Study On The Identification Of The Chinese Medicines About The Kinds Of Sedation And Tranquilization By Molecule Probe Of Melatonin Receptor

Objective:Insomnia is a common sleep disorder and the high prevalence of in the population. Not only insomnia affects people’s normal work and study, but also it aggravated or induced palpitations, dizziness, headache, apoplexy and other diseases. Currently, drugs have been used for treatment of insomnia, including zolpidem, zopiclone, zaleplon, agomelatine, fluoxetine and so on. The main mechanism of drug action is to inhibit or block GABAa receptor-mediated about contruction brainstem ascending reticular activating system, so that the cerebral cortex cells from excitability into depression, and promoting the role of sedation and sleeping. Due to the effects of GABAa receptor-targeting is wide area, easy to produce a variety of side effects and drug resistance. Chinese medicine is material base and the important means of prevention, treatment and health preservation, it is an accumulated in the long-term medical practice in the mellennium. Many kinds of traditional Chinese medicine, is widely efficacy, there are hundreds of species with practical sedative effect of traditional Chinese medicines, but play a role of traditional Chinese medicines active ingredient and mechanism of action is poorly understood. So the study of traditional Chinese medicines that sedative effect on the corresponding target is to develop the role of the active ingredient in the traditional Chinese medicines as an important means of lead compound for new drugs treatment of insomnia.The cell membrane receptor is the drug targets of more than 50%. GPCRs are a large family of transmembrane receptors on the cell surface, it has been found more than a thousand of members, involved in regulating a variety of life activities, and the drug targets are most important. Currently, GPCRs are having research methods, including isotope labeling, fluorescence labeling, antibody labeling, and so on. FRET technology as a means is a rise in recent years and the more inter-molecular interactions and widely used in studying, intramolecular structure changes. By energy the detecting of fluorescence groups transfer between the groups attached thereto the r changes of molecular in pitch as characterize, to derive changes the process of the molecular structure. Different parts of GPCR molecules are connected to two fluorophores (eYFP and turquoise) and they can energy transfer, constructing molecular probes. When the receptor combined with ligand allows the molecular structure changes, FRET signal between the two fluorophores produce changes, changes in these signals can be used fluorescence microscopy or high-throughput systems high throughput screening observe high content analysis. Use of GPCR probes, real-time monitor the impact of drugs on the change of receptor molecule structure.Currently, melatonin receptor (melatonin receptor, MTR) is commonly used to treat dyssomnia, melatonin receptor has two subtypes (melatonin to MTR, MTR conformation is changed, thus the transduction of extracellularreceptor type 1, MT1 and melatonin receptor type 2, MT2). When a ligand (or drugs) binding signals into cells and activate the downstream signal, and then exerts its biological effect. Therefore, the conformational changes of MTR have become the most direct indicators of drugs screening. Natural agonist of MTR is melatonin (melatonin, MT) in vivo, MT is primarily a neuroendocrine hormone secreted by the pineal gland, MT can bind to corresponding MTR subtype by the brain barrier, MT1 primarily inhibitory neuronal activity and regulation sleeping; MT2 primarily induced phase transition, with the regulation of circadian rhythms effect. MT primarily regulate sleep and circadian rhythm by regulating diurnal rhythm of suprachiasmatic nuclei, sleep-wake biorthythm phase switching. Natural MT has the short of half-life (about 40 min) and widespread in medicine and everyday ingredients. In the traditional Chinese medicine treatment of sleep disorders are likely to have a considerable portion of rich MT in the medicinal herbs works through MTR in human. However, traditional Chinese medicine to be effective composite components, which in addition to MT there are plenty of other active ingredients may also act on the MTR. Therefore, this study will use the MTR and its molecular probes to identification non-endogenous actives of Chinese medicine that capable of maintaining sleep efficacy, the role of mild side effect profile and don’t lead to addiction or effect of dependence sedative on the MTR.This study was first constructed MTR expression vector and its corresponding Flp-In T-Rex HEK 293 stable cell strains that induced. With different concentrations of traditional Chinese medicines treat the cell strains, the use of western blot, ELISA and FRET techniques, and comprehensive evaluation of the role traditional Chinese medicines on MTR. Construction a platform about screening of active ingredient in traditional Chinese medicines on MTR successfully, it provides new ideas for screening of the sedative active ingredient in traditional Chinese medicines.Contents of this paper：1. The plasmid of pcDNA5/FRT/TO-VSV-G-MT1/2-eYFP-Turquoise and pcDNA5/FRT/TO-VSV-G-MT1/2 that chemical synthesis of MT1/2 sequence by cloning, the conversion and sequenced was successfully constructed2. The successful plasmid of MT1/2 probe were transfected to 293T cells, eYFP fluorescence protein and turquoise fluorescence protein can successfully express by the high-speed fluorescence microscopic observation.3. Successfully, the stable cells strains of MT1/2 were constructed, and they can be expressed on the cell membrane by the high-speed fluorescence microscopic observation.4. The doxycycline induces the stable cells strains of MT1/2, and starvation treatment cells, then ramelteon treatment the stable cells, FRET curve was made by high-speed fluorescence microscope, the FRET curve of MT2 molecular probe was significant fluctuations, up to standard of drug screening. But MT1 molecular probe was not meeting the standards of drug screening.5. Selecting 28 kinds of the sedation and hypnotic effection of Chinese medicine, the different concentration of Chinese medicine solution were used to treat the stable cells strains of MT1, that detecting expression of VSV-G/ERK/p-ERK in the stable cells srtains by protein extraction, ELISA and western blot. Preliminary validate of the role of traditional Chinese medicines on MT1.This study successfully construted MT1/2 plasmids and stable cell strains, Preliminary verification the significant activity of MT2 molecular probe by FRET. Preliminary verified the significant role of Cortex Moutan, Tuber Fleeceflower Stem, Uncaria rhynchophylla (Miq.)Miq. ex Havil and Valeriana jatamansii Jones on MT1. Polygala tenuifolia Willd, Ziziphus jujubaMill and Flos Daturae itself have the rich in MT, but our studing found that they didn’t to play a role by MT1. Thus, the other ingredients of rest of Chinese medicine contain playing a role about MTR except MT.