蛇床子素对帕金森病防治作用的基础研究
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摘要
帕金森病(Parkinson's disease PD)是影响中老年人身体健康的主要神经退行性疾病,其病因仍然不清。左旋多巴一直是治疗PD的经典药物。然而,左旋多巴在连续用药后会发生诸多副作用,而且左旋多巴治疗并不能延缓多巴胺神经元的进行性死亡。因此,临床上急需研究疗效好、副作用小的治疗PD的药物。许多研究证实中药治疗PD的效果是确切的,但都为复方制剂,用量大,作用机理和有效成分均不十分清楚。因此,研究疗效可靠的中药单体药物及其作用机理具有重要的临床应用意义。
中药蛇床子是伞形科植物蛇床Cnidiumm monnier(L.)Cusson的干燥成熟果实,具有开窍祛痰、醒神益智、理气活血等功效,在治疗帕金森病的中药复方中频繁出现。现代研究表明,蛇床子可增强脑细胞耐缺氧能力、改善脑循环,并有减少自由基损害的作用。蛇床子素(Osthole,OST)是蛇床子中最重要的活性单体成分,是一种很好的天然抗氧剂,具有抗氧化、抗衰老、抗血栓、促进学习记忆的作用。但其对帕金森病的治疗作用和机理方面的研究尚未见报道。
针对临床需要,本文对蛇床子中OST进行了深入研究,为将其开发成防治PD的新药奠定了理论基础。
第一部分蛇床子中OST的提取分离方法研究
目的:建立一种方便、高效的从蛇床子中提取分离有效成分OST的方法。
方法:分别采用有机溶剂萃取法、微波萃取和浊点萃取法从蛇床子中提取OST,再取蛇床子提取物用无水乙醇进行多次重结晶纯化,最后得到OST纯品(纯度>98%)。
结果与结论:
首次采用环保型液液萃取技术-浊点萃取法提取OST,并对其进行了HPLC定量分析。经三种方法比较,浊点萃取法萃取率最高。中药传统的提取法多为有机溶剂萃取,均不同程度地存在有机溶剂用量大、处理时间长、步骤多、杂质多、环境污染严重等缺点。而浊点萃取法不使用有机溶剂,克服了以上缺点。本实验证明浊点萃取法代替传统有机溶剂提取工艺提纯OST效率和纯度高,且有较高重现性。
第二部分OST体内药代动力学的研究
目的:测定给药后大鼠血浆和脑中OST浓度的动态变化及药动学参数。考察OST透过血脑屏障的行为,为OST的临床合理用药提供可靠依据。
方法:取SD大鼠灌胃OST 40.0 mg/kg后,在给药后不同的时间点采取血浆和脑组织,经浊点萃取处理后,经HPLC测定药物含量。平均药物含量-时间数据用DAS 2.0程序拟合,按非房室模型的统计矩法进行统计。
结果:大鼠灌胃OST的药代动力学特点符合一级消除的二室开放模型,主要血浆药代动力学参数分别C_(max):2.72μg/mL;t_(1/2):5.26h;AUC_(0-t):10.49μg/h/mL;AUC_(0-∞):11.27μg/h/mL;t_(max):0.56h。脑组织中OST达峰时间为0.56h,即在脑和血浆中同时达到峰值浓度,C_(max):1.06μg/mL,且其浓度在0.5-6h内没有显著性变化,其药物下降幅度仅为48%。
结论:1.浊点萃取法应用于OST体内药代动力学的研究中样品的预处理过程方法简便、可行。
2.从时间-浓度曲线中观察到血浆中OST迅速分布,且消除较缓慢,说明OST能透过血脑屏障,且能在脑组织中停留较长时间。
第三部分OST对MPTP诱导C57BL小鼠PD的保护作用初步研究
目的:观察OST对MPTP诱发PD模型小鼠的行为学及多巴胺能神经元的影响,以探讨OST对黑质多巴胺能神经元的保护作用。
方法:小鼠随机分为模型组、美多芭组、OST高(100 mg/kg)、中(50 mg/kg)、低剂量(25 mg/k)三组,另设正常对照组。腹腔注射MPTP复制PD小鼠模型,在造模前5d灌胃OST,连续13d持续到造模结束,并观察OST对小鼠协调运动能力、TH阳性神经元细胞数及黑质多巴胺含量的影响。
结果:OST高、中剂量组、美多芭组给药第2d后小鼠的协调运动能力均有不同程度恢复,与模型组比较(P<0.05);低剂量OST对PD小鼠协调运动能力改善不显著(P>0.05),模型组小鼠与治疗前比较协调运动能力有显著降低(P<0.05),正常组小鼠行为则无变化;免疫组化显示OST高、中剂量脑黑质TH阳性神经元数量高于模型组(P<0.05),而低于美多芭组(P<0.05),但均低于正常组(P>0.05);高效液相色谱-电化学检测结果表明,模型组大鼠中脑DA、DOPAC、HVA含量均低于OST高、中剂量组、美多芭组(P<0.05或P<0.01),但均低于正常组。低剂量OST的DA、DOPAC和HVA含量与模型组比较差异不显著。
结论:OST可通过减轻PD模型小鼠脑黑质细胞的受损程度,促进其修复,并升高DA、DOPAC及HVA水平和改善协调运动能力以达到防治帕金森病的作用。
第四部分OST对PC12细胞氧化应激损伤模型保护作用研究
目的:利用H_2O_2造成PC12细胞直接氧化应激损伤,探讨OST在神经细胞中的抗氧化机制。利用观测MPP~+介导的PC12细胞间接氧化应激损伤程度,探讨OST在神经细胞中的抗凋亡机制。
方法:以神经毒素MPP~+和H_2O_2诱导PC12细胞凋亡即为PD体外细胞模型。采用形态学观察、荧光染色、透射电镜超微结构分析、流式细胞技术、DNA片断化、RT-PCR和Western blot蛋白印迹技术,在抗氧化、抗凋亡等方面,探讨OST保护PC12细胞氧化应激损伤的作用机制。
