effect increases with futokadsura stem concentration. Using the method of cell culture and RT-PCR,Enji Han also demonstrated that aqueous extract of futokadsura stem could selectively inhibit the APP gene expression in SK-N-SH cells.The inhibition effect increases with futokadsura stem concentration and the time of incubation.According to the hypothesis of amyloid protein,extracellular accumulated Aβactivates inflammation and oxidative damage,and these can cause neural degeneration.Based on these findings,we firstly want to observe if futokadsura stem has neural protective effect on dementia model rats.Then,we separated futokadsura stem,and tried to find the effective components which inhibit the expression of APP. Therefore,we assume that futokadsura stem has neural protective effect on SK-N-SH cells.What we are concerning about in this research is,whether this protective effect could happen in dementia models,and what the effective components in futokadsura stem which can inhibit APP expression are?
Objective To observe whether futokadsura stem has neural protective effect on dementia model rats,firstly,Aβwas injected to lateral ventricle of rats to establish dementia rats model.After intragastric administration with aqueous extract of futokadsura stem,the expression of Aβ,inflammatory factors TNF-αand IL-6,synaptophysin and the content of NO,NOS were detected.Secondly, using chemical method,futokadsura stem was separated and the separated components were added to SK-N-SH cells.After the action of these components,the expression of APP and Aβin SK-N-SH cells,and the content of Aβin culture medium were detected.Then we tried to determine the effective components in futokadsura stem which could inhibit the expression of APP and Aβ.
Methods and Results
1.Establishment of dementia model rats and the effect of aqueous extract of futokadsura stem for ethology and histology of dementia model rats
1.1 Aβwas injected to lateral ventricle of rats to establish dementia model rats
60 trained rats were selected,and were divided into 6 groups:normal control group,model group,sham group,positive control group(ibuprofen), high dose futokadsura stem group and low dose futokadsura stem group.Each group has 10 rats.
Normal control group received no treatment;other 5 groups were anesthetized with chloral hydrate(350mg/kg bw)by intraperitoneal injection. The heads of rats were set on the stereotaxis instrument.For the model group, positive control group,high and low dose futokadsura stern group,10μg Aβ(25-35)was injected to the lateral ventricle;for sham group,the same dose of sodium chloride were injected when the skull was opened.Each group began intragastric administration after 7 days,positive group was administered ibuprofen by 20mg/kg bw,high dose futokadsura stem group was administered aqueous extract of futokadsura stem by 140mg/100g bw,low dose group was administered aqueous extract of futokadsura stem by 46.1mg/100g bw. Normal control group,model group and sham group were administered the same dose of distilled water.
1.2 Determination of learning and memory ability of rats
Learning and memory ability of rats from each group were determined by Morris water maze experiment.The escape latencies were recorded.The shorter the escape latencies are,the better their learning and memory ability is. At the same time,we detected the spanning platform times of rats.The more times rats spanned the platform,the better their learning and memory ability is.
Morris water maze experiment results showed that,1)Compared with escape latencies of rats in model group,those in normal control group and sham group decreased obviously,but the spanning platform times increased apparently,the difference is significant(P＜0.05).There were no diffence between escape latencies of rats in normal control group and sham group.It meant that learning ability of rats decreased when Aβwas injected to their lateral ventricles.2)In the 4~(th)and 5~(th)day of Morris water maze experiment, rats in positive control group have lower escape latencies and higher spanning platform times compared with rats in model group.The difference is also significant(P＜0.05).This demonstrated that when treated with anti-inflammatory medicine ibuprofen,dementia model rats could have improved learning and memory ability.3)In the 4~(th)and 5~(th)day of Morris water maze experiment,rats in high or low dose futokadsura stem group have lower escape latencies and higher spanning platform times compared with rats in model group.The difference is significant(P＜0.05).This demonstrated that when treated with aqueous extract of futokadsura stem,dementia model rats could have improved learning and memory ability.4)However,rats in high or low dose futokadsura stem had higher escape latency and lower spanning platform times,compared with rats in normal control group.The diffence is significant(P＜0.05).This meant that even if treated with futokadsura stem, the learning and memory ability of dementia model rats could not restore to normal state.5)When rats in high and low dose futokadsura stem groups were compared,rats in high dose group had lower escape latencies and higher spanning platform times.However,the difference is not significant(P＞0.05).
1.3 Histology of rats hippocampus
HE staining of rats hippocampus showed that,in normal control group, cells in CA1 region of rats hippocampus were arranged in order,the structure of cells was integrated;in model group,cells in CA1 region of rats hippocampus were not arranged in order,the structure of cells was not integrated,the boundary of cells was not clear,the gap between cells was enlarged;in positive control group,high and low dose of futokadsura stem group,cells in CA1 region of rats hippocampus were arranged in order,the structure of cells was fairly integrated.
2.The effect of aqueous extract of futokadsura stem on the expression of Aβ,synaptophysin in frontal lobe and hippocampus neurons and on the expression of inflammatory factors in frontal lobe gliocytes of dementia model rats.
