Study On The Molecular Mechanism Of Mongolian Medicine Cistanche Verbascoside Promoting The Release Of The Neurotransmitter Acetylcholine

Objective:To study the effect of Verbascoside（OC1）on the release of neurotransmitter acetylcholine during the onset of Alzheimer’s disease（AD）.Methods:（1）The MTT method was used to detect the cell survival rate,and determine the effect dose and duration of Amyloidβ-protein(Aβ_1-42)induced PC12 cell injury to establish an in vitro model of AD.（2）Computer-aided drug design simulates the docking of OC1 with L-type and N-type calcium channels,and virtually screens the selectivity of OC1 for Ca²⁺channel subtype.（3）Detect the effect of hair OC1 on calcium ion concentration in AD cell model.（4）Quantitative omics study of whole protein phosphorylation modification to analyze the effect of OC1 on phosphorylated protein in AD cell model.（5）Western Blot was used to detect the effect of OC1 on the expression levels of p-Ca MKⅡ（Thr286）and the vesicle proteins Synapsin1,p-Synapsin1（Ser603）,Synaptotagmin-1 and Synaptophysin.（6）The kit detects the effect of OC1 on the activity of acetylcholinesterase（ACh E）in AD cell model.（7）UPLC/Q Exactive MS method was used to detect the effect of OC1 on acetylcholine（ACh）release in AD cell model.Results:（1）24 hours after Aβ_1-42（10μmol/L）exposure of PC12 cells,the cell survival rate detected by MTT reached（48.91±3.21）%,which was significantly different from the Control group（P<0.05）,indicating that AD damage was successfully established model.After drug intervention,cell survival rates increased to varying degrees.（2）The binding energies of OC1 to L-type and N-type calcium channels were-1.54 and 138.13,respectively.According to the principle that the smaller the binding energy,the easier it is to bind,OC1 proved to be more likely to affect intracellular calcium ions by binding to L-type calcium channels.（3）Compared with the Control group,the intracellular calcium fluorescence intensity of OC1（2μg/m L,10μg/m L）was significantly increased（P<0.05）;compared with the OC1 10μg/m L group,EGTA chelation was given.Extracellular calcium.It can be seen from the figure that the intracellular calcium ion fluorescence intensity is significantly reduced,which proves that OC1 can promote extracellular calcium ion influx（P<0.05）;compared with the OC1 10μg/m L group,L-type calcium channels were administered.After the blocking agent verapamil hydrochloride and the N-type calcium channel blocking agent conotoxin,the intracellular calcium ion fluorescence intensity decreased（P<0.05）.（4）Through differential protein modification analysis,it was found that the expression of 359 loci of 295 proteins had changed after OC1 administration,including 154 loci up-regulated and 205 loci down-regulated;by GO classification,KEGG pathway and protein domain three At the level of enrichment analysis,it was found that the differentially modified proteins were significantly enriched to activate calcium channels after OC1 administration.Based on the enrichment results,we performed KEGG cluster analysis on the differentially modified proteins and found that the differentially modified proteins were rich after OC1 administration.Calcium signaling pathway（mo04020 Calcium signaling pathway）was collected,in which the expression of protein p-Ca MKⅡ（Thr286）was significantly up-regulated.（5）Western Blot results showed that compared with the Control group,the expression of p-Ca MKⅡ（Thr286）and vesicle proteins Synapsin1,p-Synapsin1（Ser603）,Synaptotagmin-1,and Synaptophysin after administration of Aβ_1-42（10μmol/L）compared with the Control group.Compared with the model group,the expression of p-Ca MKⅡ（Thr286）and vesicle proteins Synapsin1,p-Synapsin1（Ser603）,Synaptophysin and Synaptotagmin-1 were increased after drug intervention.（6）The test of the kit found that compared with the Control group,the ACh E activity of the Aβ_1-42（10μmol/L）group was significantly increased（P<0.05）;the ACh E activity was decreased after the drug intervention（P<0.05）.（7）UPLC/Q Exactive MS method was established for the determination of ACh secretion from PC12 cells.ACh has a good linear relationship in the concentration range of 1-100 ng/m L,with a minimum limit of quantification of 1 ng/m L.The recovery rate of ACh is high,and the RSD is less than 5%.The sample is placed（25℃,12h）,and frozen（-80℃,30 days）,Good stability after repeated freeze-thaw cycles（-80℃,3times）.The results showed that compared with the Control group,the amount of ACh released by the Aβ_1-42（10μmol/L）group gradually decreased with time,and the ACh release increased after drug intervention,and reached a peak at 9 minutes.Conclusion:Verbascoside can promote the release of the neurotransmitter acetylcholine.The mechanism may be that Verbascoside promotes extracellular calcium ion inflow through binding with L-type calcium channels,increases the expression of p-Ca MKⅡ（Thr286）and vesicle-related proteins,and promotes acetylcholine release.In addition,OC1 can increase the action time of synaptic gap ACh by reducing the activity of ACh E.In summary,OC1 may be used in the treatment of AD by affecting the various release and hydrolysis processes of ACh.