结果:1.在H_2O_2介导的PC12细胞直接氧化应激损伤模型中,OST剂量依赖地减少了细胞中LDH的漏出;通过细胞核的形态观察以及流式细胞仪的分析,OST可以减少H_2O_2介导的PC12细胞凋亡性死亡;OST抗H_2O_2氧化应激损伤的保护作用与细胞内SOD、CAT、GSH-Px和MDA等抗氧化酶的活性密切相关。
2.在MPP~+介导的PC12细胞间接氧化应激损伤模型中,LDH分析证明能够保护质膜的完整性而抑制MPP~+介导的LDH漏出;形态观察及流式细胞仪的分析,同样证实OST可显著抑制MPP~+介导的细胞凋亡;而且,OST能够剂量依赖地降低凋亡/坏死细胞的比例。OST抑制MPP~+介导的细胞凋亡可通过阻止膜电位(MMP)的丧失,抑制ROS的产生,减少GSH的耗竭,促进SOD和CAT的激活和上调Bcl-2蛋白的表达,降低Bax/Bcl-2的比率,抑制Caspase-3激活和Cytochrome C的释放及下调核转录因子NF-кB P65蛋白。
结论:1.在受活性氧(ROS)介导的氧化应激损伤时,OST可以有效的保持PC12细胞内的抗氧化防御体系处于正常的水平,从而抑制细胞的功能衰蝎而死亡。
2. OST可能通过调节线粒体的通透性而抑制氧化应激所致的细胞凋亡。
结论和意义:
1.本课题首次将浊点萃取法应用于蛇床子中有效成分OST的提取分离和体内药代动力学研究中样品的预处理过程,并分别进行了实验优化,结果表明,该方法间便、高效、环保、适于规模操作,是一种非常有前途的分离和样品预处理方法。
2.首次证明OST对MPTP所致PD模型小鼠的行为有明显改善作用,并对MPTP所致小鼠脑中多巴胺及其代谢产物的降低有提高作用,并可保护H_2O_2和MPP~+介导的PC12细胞氧化应激引起的损伤和凋亡。通过体内、外实验研究,首次从细胞和分子水平阐明OST治疗PD的作用机制,即OST的治疗作用与维持线粒体膜电位,相应保持线粒体正常功能,抑制线粒体凋亡途径的激活及调节细胞凋亡相关蛋白的表达有关。该研究结论不仅为OST的新药创制提供实验依据,同时澄清了中药蛇床子可用于治疗PD的物质基础。
Parkinson's disease (PD) is a progressive neurodegenerative disease which defining pathological features are selective loss of dopaminergic neurons in substantia nigra pars compacta and subsequent decrease of dopamine levels in the striatum, the main target innervated by these neurons. It ranked the second common neurodegenerative disorder after Alzheimer’s disease and affected people primarily in mid and late life. Although the etiopathogenisis of PD still remains to be elucidated, the evidence strongly suggested that mitochondrial dysfunction and oxidative stress should be related to the disease. The auto-oxidation and the enzymatic oxidation of dopamine can generate ROS. Oxidative stress and/or mitochondrial impairment are believed to culminate in the activation of an apoptotic cascade which ultimately resulted in the loss of dopaminergic cells. Therefore, the regulation of intracellular ROS and modification of the apoptotic cascade may be important to prevent pathological apoptosis in PD. The L-dopamine (L-DA) is the classical strategy for the PD therapy. However, it can only improve clinical symptoms but can not retard the course of the disease. Moreover, severe side effects will appear in the long-time treatment. New drugs with good curative effect and fewer side effects are eagerly needed. Recently, a new concept for pharmecotherapy of PD called neuroprotective strategy has been proposed.
