| Objective:Type 2 diabetes mellitus(T2DM)is a serious threat to human health.Traditional Chinese Medicine has a long history in the treatment of T2DM,which Compound Chinese Traditional Medicine is the most widely used,and Coptis Chinensis Analogous Formulaes is one of them.However,the components of Coptis Chinensis Analogous Formulaes are complex,and the research on its mechanism of action in the treatment of T2DM is still in a bottleneck period.The purpose of this study is to quickly identify the chemical components in the extract of Coptis Chinensis Analogous Formulaes,to predict the targets and the signal pathways of these chemical components,to observe the curative effect of Coptis Chinensis Analogous Formulaes on zebrafish with T2DM,and to explore the common mechanism of Coptis Chinensis Analogous Formulaes in the treatment of T2DM.Then provide some data support for the clinical use of Coptis Chinensis Analogous Formulaes.Methods:1.Based on Ultra Performance Liquid Chromatography-Quadrupole-Time of Flight/Mass Spectrometry(UPLC-Q-TOF/MS),the extracts of four kinds of Coptis Chinensis Analogous Formulaes,including Antidotal Decoction of Coptis,Huanglianejiao Decoction,Rhubarb and Coptis Heart-Draining Decoction,Decoction of Pueraria,Scutellaria and coptis,were analyzed by using the method of chemomics.According to the retention time,parent ion and second order fragment ion,the chemical compositions of each Coptis C hinensis Analogous Formulaes were determined by comparing with the literature.2.In the light of the identified chemical compositions of Coptis Chinensis Analogous Formulaes,then,combining with network pharmacology and constructing the network of"Coptis Chinensis Analogous Formulaes-Traditional Chinese Medicine-Components-TargetsPathways".Firstly,selecting the targets of each chemical compositions and comparing with the targets of T2DM,and obtaining the intersection targets,in other word,the direct targets.Then,basing on these direct targets,carring out protein-protein interaction,network topology analysis and molecular docking.Finally,predicting the potential pharmacodynamics pathways of Coptis Chinensis Analogous Formulaes in the treatment of T2DM.3.Establishing the zebrafish model of T2DM by using 3%glucose solution(m/V),then set up the control group and the administration group.The administration group were given 0.05 mg/mL extract of Antidotal Decoction of Coptis,0.025 mg/mL extract of Huanglianejiao Decoction,0.1 mg/mL extract of Rhubarb and Coptis Heart-Draining Decoction,0.05 mg/mL extract of Decoction of Pueraria,Scutellaria and coptis,0.03 mg/mL metformin hydrochloride.After 48 hours of administration,the glucose content of zebrafish was detected by the kit.4.Zebrafish samples were collected and homogenized with 0.5 mL 70%methanol(V/V),centrifuged at 4? for 15 min at 12000 rpm.Then,the supernatant was extracted and analyzed by Ultra-High Performance Liquid Chromatography-Quadrupole-Exactive Orbitrap/Mass Spectrum(UHPLC-Q-Exactive Orbitrap/MS).According to the comparison between the mass spectra and the online database,the endogenous differential metabolites with variable importance in the projection(VIP)>2 and p<0.05 were screened,and the change trend of the differential metabolites after administration were observed.Finally,the pathway enrichment analysis was carried out to explain its biological significance.Results:1.A total of 160 chemical compositions were identified,including 68 Antidotal Decoction of Coptis,52 Huanglianejiao Decoction,47 Rhubarb and Coptis Heart-Draining Decoction and 65 Decoction of Pueraria,Scutellaria and coptis.It mainly includes flavonoids,alkaloids,glycosides,anthraquinones and terpenes.Dihydrooroxylin A,Groenlandicine,Berberine,5,7Dihydroxy-8,2'-Dimethoxyflavone,Palmatine,Baicalin,Epiberberine,Columbamine,Magnoflorine,Wogonin,Hispidulin,Scutevulin-7-O-?