Screening Sedative And Hypnotic Components Of Shi Changpu And Acanthopanax Senticosus With The Change Of Spatial Structure Of Histamine Receptor

Insomnia is a common sleep disorder.Not only Insomnia affects people's normal work and study,but also it caused great harm to the society.With the development of pharmaceutical development and progress,people have developed many drugs in the treatment of insomnia,but these drugs have some drawbacks,such as side effects,indistinct targets of drugs.So the study of a kind of clear targeting drugs for treating insomnia gradually become the hot attention.Experimental considerations:Histamine receptor is one of the G protein coupled receptor family member that have the structure of seven transmembrane,which involved in sleep regulation.So far,there are not drugs of the treatment of insomnia which are as the target for histamine receptor.Yunnan has rich natural medicaments resource.If we can find a class of active components from natural drugs which is the target for histamine receptor,the drugs we can use active components for lead compounds to study the drugs of treatment of insomnia,we can find a kind of drugs of treatment of insomnia which has weak side effects and clear molecular target.But awe need to overcome the technical difficulty which is how to obtain active components from the huge natural resources.By constructing the molecular probe of histamine receptors and FRET Technology,we can overcome the technical difficulty.We will get the molecular expression vector by molecular biology technology,which connects into the two fluorescent proteins YFP and Turquoise.Then we can build cell lines in which this the molecular expression vector can stable express.This cell lines can use as screening platform to screen active components.Results:The first step:PCDNA5-FRT-TO-VSV-H1R-YFP(610bp)-TURQUOISE plasmid was constructed and transfected into 293Tcells.Cells which should express two kinds of fluorescent proteins were found that YFP cannot be expressed after 36 hour later under the observation of fluorescence microscopy;The second step:PCDNA5-FRT-TO-VSV-H1R(12AA,12AA ?21AA,12AA)-YFP(720bp)-TURQUOISE and PCDNA5-FRT-TO-VSV-H3R(12AA,12AA)-YFP(720bp)-TURQUOISE plasmids were then constructed.Again,293T cells transfected and expressed these fusing proteins were found that YFP can be expressed after 36 hours.Then we construct three kinds of cell lines successfully by three plasmids.The third step:Before we use doxycycline hyclate to stimulate the three stable cell lines,we do first time and concentration gradient experiments.It makes us to know the best concentration and time efficacy.After we know the best concentration and time efficacy of stimulation,we use Doxycycline three stable cell lines to detect whether three stable cell lines can express the target gene.After we know that the three stable cell lines can express the target gene,we use Western blot to detect whether the target proteins that three stable cell lines can express has the same biological activity as H1R and H3R.The forth step:We make FRET baseline by high speed fluorescent microscope.When we make FRET baseline,we find that the corresponding wave signal of H1R Intramolecular FRET sensors are weak after agonist stimulation.So it is necessary to optimize the structure of molecular probe.We detect sedative and hypnotic components in Shi Changpu and Acanthopanax senticosus by H3R Intramolecular FRET sensorsThe fifth step:In the optimization process of the probe,we use Western blot technology to screening of traditional Chinese Medicine.Conclusions:1.When we transiently transfect into 293T cells by the molecular expression vector,then YFP and Turquoise can be expressed normally.Three kinds of stable cell lines were successfully constructed.2.We can use the optimum concentration of lOng/ml in doxycycline to stimulate cell lines,and the efficacy of doxycycline increase along with time increased.The three stable cell lines can express the target gene.Three stable cell lines can activate the downstream signaling pathway.3.The corresponding wave signal of HIR Intramolecular FRET sensors are weak after agonist stimulation.4.The signal fluctuation of H3R molecular probe is relatively strong when we use agonist to stimulate it.When we use the extracts of Shi Changpu and Acanthopanax senticosus to stimulate it,the FRET baseline can be fluctuated,it proves that Shi Changpu and Acanthopanax senticosus contains sedative and hypnotic components which effects on H3R.5.We prove that Shi Changpu and Acanthopanax senticosus contains sedative and hypnotic components which effects on H3R by the activity of ERK.