Osthole (7-methoxy-8- isopentenoxy-coumarin), which is extracted from Cnidium monnieri (Chinese name: She Chuang Zi) and has long been used in traditional Chinese medicine, has been reported to have various pharmacological properties including anti-platelet activity, anti-tumor, anti-allergic effect, anti-apoptosis, anti-oxidation, estrogen-like-effects, anti-osteoporosis, hepato- protective effect and anti-inflammation. It is a potential antioxidant that has been reported to have significant scavenging effects on oxygen free radicals and inhibit lipid peroxidation. Thus, this indicates that OST possibly has the therapeutic effect on PD.
In the present study, the protective effect of OST on dopaminergic neurons in substantia nigra pars compacta (SNC) was investigated in the PD model mice treated with MPTP in vivo. The neuroprotective effect of OST on MPP+ and H2O2-induced cytotoxicity in PC12 cells was performed in vitro. HPLC, immunohistochemistry staining, RT-PCR, Flow cytometric detection, western blot analysis, transmission electron microscope, and so on were performed in order to probe into the effect of OST on DA neurons, and identify its antioxidation and anti-apoptosis mechanism. Our studies provide strong evidences that OST is useful to the treatment of PD. Experiments were divided into five parts as follows:
Part one: the extraction and separation of OST from Cnidium monnieri
Objective:
The extract and separate technique of OST from Cnidium monnieri was improved, and the contents of OST in the extract were determined by HPLC.
Methods:
The feasibility of employing non-ionic surfactant oligoethylene glycol monoalkyl ether (Genapol X-080) as an alternative and effective solvent for the extraction of osthole in Cnidium monnieri was studied for the first time. Various experimental conditions were investigated to optimize the extraction process. Under optimum conditions, i.e. 10% Genapol X-080 (w/v), liquid/solid ratio of 100:1 (mL/g), ultrasonic-assisted extraction for 30 min, and the extraction recovery reached the highest value. For the preconcentration of osthole and imperatorin by cloud point extraction (CPE), the solution was incubated in a thermostatic water bath at 60℃for 30 min, and 2.0 moL/ L sodium chloride was added to the solution to facilitate the phase separation and increase the preconcentration factor during the CPE process.
Results:
the extraction efficiency is more than 39 percent that of conventional extraction solvent-methanol
Conclusion:
The cloud-point extraction technique is a low cost, simple and sensitive method with high cleanup effect. Finally, the method was successfully applied to separate and determine osthole from C. monnieri, respectively.
Part second: Pharmacokinetic Study of Osthole in Rat
Objective:
This research aims at systematical study on the pharmacokinetics, tissue distribution, which offers a study base to next clinical trial.
Method:
A new straightforward method based on cloud-point extraction (CPE) was developed to determine osthole in rat plasma and brain tissue by reversed phase high performance liquid chromatography. The drug concentrations were analyzed by the non-compartment model method statistical moment method of DAS 2.0 software.
Results:
The experiment results on rat implyed that the plasma concentration-time curve was fitted as second-compartmentmodel. The main pharmacokinetic parameters at dose (30 mg/kg) are as follows.
