Pharmacoloy And Mechanism Of Action Of Common Neurological Disease

Objectives:1 To construct the eukaryotic expression vector of pEGFP-N1-α-synuclein and to investigate its expression and effects on SH-SY5Y cells in vitro.2 Depression is a kind of common nervous disease, which greatly affects health-related quality of life. Hypericum perforatum extract is presumed to have anti-depression effect and be applied to patients with slight or medium depression. Generally, the main effective components of Hypericum perforatum extract in anti-depression effect are hypericin, hyperforin and so on. JingDeKang capsule(JDK) is a preparation of traditional Chinese drug from the crude drug of Hypericum perforatum extract, which mechanism is still elusive. Up to date, it is possibly regarded as acting via multi pathways and targets. A series of studies have been devoted to explore the effect of JDK and its effective constituent on the 5-HT receptor, study the signaling pathway concerned and the mechanism of the anti-depression effect of JDK as well.Methods:1 The construction of pEGFP-N1-α-synuclein vector and correlative studies on Parkinson's Disease. 1.1 To construct the eukaryotic expression vector of pEGFP-N1-α-synucleinThe special primers were designed and theα-synuclein cDNA fragment was amplified by polymerase chain reaction (PCR). And then, molecular cloning technology and enzyme digestion were used to connect the gene with the plasmid pEGFP-N1, which can be expressed in eukaryotic cells and a report gene: FLAG was already existed in the plasmid. We named the eukaryotic expression vector, which concluded our aimed geneα-synuclein as well as report gene FLAG, pEGFP-N1. The recombinant plasmids were used to transform competent E. coli DH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then the pEGFP-N1-α-synuclein was transfected to SH-SY5Y cells using LipofectamineTM2000. The expression of pEGFP-N1-α-sy nuclein was observed with fluorescence microscope and identified by Western blot.1.2 The effect of MPP⁺ on PD cell modelα-synuclein is closely related to Parkinson's disease (PD). We have constructed the vector for eukaryotic stable expression of pcDNA3.1/hisC-α-synuclein in SH-SY5Y cells. 3-methy-4-b enzyl-1,2,3,6-terathydrogenpyridine(MPTP) can be metabolized into mpp⁺ in the brain, which can produce oxygen free radical(ROS). Therefore mpp⁺ was used to produce PD model. SH-SY5Y and SH-SY5Y-α-synuclein cells were treated with MPP+ at increased concentrations for 48h. Cells survivals were detected by MTT. The cellular change in form were observed under microscope.1.3 Correlative molecular study onα-synuclein and apoptosis singal transduction pathway293T cells were transfected byα-synuclein and/or JNK3 (gene related to PD) with LipofectamineTM 2000. Caspase3 and cytochromC were detected by Western blot.2 Study on the anti-depression effect and mechanism of its active compoments in hypericum perforatum extractive2.1 The binding between JDK and its effective constituent and 5-HT1 receptor was examined by ligand-receptor binding radioassay.2.2 Chinese Hamster Ovary (CHO) cell line of stable-expressed 5-HT1a receptor was established. Different concentration of JDK incubated with CHO cells for 48h, 72h, 96h, and then proteins were extracted from the cells and expression of 5-HT1αreceptor was identified with Western Blot.2.3 Cells of stable-expressed 5-HT1αreceptor was screened and plated into 35mm confocal-special Petri dishes. When cells were confluent, add fluorochrome .After another 1h culture, wash out the fluorochrome and put the dishes on the microstat of the confocal microscope. Scanning was performed under the 20×object lens using Time Series. After determining the parameters, multiple cells with good vitality, morphology and loading were selected to scan, then ionomycin(positive control), hypericin, JDK were added respectively. Curves reflecting alteration of fluorescence intensity were also drawn.2.4 Cells of stable-expressed 5-HT1a rceptor was selected and plated into 60mm Petri dishes. After confluent, different concentrations