The Study On Protective Effects Of Osthole In Models For Parkinson's Disease

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 monnieriObjective: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-methanolConclusion: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 RatObjective: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 MPTPObjective:The protective effect of OST on dopaminergic neurons in substantia nigra pars compacta was investigated in the PD model mice treated with MPTPMethod: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 H₂O₂ induced injury in dopaminergic PC12cellsObjective:To study the protect effects of OST on MPP+ and H₂O₂ induced injury in dopaminergic PC12cells.Method:The neuroprotective effects of OST on MPP+ and H₂O₂-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 H₂O₂-induced direct oxidative stress in cultured PC12 cells were investigated. Exposure of PC12cells to H₂O₂ 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 H₂O₂-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 significance1. 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 H₂O₂-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.