C_(max): 2.72μg/mL, t_(1/2): 5.26h, AUC_(0-t): 10.49μg h mL~(-1), AUC_(0-∞): 11.27μg h mL~(-1), t_(max): 0.56h. The peak concentration of OST in mouse plasma was 1.06μg/mL. The concentration of OST in mouse brain at 0.5-6h no significant changes in their drug drop of only 48%.
Conclusion:
The results indicated that OST readily penetrated the blood-brain barrier and could be stay in the brain for a long time.
Part three: Protective effect of OST on PD model mice treated with MPTP
Objective:
The protective effect of OST on dopaminergic neurons in substantia nigra pars compacta was investigated in the PD model mice treated with MPTP
Method:
Behaviors, HPLC, immunohischemistry technique were used to observe the damage of substantia nigral neurons respectively.
Results:
1. In the coordination exercise test, the model group mice demonstrated dyskinesia, scores obtained from the behavioral tests were decreased significantly compared to control group (P<0.05), while the scores in pretreatment groups (hight, middle) showed a marked increase to some degree (P<0.05).
2. The level of DA, DOPAC and HVA in the OST high dose group, middle dose group and madopar group were higher than that of the model group (P<0.05or P<0.01), but lower than that of normal group,the level of DA, DOPAC and HVA in OST low dose group were not significantly changed.
Conclusion:
The results suggest that OST may provide a protective effect against MPTP-induced neuronal death in parkinsonian model by reduceing DA neuron loss and increasing the level of DA, DOPAC and HVA.
Part four: Protectvie effect of OST on MPP+ and H_2O_2 induced injury in dopaminergic PC12cells
Objective:
To study the protect effects of OST on MPP+ and H_2O_2 induced injury in dopaminergic PC12cells.
Method:
The neuroprotective effects of OST on MPP+ and H_2O_2-induced oxidative stress in cultured PC12 cells were investigated. HPLC, immunohisto- chemistry staining, RT-PCR, Flow cytometric detection, western blot analysis, transmission electron microscope, and so on were performed in order to probe into the effect of OST on DA neurons, and identify its antioxidation and anti-apoptosis mechanism.
Results:
1. The neuroprotective effects of OST on H_2O_2-induced direct oxidative stress in cultured PC12 cells were investigated. Exposure of PC12cells to H_2O_2 induced a leakage of lactate dehydrogenase (LDH), which was accordant in cell viability denoted by MTT assay. By observing the nuclear morphological changes and flow cytometric analysis, pretreatment with OST markedly elevated H_2O_2-induced cell survival and antioxidant enzyme activities (GSH-Px、SOD and CAT) and decreased the level of MDA in a dose-dependent manner.
2. The neuroprotective effects of OST on MPP+-induced indirect oxidative stress in cultured PC12 cells were investigated. Treatment with OST significantly protected against MPP+-induced apoptosis observed the nuclear morphological changes and measured using flow cytometric analysis. The apoptosis in MPP+-induced PC12cells was associated with loss of mitochondrial merebrane potential, the formation of ROS, GSH depletion, suppressions of SOD and CAT, activation of caspase-3 and Cytochrome c, down-regulation of NF-кB P65、Bcl-2 and the increase in Bax/Bcl-2 ratio, and the generation of intracellular reactive oxygen species. In contrast, treatment of PC12 cells with OST remarkably reversed above-mentioned mitochondrial dysfunction.
Conclusion:
1. These results suggest that OST is very effective in preventing oxidative stress triggered by deterioration of cellular functions to reduce ROS levels. OST can protect PC12cells against oxidative stress probably due to stimulating endogenous antioxidant defense mechanisms.
2. These results suggest that OST may have the capacity to counteract the toxicity of MPP+ by attenuating change of the mitochondrial membrane permeability that is associated with oxidative stress damage.
Conclusion and significance
1. The method of cloud point extraction was used to the extraction separation of OST and the sample pretreatment of pharmacokinetics study in first time. Experimental results show that the method was convenience, efficience, environment protection, and for the scale of operation. It is a very promising method of separation and sample preparation.