Using immuno-fluorescence staining combined with image analysis,we observed the expression of Aβ,TNF-α,IL-6 and synaptophysin in frontal lobe and hippocampus of dementia model rats which was established by lateral ventricle Aβinjection.
The results of Aβexpression in hippocampus:1)Rats in model group had higher fluorescence intensity of Aβin the hippocampus than rats in normal control group and sham group had.The mean positive ratios were of significant difference(P＜0.05).This demonstrated that dementia model rats established by lateral ventricle Aβinjection could increase the expression of Aβin the hippocampus;2)Rats in positive control group had lower fluorescence intensity of Aβin the hippocampus compared with rats in model group.The mean positive ratios were of significant difference(P＜0.05).This meant that anti-inflammatory medicine ibuprofen could decrease the expresson of Aβin the hippocampus of dementia model rats;3)Compared with rats in model group,rats in high or low dose futokadsura stem group had lower fluorescence intensity of Aβin hippocampus,The mean positive ratios were of significant difference(P＜0.05),This meant that aqueous extract of futokadsura stern could decrease the expression of Aβin the hippocampus of dementia model rats;4)When rats in high and low dose futokadsura stem groups were compared,rats in high dose group had lower fluorescence intensity of Aβin hippocampus,but the mean positive ratios were of no significant difference.It showed that high dose futokadsura stern had similar treatment effect than low dose in decreasing the expression of Aβ.
The results of TNF-α、IL-6 expression in frontal lobe:1)Rats in model group had higher fluorescence intensity of TNF-α、IL-6 in the frontal lobe gliocytes than rats in normal control group and sham group had.The mean positive ratios were of significant difference(P＜0.05).This demonstrated that dementia model rats established by lateral ventricle Aβinjection could increase the expression of TNF-α、IL-6 in the frontal lobe gliocytes;2)Rats in positive control group had lower fluorescence intensity of TNF-α、IL-6 in the frontal lobe gliocytes compared with rats in model group.The mean positive ratios were of significant difference(P＜0.05).This meant that anti-inflammatory medicine ibuprofen could decrease the expresson of TNF-α、IL-6 in the frontal lobe gliocytes of dementia model rats;3)Compared with rats in model group,rats in high dose futokadsura stem group had lower fluorescence intensity of TNF-α、IL-6 in frontal lobe gliocytes,The mean positive ratios were of significant difference(P＜0.05);4)Although rats in low dose futokadsura stem groups had lower fluorescence intensity of TNF-α、IL-6 in frontal lobe gliocytes,the mean positive ratios were of no significant difference(P＞0.05).It showed that aqueous extract of futokadsura stem could decrease the expression of TNF-α、IL-6 in frontal lobe gliocytes,the high dose group had better treatment effect than low dose group in decreasing the expression of TNF-α、IL-6.
The detection of NO,NOS content in rats brain:1)Rats in model group had higher content of NO,NOS in the brain than rats in normal control group and sham group had.It was of significant difference(P＜0.05).This demonstrated that dementia model rats established by lateral ventricle Aβinjection could increase the expression of NO,NOS in the brain of dementia model rats;2) Rats in positive control group had lower content of NO,NOS in the brain compared with rats in model group.It was of significant difference(P＜0.05). This meant that anti-inflammatory medicine ibuprofen could decrease the expresson of in the brain of dementia model rats;3)Compared with rats in model group,rats in high dose futokadsura stem group had lower content of NO,NOS in the brain,It was of significant difference(P＜0.05);4)Although rats in low dose futokadsura stem groups had lower content of NO,NOS in the brain,It was of no significant difference(P＞0.05).It showed that aqueous extract of futokadsura stem could decrease the expression of NO,NOS in the brain,the high dose group had better treatment effect than low dose group in decreasing the expression of NO,NOS.
The results of synaptophysin(SYP)expression in rats frontal lobe and hippocampus:1)Rats in model group had lower fluorescence intensity of SYP in the frontal lobe and hippocampus than rats in normal control group and sham group had.The mean positive ratios were of significant difference(P＜0.05).This demonstrated that dementia model rats established by lateral ventricle Aβinjection could decrease the expression of SYP in the frontal lobe and hippocampus;2)Rats in positive control group had higher fluorescence intensity of SYP in the frontal lobe and hippocampus compared with rats in model group.The mean positive ratios were of significant difference(P＜0.05). This meant that anti-inflammatory medicine ibuprofen could increase the expresson of SYP in the frontal lobe of dementia model rats;3)Compared with rats in model group,rats in high dose futokadsura stem group had higher fluorescence intensity of SYP in frontal lobe and hippocampus,The mean positive ratios were of significant difference(P＜0.05),It showed that aqueous extract of futokadsura stem could increase the expression of SYP in frontal lobe and hippocampus,4)Compared with rats in model group,rats in low dose futokadsura stem group had higher fluorescence intensity of SYP in hippocampus,The mean positive ratios were of significant difference(P＜0.05),5)Rats in low dose futokadsura stem groups did not have higher fluorescence intensity of SYP in frontal lobe,the mean positive ratios were of no significant difference(P＞0.05).It meant that low dose aqueous extract of futokadsura stem could only increase the expression of SYP in the hippocampus,not the frontal lobe of dementia model rats.
