Effect of Panax notoginseng saponins on the expression of beta-amyloid protein in the cortex of the parietal lobe and hippocampus, and spatial learning and memory in a mouse model of senile dementia

BACKGROUND： The pharmacological actions of Panax notoginseng saponins （PNS） lie in removing free radicals, anti-inflammation and anti-oxygenation. It can also improve memory and behavior in rat models of Alzheimer＇s disease. OBJECTIVE： Using the Morris water maze, immunohistochemistry, real-time PCR and RT-PCR, this study aimed to measure improvement in spatial learning, memory, expression of amyloid precursor protein （App） and β -amyloid （A β ）, to investigate the mechanism of action of PNS in the treatment of AD in the senescence accelerated mouse-prone 8 （SAMP8） and compare the effects with huperzine A. DESIGN, TIME AND SETTING： A completely randomized grouping design, controlled animal experiment was performed in the Center for Research ＆ Development of New Drugs, Guangxi Traditional Chinese Medical University from July 2005 to April 2007. MATERIALS： Sixty male SAMP8 mice, aged 3 months, purchased from Tianjin Chinese Traditional Medical University of China, were divided into four groups： PNS high-dosage group, PNS low-dosage group, huperzine A group and control group. PNS was provided by Weihe Pharmaceutical Co., Ltd. （batch No.： Z53021485, Yuxi, Yunan Province, China）. Huperzine A was provided by Zhenyuan Pharmaceutical Co., Ltd. （batch No.： 20040801, Zhejiang, China）. METHODS： The high-dosage group and low-dosage group were treated with 93.50 and 23.38 mg/kg PNS respectively per day and the huperzine A group was treated with 0.038 6 mg/kg huperzine A per day, all by intragastric administration, for 8 consecutive weeks. The same volume of double distilled water was given to the control group. MAIN OUTCOME MEASURES： After drug administration, learning and memory abilities were assessed by place navigation and spatial probe tests. The recording indices consisted of escape latency （time-to-platform）, and the percentage of swimming time spent in each quadrant. The number of A β 1-40, A β 1-42 and App immunopositive neurons in the brains of SAMP8 mice was analyzed by immunohistochemistry. The mRNA content ofApp, tau, acetylcholinesterase, and synaptophysin （Syp） was tested by real time PCR and RT-PCR. RESULTS： The PCR results show that PNS can downregulate the expression of the App gene and upregulate the expression of the Syp gene in the parietal cortex and hippocampus of SAMP8 mice. The therapeutic effects of the PNS high-dosage group were greater than those of the PNS low-dosage group and the huperzine A group （P 〈 0.05）. The results of the Morris water maze and immunohistochemistry indicated that PNS can improve the capacity for spatial learning and memory in SAMP8 mice, and reduce the content of A β 1-40, A β 1-42 and expression of App in the brains of SAMP8 mice. The therapeutic effects of the PNS high-dosage group were greater than that of the PNS low-dosage group and the huperzine A group （P 〈 0.05）. CONCLUSION： These results support the hypothesis that PNS plays a therapeutic and protective role on the pathological lesions and learning dysfunction of Alzheimer＇s disease. The therapeutic effects of PNS for Alzheimer＇s disease are possibly achieved through downregulating the expression of the App gene and upregulating the expression of the Syp gene. The therapeutic effects of PNS are dose-dependent and are greater than the effect of huperzine A.

Notoginseng Saponin Rg1 Prevents Cognitive Impairment through Modulating APP Processing in Aβ1-42-injected Rats