2. The first demonstration that OST given orally significantly improved the abnormal behavior and increased the content of DA and its metabolites in striatum in MPTP model mice. Moreover, OST also has remarkably protective effects on MPP+ and H_2O_2-induced injury in PC12cells may be ascribed to its anti-oxidative properties, anti-apoptotic activity via maintaining normal mitochondrial function, inhibiting the activation of mitochondrial pathways of apoptosis and regulating apoptosis-related protein expression. These results indicated that OST might provide a valuable therapeutic strategy for the treatment of progressive neurodegenerative disease such as Parkinson's disease.
中药蛇床子是伞形科植物蛇床Cnidiumm monnier(L.)Cusson的干燥成熟果实,具有开窍祛痰、醒神益智、理气活血等功效,在治疗帕金森病的中药复方中频繁出现。现代研究表明,蛇床子可增强脑细胞耐缺氧能力、改善脑循环,并有减少自由基损害的作用。蛇床子素(Osthole,OST)是蛇床子中最重要的活性单体成分,是一种很好的天然抗氧剂,具有抗氧化、抗衰老、抗血栓、促进学习记忆的作用。但其对帕金森病的治疗作用和机理方面的研究尚未见报道。
针对临床需要,本文对蛇床子中OST进行了深入研究,为将其开发成防治PD的新药奠定了理论基础。
第一部分蛇床子中OST的提取分离方法研究
目的:建立一种方便、高效的从蛇床子中提取分离有效成分OST的方法。
方法:分别采用有机溶剂萃取法、微波萃取和浊点萃取法从蛇床子中提取OST,再取蛇床子提取物用无水乙醇进行多次重结晶纯化,最后得到OST纯品(纯度>98%)。
结果与结论:
首次采用环保型液液萃取技术-浊点萃取法提取OST,并对其进行了HPLC定量分析。经三种方法比较,浊点萃取法萃取率最高。中药传统的提取法多为有机溶剂萃取,均不同程度地存在有机溶剂用量大、处理时间长、步骤多、杂质多、环境污染严重等缺点。而浊点萃取法不使用有机溶剂,克服了以上缺点。本实验证明浊点萃取法代替传统有机溶剂提取工艺提纯OST效率和纯度高,且有较高重现性。
第二部分OST体内药代动力学的研究
目的:测定给药后大鼠血浆和脑中OST浓度的动态变化及药动学参数。考察OST透过血脑屏障的行为,为OST的临床合理用药提供可靠依据。
方法:取SD大鼠灌胃OST 40.0 mg/kg后,在给药后不同的时间点采取血浆和脑组织,经浊点萃取处理后,经HPLC测定药物含量。平均药物含量-时间数据用DAS 2.0程序拟合,按非房室模型的统计矩法进行统计。
结果:大鼠灌胃OST的药代动力学特点符合一级消除的二室开放模型,主要血浆药代动力学参数分别C_(max):2.72μg/mL;t_(1/2):5.26h;AUC_(0-t):10.49μg/h/mL;AUC_(0-∞):11.27μg/h/mL;t_(max):0.56h。脑组织中OST达峰时间为0.56h,即在脑和血浆中同时达到峰值浓度,C_(max):1.06μg/mL,且其浓度在0.5-6h内没有显著性变化,其药物下降幅度仅为48%。
结论:1.浊点萃取法应用于OST体内药代动力学的研究中样品的预处理过程方法简便、可行。
2.从时间-浓度曲线中观察到血浆中OST迅速分布,且消除较缓慢,说明OST能透过血脑屏障,且能在脑组织中停留较长时间。
第三部分OST对MPTP诱导C57BL小鼠PD的保护作用初步研究
目的:观察OST对MPTP诱发PD模型小鼠的行为学及多巴胺能神经元的影响,以探讨OST对黑质多巴胺能神经元的保护作用。
方法:小鼠随机分为模型组、美多芭组、OST高(100 mg/kg)、中(50 mg/kg)、低剂量(25 mg/k)三组,另设正常对照组。腹腔注射MPTP复制PD小鼠模型,在造模前5d灌胃OST,连续13d持续到造模结束,并观察OST对小鼠协调运动能力、TH阳性神经元细胞数及黑质多巴胺含量的影响。
结果:OST高、中剂量组、美多芭组给药第2d后小鼠的协调运动能力均有不同程度恢复,与模型组比较(P<0.05);低剂量OST对PD小鼠协调运动能力改善不显著(P>0.05),模型组小鼠与治疗前比较协调运动能力有显著降低(P<0.05),正常组小鼠行为则无变化;免疫组化显示OST高、中剂量脑黑质TH阳性神经元数量高于模型组(P<0.05),而低于美多芭组(P<0.05),但均低于正常组(P>0.05);高效液相色谱-电化学检测结果表明,模型组大鼠中脑DA、DOPAC、HVA含量均低于OST高、中剂量组、美多芭组(P<0.05或P<0.01),但均低于正常组。低剂量OST的DA、DOPAC和HVA含量与模型组比较差异不显著。
结论:OST可通过减轻PD模型小鼠脑黑质细胞的受损程度,促进其修复,并升高DA、DOPAC及HVA水平和改善协调运动能力以达到防治帕金森病的作用。
第四部分OST对PC12细胞氧化应激损伤模型保护作用研究
目的:利用H_2O_2造成PC12细胞直接氧化应激损伤,探讨OST在神经细胞中的抗氧化机制。利用观测MPP~+介导的PC12细胞间接氧化应激损伤程度,探讨OST在神经细胞中的抗凋亡机制。