3.Inhibition of APP gene transcription and protein expression in SK-N-SH cells by piperlonguminineldihydropiperlonguminine components separated from futokadsura stem
3.1 Preparation of futokadsura stem monomers components
Futokadsura stem from Fujian province was first extracted with water,after concentration,we got the extract.Then,it was extracted with petroleum ether, acetic ether and normal butyl alcohol in turn.The acetic ether extract phase was dissolved by 95%ethanol,then subject to silica gel column chromatography,chloroform-acetone gradient elution.Chloroform-acetone(9:1) elution phase Ft.1 was repeatedly crystallized by petroleum ether-acetone, and HFT-1 was obtained.
According to the data of ~1HNMR and ~(13)CNMR spectra,referring to related literature,we determined the crystal was composed of piperlonguminine(A) and dihydropiperlonguminine(B),the ratio of A to B is 1:0.8.The structures of piperlonguminine and dihydropiperlonguminine are showed below:
A:piperlonguminine
B:dihydropiperlonguminine
3.2 Pharmacological effect of piperlonguminine/ dihydropiperlonguminine components separated from futokadsura stem
SK-N-SH cells were divided into normal control group,DMSO group(1‰DMSO),aqueous extract group(15g/I),high dose of monomers components group(13.13μg/ml),middle dose of monomers components group(6.56μg/ml), low dose of monomers components group(3.28μg/ml).After 22 hours of treatment,different indexes were detected.6 reproducible experiments were performed.
Using the method of MTT,SK-N-SH cells proliferation assay was performed.The results showed that,after 22 hours different treatments,no significant difference was found for SK-N-SH cells proliferation among different groups.
Using the method of RT-PCR,APP gene expression was detected in SK-N-SH cells.After electrophoresis,the fluorescence intensity ratio between APP andβ-Actin was used to represent the expression of APP mRNA.The results showed that the fluorescence intensity ratios in aqueous extract group and high dose of monomers components group were apparently lower than the ratios in normal control group.The difference was significant.However,the ratios of DMSO group,middle dose and low dose of monomers components group had no significant difference compared to the ratios of normal control group.This demonstrated that aqueous extract of futokadsura stem(15g/I)and piperlonguminine/dihydropiperlonguminine components separated from futokadsura stem(13.13μg/ml)could reduce the expression of APP mRNA.
Using the method of Western blot,amyloid precursor protein(APP) expression was detected in SK-N-SH cells.The fluorescence intensity ratio between APP andβ-Actin was used to represent the expression of amyloid precursor protein.The results showed that the fluorescence intensity ratios in aqueous extract group and high dose of monomers components group were apparently lower than the ratios in normal control group.The difference was significant.However,the ratios of DMSO group,middle dose and low dose of monomers components group had no significant difference compared to the ratios of normal control group.This demonstrated that aqueous extract of futokadsura stem(15g/I)and piperlonguminine/dihydropiperlonguminine components separated from futokadsura stem(13.13μg/ml)could reduce the expression of amyloid precursor protein.
Using the method of immuno-fluorescence staining combined with image analysis,Aβexpression was detected in SK-N-SH cells.The results showed that the fluorescence intensity of Aβin aqueous extract group and high dose of monomers components group was apparently lower than that in normal control group.The mean positive ratios were of significant difference.However,the mean positive ratios in DMSO group,middle dose and low dose of monomers components group had no significant difference compared to the ratios of normal control group.This demonstrated that aqueous extract of futokadsura stem(15g/I)and piperlonguminine/dihydropiperlonguminine components separated from futokadsura stem(13.13μg/ml)could reduce the expression of Aβ.
Conclusion
1.Aβlateral ventricle injection to rats could decrease the their learning and memory ability,damage the neurons in hippocampus.It is a fairly ideal AD animal model.Aqueous extract of futokadsura stem could ameliorate the learning and memory ability of dementia model rats,protect the damaged neurons in hippocampus.
2.Aqueous extract of futokadsura stem could decrease the expression of Aβin neurons,reduce the expression of inflammatory factors TNF-α,IL-6 and NO, NOS in gliocytes,ameliorate the chronic inflammatory response in the brain of Aβlateral ventricle injection AD model rats.Aqueous extract of futokadsura stem could also increase the expression of synaptophysin in the neurons of AD model rats,improve the synapse damage caused by chronic inflammatory response.
3.Piperlonguminine/dihydropiperlonguminine components separated from Futokadsura stem could inhibit the expression of APP mRNA and APP in SK-N-SH.
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