Screening Sedative And Hypnotic Ingredients Of Panax Notoginseng、Schisandra Chinensis With The Changes Of Spatial Structure Of Adenosine Receptors

Insomnia seriously endangers health and well-being in many people,it not only affects the quality of life,but also is closely related to diseases such as depression and anxiety.Presently,there are many insurmountable difficulties for the treatment of insomnia because of the non-specificity of drugs’ action,strong side effects and drug resistance.So to find sleep-promoting drugs with specific targets may provide clues to develop new drugs with minimal side effects.G protein coupled receptors are the main receptors for regulating the sleep of humans,two of which,adenosine receptors A1 and A2A play a crucial role in sleep regulation,and they can be considered as specific targets for screening sleep-promoting drugs.For a long time,traditional Chinese medicine provided the important resources for new drug research,which include Panax pseudoginseng and Schisandra.But which ingredient in the traditional Chinese medicine and which targets it woks is still unknown.Utill today,we are still lack of the effective and accurate platform to creen and verify the effective ingredients.In this paper,we screen the sedative and hypnotic ingredients of Panax Notoginseng.Schisandra Chinensis with the changes os spatial structure of adenosine receptor to provide the platform to screen the ingredients.In this study,we firstly constructs adenosine A1 and A2A receptors molecular probes contain the enhanced yellow(eYFP)and cyan fluorescence(Turquoise)group,which locate respectively in the intracellular third loop and C-terminal tail.The two groups can be fluorescence resonance energy transfer(FRET).When the receptor can be effected to have the spatial structure changes by the ligand,we can monitor the change of FRET baseline fluctuation.We can monitor the ingredients which can work on the adenosion receptor A1 and A2A to complete the preliminary screening on Panax pseudoginseng and Schisandra.Contents and Conclusion:The first step:Construct plasmids of pcDNA5/FRT/TO-VSV-adenosine receptor Al/A2A-eYFP-Turquoise molecular probes,and detect the fluorescence normal expression by the fluorescence microscope.Because the agonists(CCPA and CGS-21680)can activate downstream signaling pathway and cause the ERK phosphorylation,we can identify whether the adenosion can activate the downstream signaling by detect the change of pERK.At the same time,the validity of the molecular probe is proved and the stable cell line of the molecule probes are constructed too.The second step:The adenosine receptor agonists(CCPA,CGS-21680)work on the stable cell lines expressing the induced molecular probes,and monitor the changes of FRET curve with the high-speed fluorescence microscope to verify the effectiveness of the fluorescent molecular probes.Then we treat the stable cells which can expression of the molecules probe with the extaction of Panax pseudoginseng and Schisandra to prove that these drugs have the effectiveness to the adenosine A1 and A2A receptors under the monitor of the fluorescent molecular probes.The third step:Construct the adenosine Al and A2A receptors plasmids which do not contain the fluorophores gene and establish the stable cell lines.Using Western blot to detect the pERK level after traditional Chinese medicine’ treatment to confirm the ingredients in Panax pseudoginseng and Schisandra.Conclusion:eYFP and turquoise fluorescent groups were inserted in the third inner loop and C-terminus of adenosine receptors A1 and A2A,respectively,to form molecular probes by using FRET technique to detect the changes of receptors spatial structure.FRET signal was successfully recorded after treating the cells which express molecular probes with specific agonists,so it indicated that we initially established a drug screening platform based on the changes of spatial structure of adenosine receptors A1 and A2A.Later,this platform was applied to screen the extracts of Panax pseudoginseng and Schisandra,we firstly prove that the extracts of Panax pseudoginseng and Schisandra contain components which specifically target adenosine receptors A1 and A2A.