方法:以神经毒素MPP~+和H_2O_2诱导PC12细胞凋亡即为PD体外细胞模型。采用形态学观察、荧光染色、透射电镜超微结构分析、流式细胞技术、DNA片断化、RT-PCR和Western blot蛋白印迹技术,在抗氧化、抗凋亡等方面,探讨OST保护PC12细胞氧化应激损伤的作用机制。
结果:1.在H_2O_2介导的PC12细胞直接氧化应激损伤模型中,OST剂量依赖地减少了细胞中LDH的漏出;通过细胞核的形态观察以及流式细胞仪的分析,OST可以减少H_2O_2介导的PC12细胞凋亡性死亡;OST抗H_2O_2氧化应激损伤的保护作用与细胞内SOD、CAT、GSH-Px和MDA等抗氧化酶的活性密切相关。
2.在MPP~+介导的PC12细胞间接氧化应激损伤模型中,LDH分析证明能够保护质膜的完整性而抑制MPP~+介导的LDH漏出;形态观察及流式细胞仪的分析,同样证实OST可显著抑制MPP~+介导的细胞凋亡;而且,OST能够剂量依赖地降低凋亡/坏死细胞的比例。OST抑制MPP~+介导的细胞凋亡可通过阻止膜电位(MMP)的丧失,抑制ROS的产生,减少GSH的耗竭,促进SOD和CAT的激活和上调Bcl-2蛋白的表达,降低Bax/Bcl-2的比率,抑制Caspase-3激活和Cytochrome C的释放及下调核转录因子NF-кB P65蛋白。
结论:1.在受活性氧(ROS)介导的氧化应激损伤时,OST可以有效的保持PC12细胞内的抗氧化防御体系处于正常的水平,从而抑制细胞的功能衰蝎而死亡。
2. OST可能通过调节线粒体的通透性而抑制氧化应激所致的细胞凋亡。
结论和意义:
1.本课题首次将浊点萃取法应用于蛇床子中有效成分OST的提取分离和体内药代动力学研究中样品的预处理过程,并分别进行了实验优化,结果表明,该方法间便、高效、环保、适于规模操作,是一种非常有前途的分离和样品预处理方法。
2.首次证明OST对MPTP所致PD模型小鼠的行为有明显改善作用,并对MPTP所致小鼠脑中多巴胺及其代谢产物的降低有提高作用,并可保护H_2O_2和MPP~+介导的PC12细胞氧化应激引起的损伤和凋亡。通过体内、外实验研究,首次从细胞和分子水平阐明OST治疗PD的作用机制,即OST的治疗作用与维持线粒体膜电位,相应保持线粒体正常功能,抑制线粒体凋亡途径的激活及调节细胞凋亡相关蛋白的表达有关。该研究结论不仅为OST的新药创制提供实验依据,同时澄清了中药蛇床子可用于治疗PD的物质基础。
Parkinson's disease (PD) is a progressive neurodegenerative disease which defining pathological features are selective loss of dopaminergic neurons in substantia nigra pars compacta and subsequent decrease of dopamine levels in the striatum, the main target innervated by these neurons. It ranked the second common neurodegenerative disorder after Alzheimer’s disease and affected people primarily in mid and late life. Although the etiopathogenisis of PD still remains to be elucidated, the evidence strongly suggested that mitochondrial dysfunction and oxidative stress should be related to the disease. The auto-oxidation and the enzymatic oxidation of dopamine can generate ROS. Oxidative stress and/or mitochondrial impairment are believed to culminate in the activation of an apoptotic cascade which ultimately resulted in the loss of dopaminergic cells. Therefore, the regulation of intracellular ROS and modification of the apoptotic cascade may be important to prevent pathological apoptosis in PD. The L-dopamine (L-DA) is the classical strategy for the PD therapy. However, it can only improve clinical symptoms but can not retard the course of the disease. Moreover, severe side effects will appear in the long-time treatment. New drugs with good curative effect and fewer side effects are eagerly needed. Recently, a new concept for pharmecotherapy of PD called neuroprotective strategy has been proposed.