With the intensification of the aging process of the world,Alzheimer's disease(AD),which is the main type of senile dementia,has become a primary problem in the present society.Lots of strategies have been used to prevent and treat AD in animal nlodels and clinical trials,but most of them ended in failure.Panax notoginseng saponins(PNS)contain a variety of monomer compositions which have been separated and identified.Among of the monomer compositions,notoginseng saponin Rg1(Rg1)accounts for 20%of the cultivation of panax notoginseng roots.And now PNS have been reported to be widely used to treat cardicerebrovascular diseases and have neuroprotective effects to restrain theβ-amyloid peptide(Aβ)25-35-niediated apoptosis.Moreover,it is reported that PNS could accelerate the growth of nerve cells,increase the length of axons and promote synaptic plasticity.Whether Rg1 can ameliorate the cognitive impairment and the underlying mechanism has not been elucidated.To study the preventive effect of Rg1 on cognitive impairment and the possible mechanism,we established the cognitive impairment model in rats through Aβ1-42(2.6μg/μL,5μL)injection and then treated the rats with Rg1(25,50 and 100 mg/kg)administered intragastrically for 4 weeks.We observed that Aβ1-42 could induce spatial learning and memory deficits in rats.Simultaneously,Aβ1-42 injection also resulted in the reduced neuron number in comuammonis 1(CA1)and dentate gyrus(DG)of hippocampus,as well as the increased level of hyperphosphorylatedβ-amyloid precursor protein(APP)at Thr668 site with up-regulation ofβ-APP cleaving enzyme 1(BACE1)and presenilin 1(PS1)and down-regulation of a disintegrin and metalloprotease domain-containing protein 10(ADAM 10)and insulindegrading enzyme(IDE).Administration of Rg1 effectively rescued the cognitive impairment and neuronal loss,and inhibited theβ-secretase processing of APP through reducing APP-Thr668 phosphorylation and BACE1/PS1 expression,and increasing the expression of ADAM 10 and IDE.We concluded that Rg1 might have neuroprotective effects and could promote learning and memory ability,which might be a viable candidate in AD therapy probably through reducing the generation of Aβand increasing the degradation of Aβ.

Panax notoginseng saponins promotes cerebral recovery from ischemic injury by downregulating LINGO-1 and activating the EGFR/PI3K/AKT signaling pathways in vivo

Objective:Panax notoginseng saponins (PNS),extracted from rhizome of the herb Radix et Rhizoma Notoginseng (Panax notoginseng (Burk.) F.H.Chen),was recently discovered to have beneficial effects against neurological damage.This study investigated the effects of PNS on cerebral ischemia and elucidated the molecular mechanisms underlying these effects.Methods:Middle cerebral artery occlusion rats were treated with PNS (3.6 mg/100 g or 7.2 mg/100 g per day) for 7 days,the gene of LINGO-1 was measured and the expression of protein synaptophysin,postsynaptic density protein 95,LINGO-1 and p-EGFR/p-PI3K/p-AKT were investigated.The weight and mNSS score of Sprague-Dawley rats in each group were recorded every day during the 7 days.Results:PNS promoted middle cerebral artery occlusion rats' weight and the recovery of neural function.PNS significantly decreased ischemia-induced LINGO-1 protein expression.PNS also elevated EGFR/PI3K/AKT phosphorylation levels.Conclusion:PNS promoted cerebral recovery from ischemic injury by accelerating synapse reconstruction and inhibiting the neuron growth negative regulatory protein LINGO-1 and activating the epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway in vivo.

三七皂甙单体Rb_1对心肌细胞Ca^（2＋）内流作用的研究

应用Ｆｕｒａ－２荧光技术及放射配基结合实验探讨三七皂甙单体Ｒｂ１对心肌细胞胞外Ｃａ２＋内流的作用。０．０４ｍｍｏｌ·Ｌ－１Ｒｂ１可以抑制高钾引起的大鼠心肌细胞胞浆Ｃａ２＋浓度的升高，且呈浓度依赖性；可以完全阻断高钾基础上的异丙肾上腺素的作用。其作用与钙通道拮抗剂维拉帕米类似。Ｒｂ１对大鼠心肌细胞膜上β受体亲和力和受体数均无明显影响。结果提示：Ｒｂ１对心肌细胞电压依赖性钙通道开放引起的胞内钙升高有抑制作用；对β受体相关联的钙通道开放引起的胞浆Ｃａ２＋升高也有抑制作用。而不影响β受体本身。