of JDK diluted with culture medium were incubated for different time in CHO cells. According to the instructions of the Cyclic AMP EIA Kit, the content of cAMP in cells was assayed and the changes were analyzedResults:1 The construction of pEGFP-N1-α-synuclein vector and correlative studies on Parkinson's Disease. The eukaryotic expression vector for the gene encodingα-synuclein is constructed successfully. The fusion protein molecular mass is 47kd. After treatment with mpp+ for 48h, the survival rate decreased. Cellular aberrant morphogenesis was observed. SH-SY5Y-α-synuclein cells were much more sensitive than SH-SY5Y cells. Caspase3 was activated in cells cotransfected withα-synuclein and JNK3. The content of cytochromC increased in the endochylema.2 Study on the anti-depression effect and mechanism of its active compoments in hypericum perforatum extractive.2.1 Hypericum perforatum extract and JDK both have a high affinity with 5-HT1 receptors. Their binding rates are 79.74% and 77.23%. Hypericin has no affinity with 5-HT1 receptors.2.2 JDK can up-regulate 5-HT1a receptors and increase the sensitivity of 5-HT1a receptors. The 1μg·ml^-1 group has the most powerful effect. 2.3 Flou-3 can be used to detect the intracellular free calcium. There is no change of free calcium in placebo group; Ionomycin, a positive control, can increase the concentration of intracellular free calcium and then decrease the concentration to 20% of the basal level. JDK can increase the concentration of free calcium by 4-fold. Hypericin can increase the concentration of free calcium by 5-fold. 5-HT1a-CHO cells were treated with different concentrations of JDK. The content of cAMP increased by 23.49% in the 1μg·ml-1 group after 2h. The content of cAMP decreased by 21.64% in the 10μg·ml-1 group after 15min.Significant statistical analysis is indicated P<0.05.Conclusions:1 The construction of pEGFP-N1-α-synuclein vector and correlative studies on Parkinson's Disease.1.1 We constructed successfully the eukaryotic expression vector of pEGFP-N1-α-synuclein.1.2 We verified thatα-synuclein could promote the release of cytochromC from cytochondriome to endochylema. Caspase3 was also activated in the cells cotransfected withα-synuclein and JNK3. We found thatα-synuclein played an pivotal role in cell apoptosis. Compared with SH-SY5Y, SH-SY5Y-α-synuclei n cells were much more sensitive to oxidative stress. Thereby, SH-SY5Y-α-synuclein can be used as a cellular model of Parkinson's disease.2 Study on the anti-depression effect and mechanism of its active compoments in hypericum perforatum extractive. 2.1 We constructed CHO cell line transfected with 5-HT1a rceptor, which contributed to screen for new antidepressants and provided an important cell model for study the mechanism of new antidepressants.2.2 Ligand-receptor binding radioassay indicated that the anti-depression mechanism of Hypericum perforatum extract and JDK was possible to activate 5-HT1 receptors and its downstream targets.2.3 A line of evidences showed that Hypericum perforatum extract could up-regulate 5-HT1a receptors. Our experiments indicated that JDK could increase 5-HT1a receptors in 5-HT1a-CHO cells. The results implied that up-regulating receptors and enhancing the sensitiveness of receptors may be the mechanism of anti-depression drug.2.4 The intracellular concentration of free calcium can influence the release of neurotransmitters. During our experiments, we found that JDK and hypericin could increase the concentration of free calcium. Therefore JDK may play a pivotal role in the release of neurotransmitters.cAMP is an important molecule in the signal transduction. In our experiments, after treatment with high concentration of JDK, the intracellular cAMP content decreased. This result indicated that JDK may inhibit adenyl cyclase (AC) through 5-HT1 receptors and then decrease the cAMP content. After treatment with 1μg·ml-1 of JDK for 2h, the cAMP content increased. It is concluded that different concentrations of JDK influence the intracellular signal transduction in different ways.