Osthole (7-methoxy-8- isopentenoxy-coumarin), which is extracted from Cnidium monnieri (Chinese name: She Chuang Zi) and has long been used in traditional Chinese medicine, has been reported to have various pharmacological properties including anti-platelet activity, anti-tumor, anti-allergic effect, anti-apoptosis, anti-oxidation, estrogen-like-effects, anti-osteoporosis, hepato- protective effect and anti-inflammation. It is a potential antioxidant that has been reported to have significant scavenging effects on oxygen free radicals and inhibit lipid peroxidation. Thus, this indicates that OST possibly has the therapeutic effect on PD.
In the present study, the protective effect of OST on dopaminergic neurons in substantia nigra pars compacta (SNC) was investigated in the PD model mice treated with MPTP in vivo. The neuroprotective effect of OST on MPP+ and H2O2-induced cytotoxicity in PC12 cells was performed in vitro. HPLC, immunohistochemistry staining, RT-PCR, Flow cytometric detection, western blot analysis, transmission electron microscope, and so on were performed in order to probe into the effect of OST on DA neurons, and identify its antioxidation and anti-apoptosis mechanism. Our studies provide strong evidences that OST is useful to the treatment of PD. Experiments were divided into five parts as follows:
Part one: the extraction and separation of OST from Cnidium monnieri
Objective:
The extract and separate technique of OST from Cnidium monnieri was improved, and the contents of OST in the extract were determined by HPLC.
Methods:
The feasibility of employing non-ionic surfactant oligoethylene glycol monoalkyl ether (Genapol X-080) as an alternative and effective solvent for the extraction of osthole in Cnidium monnieri was studied for the first time. Various experimental conditions were investigated to optimize the extraction process. Under optimum conditions, i.e. 10% Genapol X-080 (w/v), liquid/solid ratio of 100:1 (mL/g), ultrasonic-assisted extraction for 30 min, and the extraction recovery reached the highest value. For the preconcentration of osthole and imperatorin by cloud point extraction (CPE), the solution was incubated in a thermostatic water bath at 60℃for 30 min, and 2.0 moL/ L sodium chloride was added to the solution to facilitate the phase separation and increase the preconcentration factor during the CPE process.
Results:
the extraction efficiency is more than 39 percent that of conventional extraction solvent-methanol
Conclusion:
The cloud-point extraction technique is a low cost, simple and sensitive method with high cleanup effect. Finally, the method was successfully applied to separate and determine osthole from C. monnieri, respectively.
Part second: Pharmacokinetic Study of Osthole in Rat
Objective:
This research aims at systematical study on the pharmacokinetics, tissue distribution, which offers a study base to next clinical trial.
Method:
A new straightforward method based on cloud-point extraction (CPE) was developed to determine osthole in rat plasma and brain tissue by reversed phase high performance liquid chromatography. The drug concentrations were analyzed by the non-compartment model method statistical moment method of DAS 2.0 software.
Results:
The experiment results on rat implyed that the plasma concentration-time curve was fitted as second-compartmentmodel. The main pharmacokinetic parameters at dose (30 mg/kg) are as follows.
C_(max): 2.72μg/mL, t_(1/2): 5.26h, AUC_(0-t): 10.49μg h mL~(-1), AUC_(0-∞): 11.27μg h mL~(-1), t_(max): 0.56h. The peak concentration of OST in mouse plasma was 1.06μg/mL. The concentration of OST in mouse brain at 0.5-6h no significant changes in their drug drop of only 48%.