三七皂苷中人参皂苷Rg_1与Rb_1口服吸收及其体内药代动力学的研究和比较

分别考察三七总皂苷(PNS)在大鼠灌胃和静脉给药后的人参皂苷Rg1(Rg1)、人参皂苷Rb1(Rb1)的胆汁排泄;采用平衡透析法测定药物的血浆蛋白结合率;并结合药代动力学的实验结果,研究和比较Rg1与Rb1的口服吸收及其体内药代动力学性质。结果表明,静脉给药后10 h,Rg1及Rb1胆汁排泄累积量分别为给药剂量的(61.48±18.30)%和(3.94±1.49)%;灌胃给药后12 h,Rg1及Rb1胆汁排泄累积量分别为给药剂量的(0.91±0.51)%和(0.055±0.02)%。Rg1及Rb1的血浆蛋白结合率分别为6.56%~12.74%和80.11%~89.69%。Rg1的胃、肠和肝的通过率(FS,FI和FH)分别为49.85%,13.05%和50.56%;Rb1分别为25.82%,4.18%和65.77%。因此,肠壁吸收差是Rg1和Rb1生物利用度低的主要原因。Rg1具有较高的胆汁排泄和较低的血浆蛋白结合率,Rb1的胆汁排泄较低,而血浆蛋白结合率较高。Rb1的肠黏膜透过性和体内消除速度都低于Rg1,但前者的平均滞留时间(MRT)和血药浓度曲线下面积(AUC)均大于后者。

HPLC法同时测定三七总皂苷中人参皂苷Rg_1与Rb_1的含量

目的 建立 HPL C同时测定三七总皂苷中人参皂苷 Rg1 与 Rb1 的方法。方法 采用 HPL C法 ,以茶碱为内标 ,氨基键合相为固定相 ,流动相为乙腈 -水 (80∶ 2 0 ) ,检测波长 2 0 3nm。结果 三七总皂苷中人参皂苷 Rg1 和 Rb1与其他成分分离良好 ,保留时间分别约为 5 .7和 2 1.5 min。人参皂苷 Rg1 在 80～ 2 80μg/ m L (r=0 .9995 ) ,Rb1 在80～ 2 4 0 μg/ m L(r=0 .9993)线性关系良好 ,Rg1 和 Rb1 加样回收率分别为 97.1%和 98.4 %,RSD分别为 2 .4 4 %和 2 .35 %(n=9)。结论 本法操作简便 ,准确 ,重现性好 ,可用于人参及三七的质量控制。

三七皂甙Rg_1、Rb_1对脂多糖引起的大鼠肝细胞凋亡及相关基因表达的保护作用

目的:探讨三七皂甙对急性肝损害肝细胞凋亡及凋亡相关基因表达的影响。方法:采用脂多糖(LPS)造成大鼠急性肝损伤模型,用 TUNEL 染色法检测肝细胞凋亡,用免疫组化法测定 bcl-2、bax、Fas、FasL 凋亡相关基因在肝内的表达,观察三七皂甙 Rg_1和 Rb_1对上述指标的作用。结果:TUNEL 法检测出细胞凋亡高峰时间在 LPS 处理后12～15 h。免疫组化法检测出 bcl-2、bax、Fas、FasL 凋亡相关基因在肝内表达的时相性变化。Rg_1、Rb_1对 bcl-2没有明显影响,而显著抑制促凋亡基因 bax、Fas、FasL 的表达。结论:LPS处理启动和促进了肝细胞凋亡的相关基因的表达,而 Rg_1、Rb_1通过抑制促凋亡基因表达而减少肝细胞凋亡。

LSCM观察三七总皂甙及其组分对游离钙离子影像变化的影响

用盖玻片或ＭａｔＴｅｋ培养皿培养猪主动脉内皮细胞（ａＥＣ）约１８ｈ后，以荧光探针（ｆｌｕｏ－３／ＡＭ终浓度１０μｍｏｌ·Ｌ－１）负载，当细胞内游离钙离子（Ｃａ２＋）与ｆｌｕｏ－３结合后，通过激光扫描共聚焦显微镜（ＩｎＳＩＧＨＴ－ｐｌｕｓＩＱ型）观察细胞的荧光影像变化。基于三七总皂甙（ｔ－ＰＮＳ）对ａＥＣ所产生的生物活性物质有明显的作用，本拟进一步探索上述药物对ａＥＣ中［Ｃａ２＋］；的影响。实验结果显示：在ＡＴＰ作用后，ｔ－ＰＮＳ可使ａＥＣ内的荧光强度在２０～８０ｓ内急剧升高至峰值。但其组分Ｒｂ１及Ｒｇ１使荧光强度有下降或无明显变化。提示ｔ－ＰＮＳ有使内皮细胞［Ｃａ２＋］；升高的作用。