-D-Glucuronide,Coptisine,Oroxylin A,Wogonoside,Jatrorrhizine are common chemical constituents of Coptis Chinensis Analogous Formulaes.2.In the network pharmacology part,the chemical composition targets were mapped with T2DM targets,and 63 direct targets of Antidotal Decoction of Coptis,45 direct targets of Huanglianejiao Decoction,55 direct targets of Rhubarb and Coptis Heart-Draining Decoction and 59 direct targets of Decoction of Pueraria,Scutellaria and coptis were obtained.Through the direct targets of Coptis Chinensis Analogous Formulaes,20 indirect targets were obtained for each class.Molecular docking showed that the targets obtained by virtual screening had certain credibility.DPP-4,CASP3,NOS2,NOS3 and other 37 targets were the common targets of Coptis Chinensis Analogous Formulaes.AGE-RAGE signaling pathway in diabetic complications,MAPK signal pathway and HIF-1 signal pathway were the important pathways for Coptis Chinensis Analogous Formulaes to treat T2DM.This suggested that Coptis Chinensis Analogous Formulaes could treat T2DM by acting on the same targets and pathways.3.After cultured Zebrafish with glucose solution,compared with the control group,the glucose content in the model group were increased(p<0.001).Compared with the model group,the glucose content in the positive drug group and Decoction of Pueraria,Scutellaria and coptis group were decreased(p<0.001).There was no significant difference in glucose content among Antidotal Decoction of Coptis group,Huanglianejiao Decoction group and Rhubarb and Coptis Heart-Draining Decoction group.But all of them had the tendency of hypoglycemic.4.Basing on non-targeted metabolomics,we obtained 25 endogenous metabolites.The contents of L-valine,L-sorbose,uric acid,guanine,LysoPE(0:0/18:2(9Z,12Z)),LysoPE(P16:0/0:0),androsterone,LysoPI(18:1(9Z)/0:0),arachidonic acid,oleamide,sphinganine,histidinol were significantly increased in the model group(p<0.05).The contents of D-arginine,isopropylmaleic acid,L-tyrosine,allopurinol riboside,leucine,L-phenylalanine,2hydroxyphenethylamine,LysoPC(22:5(4Z,7Z,10Z,13Z,16Z)/0:0),LysoPE(16:0/0:0),LysoPC(P-16:0/0:0),LysoPE(18:0/0:0),docosahexaenoic acid,tridecyl phloretate were significantly decreased in the model group(p<0.05).Antidotal Decoction of Coptis could recall 21 differential metabolites(p<0.01),Huanglianejiao Decoction could recall 22 differential metabolites(p<0.05),Rhubarb and Coptis Heart-Draining Decoction could recall 14 differential metabolites(p<0.05),Decoction of Pueraria,Scutellaria and coptis could recall 20 differential metabolites(p<0.05),Positive drug could recall 19 differential metabolites(p<0.01).The pathway enrichment analysis of different metabolites in each group was related to phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,arachidonic acid metabolism,sphingolipid metabolism,tyrosine metabolism and other metabolic pathways,indicating that Coptis Chinensis Analogous Formulaes may improve T2DM through these metabolic pathways.Conclusion:Our research shows that 0.05 mg/mL Antidotal Decoction of Coptis,0.025 mg/mL Huanglianejiao Decoction,0.1 mg/mL Rhubarb and Coptis Heart-Draining Decoction and 0..05 mg/mL Decoction of Pueraria,Scutellaria and coptis have a hypoglycemic tendency.The mechanism of Coptis Chinensis Analogous Formulaes in improving T2DM is as follows:Berberine,Baicalin,Coptidine and other chemical components can directionally regulate DPP4,CASP3,NOS2,NOS3 and other targets,which are mediated by AGE-RAGE signaling pathway in diabetic complications,MAPK signaling pathway and HIF-1 signaling pathway.The content of endogenous metabolites such as L-valine and L-sorbitose changes,and further regulates the metabolism of amino acid metabolism,lipid metabolism,purine metabolism,sphingosine metabolism and arachidonic acid metabolism,so as to play a role in regulating glycolipid metabolism,improving IR,inhibiting cell apoptosis,anti-oxidation and antiinflammation,and finally improving T2DM. |
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