Study On The Mechanisms Of Drug Screening, Metabolism And Action Of Caenorhabditis Elegans Model

Background:Caenorhabditis elegans(C.elegans)turns into an effective model for screening HD drugs because of its complete nervous system,special physiological structure and making model easily.For example,Galantamine,disaccharide of thalassophyte and Gengnianchun etc has been found to play a role in the treatment of neurodegenerative diseases by the C.elegans model.But,the mothed using nematode model to screen traditional Chinese medicine is not perfect.In recent years,Caenorhabditis elegans has been gradually applied to study the mechanism of drug metabolism and drug action,however,the metabolism of compounds in Caenorhabditis elegans is less.And the process of separation and preparation of Harmine has been completed and the action for ameliorating Huntington’s disease(HD)of Peganum harmala L.polysaccharide(PHPS)on the basis of the early work of the laboratory.Huntington’s disease is one of the neurodegenerative disease with autosomal dominant inheritance and caused by the N-terminal fragments contain the polyQ protein,which is the result of transcription and translation from it-15 gene mutation.The mechanism of polyQ causing neurocytopathic lesions is not yet clear and clinical commonly medicine are directed against HD’s clinical symptoms without the recurred drugs appearing on the market at present.In recent years,the drug screening targeting polyQ protein for the HD is becoming a hot topic.Based on this,this project is to improve the method using of Caenorhabditis elegans model to screen drugs for HD and the metabolism mechanism of traditional Chinese medicine components in worms is studied taking Harmine as an example.Activity and mechanism of action of PHPS is conducted further research using polyQ model of C.elegans on the basis of drug screening.Objective:To consummate the method screening effective components of traditional Chinese medicine for HD using Caenorhabditis elegans model;And the method about metabolism of Chinese herbal medicine in vivo of Caenorhabditis elegans will be established taking Harmine as the example.Inhibition of polyQ protein aggregation of PHPS will be carried out a comprehensive evaluation by the AM141;To explore the application of Caenorhabditis elegans model in mechanism of drug action using Caenorhabditis elegans.Method:1.The drug screening method for the traditional Chinese medicine inhibiting the aggregation of polyQ protein is established with the AM 141 model and solid medium.The evaluation index is based on the solubility of the drug,the growth state of the nematode,the growth of aseptic growth and the difficulty of operation and the drug source are component,polysaccharide and monomer of Chinese herbal medicine.The pharmaceutical analysis components extracted from AM141,treating with harmine,using methanol:water(3:1)are analyzed by chromatographic technologies,in which fragmentation pattern of harmine in electrospray positive ion mode is summarized and metabolic products in worm of harmine are studied.2.A comprehensive evaluation of the PHPS to inhibit the aggregation of polyQ protein is in progress by phenotypic evaluation(spawning,longevity and molity performance evaluation)and fluorescence bleaching recovery method,and determining the optimum concentration of drug delivery by establishing the efficacy-concentration curve.Different state of survival E.coli OP50(live,dead and Incubation in 37 ℃)and PHPS are mixed to study different pesticide effect,and analysing the change of PHPS under the influence of E.coli OP50 using the UPLC-Q-TOF/MSE.3.After hydrolysis of three fluoroacetic acid(TFA),the PHPS and monosaccharide are derived with the 1-phenyl-3-methyl-5-pyrazolone(PMP)and the standard products are analysed using HPLC-MS.What is more,we establish and perfect the fragmentation pattern of monosaccharide derivatization products in electrospray ionization mode,and analyze the monosaccharide composition of PHPS.4.Using the western blot and qRT-PCR to analyse the expression level of polyQ protein and Htn gene.On this basis,we used different models of Caenorhabditis elegans to explore the action mechanism of PHPS.Result:1.The extraction secretion are all produced flocculatidn precipitate in liquid nutrient medium,except the liquid extracts of traditional Chinese medicine,but it is not in solid medium.Through practice we find that 12 hole plate is more suitable for the efficacy screening of Chinese herbal extracts.And after screening the PHPS has the ability to change the AM141 phenotype-polyQ protein aggregation.In the electrospray positive ion mode,the Harmine loses of methyl,CO,acetylene and imino group and RDA and alpha lysis are the main splitting decomposition;The m/z 229.0961and m/z 199.0857[M+H]+ are metabolic products in urine.of n1046(gf)about Harmine,respectively hydroxylation and demethylation.2.The number of fluorescence dots in body wall muscle cells of AM141 decreases about 30%and the survival of ASH neuron in the head of HA759 Obviously improved under the action of PHPS.And the amount of laying eggs and body bends of AM141 is higher than control after administration of PHPS,but the longevity is not changing.The inhibition rate of the mixture of PHPS and E.coli OP50 with different survival conditions was incubation at 37 ℃>live>dead.And the PHPS is decomposed into stachyose,verbascose and trisaccharide and tetrasccharide composed of aldohexose under the influence of E.coli OP50 using UPLC-Q-TOF/MSE.3.The monosaccharide derivatives are mainly dominated by alpha and beta under the electrospray negative ionization mode.There are m/z 244.8,m/z 172.7 fragments belonging to aldehyde acid monosaccharide derivatives and m/z 214.7,m/z 186.7 belonging to six reductive aldoses.The PHPS consists of mannose,rhamnose,galactohydric acid,galactose,arabia sugar,xylose and glucose.4.After normalization of polyQ protein and gene expression in the PHPS and control group of AM 141,the expression level of polyQ protein in PHPS is lower than the control group,and there is no significant difference between the two groups of gene expression.The autofluorescence of PHPS and E.coli OP50 mix interferes observation of phenotypes about nematode OP683 and VK1243,but the DLM11.Conclusion:This project consummates the method that using Caenorhabditis elegans models to find ingredients of traditional Chinese medicine curing HD and the research method of metabolism in vivo of Caenorhabditis elegans about herbal compounds taking Harmine as example.The PHPS is found to inhibit polyQ protein aggregation remarkably using the established drug screening method,it can inhibit toxic of polyQ protein aggregation and improve the phenotype of worms.And the action of PHPS is better through decomposed of E.coli OP50 and it is decomposed into stachyose,verbascose,trisaccharide and tetrasccharide composed of homogeneous galacturonic acid.The mechanism of the action mechanism of PHPS was explored by using the Caenorhabditis elegans models,which provided a feasible idea for the study of the mechanism of drug action,and laid the foundation for the wide application of C.elegans in the screening of Chinese medicine.