三七皂苷R_1、人参皂苷R_d对微循环及凝血作用的影响

目的 研究三七皂苷 R1 (R1 )和人参皂苷 Rd(Rd)的活血化瘀作用 ,以证实二者为三七中的有效成分。方法 显微电视放大系统定量观测 R1 和 Rd 对正常及去甲肾上腺素 (NA)所致耳廓微循环障碍小鼠耳廓微循环的影响 ;血浆复钙试验测定 R1 和 Rd 的抗凝作用。结果 R1 和 Rd 能显著促进或改善正常及 NA所致耳廓微循环障碍小鼠耳廓的微循环 ;亦可延长血浆复钙时间。结论 R1 和 Rd 均能显著改善微循环并适度延长凝血时间 。

三七总皂苷对大鼠脑缺血再灌注后Caspase表达的影响

目的研究大鼠局灶性脑缺血再灌注后脑组织中天冬氨酸特异性半胱氨酸蛋白酶(Caspase-1、Caspase-3、Caspase-8)蛋白表达的变化,以及三七总皂苷(PNS)对其表达的影响。方法采用线栓法建立大鼠大脑中动脉栓塞局灶性脑缺血再灌注模型。实验动物随机分为假手术组、模型组、三七总皂苷组和尼莫地平对照组,于缺血前5 m in、缺血后12、24、36 h给药,共给药4次。缺血2 h再灌注46 h后,采用免疫组织化学方法检测脑组织中Caspase-1、Caspase-3、Caspase-8蛋白表达的变化。结果缺血2 h再灌注46 h后,可诱导脑组织Caspase-1、Caspase-3表达增强,PNS能下调Caspase-1、Caspase-3蛋白的表达;但对Caspase-8的表达无影响。结论PNS能抑制脑缺血再灌注后Caspase-1、Caspase-3蛋白的表达,这可能是其促进脑缺血后神经元存活及损伤后修复的机制之一。

基于Nrf2信号通路的三七总皂苷对Aβ_(25-35)诱导PC12细胞凋亡的保护作用机制研究

目的探讨三七总皂苷对Aβ_(25-35)诱导的PC12细胞凋亡的保护作用及分子机制。方法利用Aβ_(25-35)诱导PC12细胞损伤模型,MTT法检测PC12细胞活力,试剂盒测定LDH漏出量、ROS水平、MDA含量、Caspase-3活力以及抗氧化酶SOD、CAT和GHS-Px活力,TUNEL染色检测细胞凋亡,JC-1染色观察线粒体膜电位,Western blot检测相关蛋白HO-1、Lamin-B、细胞核内Nrf2及总Nrf2的蛋白表达。结果三七总皂苷能够明显抑制Aβ_(25-35)诱导的PC12细胞活力下降(P<0.01)和LDH漏出(P<0.01),减少ROS和MDA的产生(P<0.01),增加SOD、CAT和GSH-Px活力(P<0.01),稳定线粒体膜电位,抑制caspase-3的活化(P<0.01)。而且,三七总皂苷还能够上调PC12细胞细胞核Nrf2、总Nrf2和细胞质中HO-1的蛋白表达。HO-1抑制剂Sn PP能够抑制三七总皂苷的神经保护作用。结论三七总皂苷可能通过上调Nrf2/HO-1信号通路,从而抑制Aβ25-35诱导PC12细胞氧化应激和凋亡。

三七皂甙Rg_1对大鼠实验性血栓形成,血小板聚集率及血小板内游离钙水平的影响

三七皂甙Ｒｇ１可明显降低实验性血栓形成，并且以剂量依赖方式抑制凝血酶诱导的血小板聚集．此外，Ｒｇ１还可抑制凝血酶诱导的正常血压及肾性高血压大鼠血小板内游离钙（［Ｃａ２＋］ｉ）升高．表明Ｒｇ１的抗血栓形成和抗血小板聚集作用可能与抑制血小板［Ｃａ２＋］ｉ升高有关．