Conclusion:
The results indicated that OST readily penetrated the blood-brain barrier and could be stay in the brain for a long time.
Part three: Protective effect of OST on PD model mice treated with MPTP
Objective:
The protective effect of OST on dopaminergic neurons in substantia nigra pars compacta was investigated in the PD model mice treated with MPTP
Method:
Behaviors, HPLC, immunohischemistry technique were used to observe the damage of substantia nigral neurons respectively.
Results:
1. In the coordination exercise test, the model group mice demonstrated dyskinesia, scores obtained from the behavioral tests were decreased significantly compared to control group (P<0.05), while the scores in pretreatment groups (hight, middle) showed a marked increase to some degree (P<0.05).
2. The level of DA, DOPAC and HVA in the OST high dose group, middle dose group and madopar group were higher than that of the model group (P<0.05or P<0.01), but lower than that of normal group,the level of DA, DOPAC and HVA in OST low dose group were not significantly changed.
Conclusion:
The results suggest that OST may provide a protective effect against MPTP-induced neuronal death in parkinsonian model by reduceing DA neuron loss and increasing the level of DA, DOPAC and HVA.
Part four: Protectvie effect of OST on MPP+ and H_2O_2 induced injury in dopaminergic PC12cells
Objective:
To study the protect effects of OST on MPP+ and H_2O_2 induced injury in dopaminergic PC12cells.
Method:
The neuroprotective effects of OST on MPP+ and H_2O_2-induced oxidative stress in cultured PC12 cells were investigated. HPLC, immunohisto- chemistry staining, RT-PCR, Flow cytometric detection, western blot analysis, transmission electron microscope, and so on were performed in order to probe into the effect of OST on DA neurons, and identify its antioxidation and anti-apoptosis mechanism.
Results:
1. The neuroprotective effects of OST on H_2O_2-induced direct oxidative stress in cultured PC12 cells were investigated. Exposure of PC12cells to H_2O_2 induced a leakage of lactate dehydrogenase (LDH), which was accordant in cell viability denoted by MTT assay. By observing the nuclear morphological changes and flow cytometric analysis, pretreatment with OST markedly elevated H_2O_2-induced cell survival and antioxidant enzyme activities (GSH-Px、SOD and CAT) and decreased the level of MDA in a dose-dependent manner.
2. The neuroprotective effects of OST on MPP+-induced indirect oxidative stress in cultured PC12 cells were investigated. Treatment with OST significantly protected against MPP+-induced apoptosis observed the nuclear morphological changes and measured using flow cytometric analysis. The apoptosis in MPP+-induced PC12cells was associated with loss of mitochondrial merebrane potential, the formation of ROS, GSH depletion, suppressions of SOD and CAT, activation of caspase-3 and Cytochrome c, down-regulation of NF-кB P65、Bcl-2 and the increase in Bax/Bcl-2 ratio, and the generation of intracellular reactive oxygen species. In contrast, treatment of PC12 cells with OST remarkably reversed above-mentioned mitochondrial dysfunction.
Conclusion:
1. These results suggest that OST is very effective in preventing oxidative stress triggered by deterioration of cellular functions to reduce ROS levels. OST can protect PC12cells against oxidative stress probably due to stimulating endogenous antioxidant defense mechanisms.
2. These results suggest that OST may have the capacity to counteract the toxicity of MPP+ by attenuating change of the mitochondrial membrane permeability that is associated with oxidative stress damage.
Conclusion and significance
1. The method of cloud point extraction was used to the extraction separation of OST and the sample pretreatment of pharmacokinetics study in first time. Experimental results show that the method was convenience, efficience, environment protection, and for the scale of operation. It is a very promising method of separation and sample preparation.
2. The first demonstration that OST given orally significantly improved the abnormal behavior and increased the content of DA and its metabolites in striatum in MPTP model mice. Moreover, OST also has remarkably protective effects on MPP+ and H_2O_2-induced injury in PC12cells may be ascribed to its anti-oxidative properties, anti-apoptotic activity via maintaining normal mitochondrial function, inhibiting the activation of mitochondrial pathways of apoptosis and regulating apoptosis-related protein expression. These results indicated that OST might provide a valuable therapeutic strategy for the treatment of progressive neurodegenerative disease such as Parkinson's disease.
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