三七总甙对人肾成纤维细胞的影响

目的 :研究三七总甙 (PNS)对人肾成纤维细胞 (KFB)增殖、分泌I型胶原、表达整合素 β1的影响。方法 :体外培养KFB ,分别采用四甲基偶氮唑蓝 (MTT)法、ELISA法、流式细胞仪检测PNS对KFB增殖、分泌Ⅰ型胶原及表达整合素β1的影响。结果 :PNS在最佳浓度范围和作用时间内可明显抑制KFB细胞增殖及分泌Ⅰ型胶原 (与空白对照组比较 ,P <0 0 5 ) ,同时显著降低了KFB上整合素β1的表达 (与空白对照组比较 ,P <0 0 5 )。结论 :PNS可成为防治肾间质纤维化的有效药物。

三七皂苷Rg_1对失血性休克大鼠肠上皮细胞线粒体损伤保护作用的研究

目的 观察大鼠失血性休克后肠上皮细胞线粒体编码基因细胞色素氧化酶 (COXⅠ ,COXⅡ ,COXⅢ )mRNA的表达变化 ,并探讨三七皂苷Rg1(Rg1)对其保护作用的分子机制 ,为休克的防治提供实验依据。方法 采用失血性休克模型 ,分离肠上皮细胞后进行RNA的提取 ,应用RT PCR检测大鼠失血性休克后及用Rg1治疗后COXⅠ ,COXⅡ ,COXⅢmRNA的表达变化情况。结果 大鼠失血性休克 1h ,COXⅠ ,COXⅡ基因表达开始增强 ,2h表达最强 ,以后随着休克时间延长 ,表达又逐渐减弱 ,失血性休克晚期 5h表达显著低于正常对照鼠 (P <0 .0 1) ;Rg1(5mg·kg-1)治疗后 ,可使COXⅠ ,COXⅡmRNA表达显著增强 (P <0 .0 1)。结论 失血性休克可引起大鼠肠上皮细胞线粒体编码基因COXⅠ ,COXⅡmRNA的明显改变 ,Rg1可提高其表达 ,对失血性休克肠上皮细胞线粒体损伤有明显的保护作用。

三七总皂苷对肝纤维化小鼠血清中转化生长因子-β1及白介素-1的影响

目的观察三七总皂苷对肝纤维化小鼠血清中TGF-β1及IL-1的影响,探讨三七总皂苷抗肝纤维化可能的免疫学机理。方法用CC l4复制小鼠中毒性肝纤维化模型,同时给与三七总皂苷治疗,酶联免疫吸附实验(ELISA法)测定血清中TGF-β1及IL-1的水平,并行肝脏病理组织切片检测。结果三七总皂苷能显著降低肝纤维化小鼠血清中TGF-βl及IL-1的水平,减轻肝纤维化程度。结论三七总皂苷具有抗肝纤维化作用,其机理可能与降低血清中TGβ-p1及IL-1的水平有关。

三七总皂苷对大鼠脑梗死区ICAM-1表达和中性粒细胞浸润的影响

目的:研究三七总皂苷对大鼠脑梗死区细胞间粘附分子-1(ICAM- 1)表达和中性粒细胞浸润的影响。方法:大鼠大脑中动脉短暂阻塞制成局灶性脑缺血2h再灌注24h模型。再灌注后2h腹腔注射三七总皂苷25或50mg/kg。再灌注24h后分光光度法测定缺血区大脑皮层和尾壳核髓过氧化物酶(MPO)活性,免疫组织化学染色测定大脑皮层和尾壳核细胞间粘附分子1(ICAM -1)的表达。结果:大鼠局灶性脑缺血再灌注后,缺血区大脑皮层和尾壳核MPO活性和ICAM -1表达明显增加,三七总皂苷50mg/kg治疗用药能降低MPO活性和抑制ICAM- 1表达。结论:三七总皂苷可抑制大鼠局灶性脑缺血再灌注后ICAM- 1表达和中性粒细胞浸润,减轻脑梗死区炎症反应。

正交试验优化超声辅助法提取三七有效成分工艺

研究超声辅助法提取三七有效成分的工艺。通过单因素试验研究乙醇溶液体积分数、料液比、超声波频率和提取时间对三七人参皂苷Rg1提取率的影响。在单因素试验基础上对4个因素安排L9(34)正交试验,得出影响三七有效成分人参皂苷Rg1提取率的先后次序为:乙醇体积分数>提取时间>料液比>超声频率。选出最佳提取工艺为:乙醇体积分数70%、料液比1:12(g/mL)、超声频率59kHz、提取时间60min。在此最佳工艺下,得到三七干燥后的人参皂苷Rg1含量为1.85%。

三七总皂苷对β-淀粉样蛋白42损伤脑微血管内皮细胞ZO-1表达的影响

目的观察三七总皂苷对β-淀粉样蛋白(Aβ)42损伤脑微血管内皮细胞ZO-1表达的影响,为阿尔茨海默病的对因治疗提供新的依据。方法细胞分为正常对照组、模型组和三七总皂苷低、中、高剂量组,37℃、5%CO2培养箱中培养24 h后,采用MTT比色法检测细胞活力,Western blot检测ZO-1蛋白表达。结果 Aβ42刺激后微血管内皮细胞活力降低(P<0.01),ZO-1蛋白表达被抑制(P<0.01);与模型组比较,三七总皂苷低、中、高剂量组微血管内皮细胞活力增加,其中三七总皂苷中、高剂量组增加较为明显,差异有统计学意义(P<0.05),ZO-1蛋白表达增加。结论三七总皂苷通过抑制Aβ42所致微血管内皮细胞活力降低,恢复血脑屏障的部分屏障功能。

三七总皂甙对移植肝缺血再灌注后核因子-κB、ICAM-1表达的影响

目的研究三七总皂甙(PNS)对大鼠移植肝缺血再灌注损伤中核因子-κB、ICAM-1表达的影响。方法改良Kamada法建立同种大鼠原位肝脏移植模型,将动物模型随机分为3组:生理盐水对照组(N组)、三七总皂甙预处理组(P组)和假手术组(S组)。3组分别于供肝再灌注后2、6和24h时相点,用免疫组化检测NF-κB及ICAM-1的表达;用髓过氧化酶法(MPO)定量测定肝组织中中性粒细胞浸润,并抽血检测血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)水平。结果N组供肝再灌注后2hNF-κB的活性显著升高,之后逐渐下降;肝组织ICAM-1的表达在再灌注后2h开始增高,6h达到高峰,与中性粒细胞的浸润增加和肝损伤有相关性。但P组明显低于N组(P<0.05)。结论大鼠移植肝缺血再灌注时刺激NF-κB的活化,启动ICAM-1的表达参与肝脏缺血-再灌注损伤的发生过程,三七总皂甙能显著降低再灌注时供肝组织中炎症介质的转录表达,并有效改善供肝的再灌注损伤。

HPLC同时测定陇中损伤胶囊中3种皂苷含量

目的建立同时测定陇中损伤胶囊中三七皂苷R1、人参皂苷Rg1、人参皂苷Rb1含量的高效液相色谱梯度洗脱方法。方法采用C18柱(250 mm×4.6 mm,5μm);流动相为乙腈(A)-0.05%磷酸溶液(B);梯度洗脱0～32 min(A23%～23%),32～42 min(A 23%～30%),42～67 min(A 30%～46%);检测波长203 nm;流速1.0 mL/min。结果三七皂苷R1、人参皂苷Rg1、人参皂苷Rb1进样量分别在1.02～8.50μg(r=0.9995)、0.90～7.50μg(r=0.9990)、1.17～9.75μg(r=0.999 6)范围内与峰面积线性关系良好,平均加样回收率为100.98%(RSD=2.71%)、97.73%(RSD=1.98%)、99.57%(RSD=1.62%)。结论所用方法简单易行、准确可靠,可作为陇中损伤胶囊的质量控制方法。