Pharmacodynamic Effects And Mechanisms Of Artesunate On Ulcerative Colitis Based On Network Pharmacology And Molecular Docking

Objective1.To predict the potential targets of artesunate on ulcerative colitis based on network pharmacology approach.2.To further screen the potential targets of artesunate on ulcerative colitis based on molecular docking.3.To observe the efficacy of artesunate on 3%DSS-induced experimental ulcerative colitis.4.To verify the expression of the key targets screened by network pharmacology and molecular docking in experimental colitis and to study the effect of artesunate on it.5.To investigate the pathogenesis of ulcerative colitis and potential mechanism of artesunate on ulcerative colitis through the perspective of apoptosis.Methods1.In this study,potential targets of artesunate were predicted by ChEMBL database,Swiss Target Prediction database,Target-Prediction database and PharmMapper online server.Disease targets of ulcerative colitis were retrieved from databases,text mining tools and GEO chip analysis.After that,the protein-protein interaction(PPI)network of artesunate and ulcerative colitis targets was constructed and cluster analysis was carried out to obtain key targets of artesunate and ulcerative colitis.Furthermore,intersection of the key targets of artesunate and ulcerative colitis was conducted to obtain the potential targets of artesunate in the treatment of ulcerative colitis and PPI network was constructed.Finally,gene ontology analysis(GO Analysis)and pathway enrichment analysis were carried out to explore the potential mechanism of artesunate on ulcerative colitis.2.Molecule structure of artesunate was obtained from DrugBank database and was used as docking ligand.Crystal structures of the potential targets were obtained from PDB database and were used as docking receptors.Active sites of the target proteins were predicted by online protein active site prediction system POCOSA1.1.After that,preparations of ligands and receptors and docking parameters were settled by AutoDockTools.Then molecular docking and re-docking of artesunate and 20 target proteins were completed by Autodock4.0.Finally,visual display of the results was completed by Pymol.Hydrogen bonds and hydrophobic interactions analysis of the ligand-receptor complexes were analyzed by Ligplot.3.72 C57 mice were randomly divided into one control group and five experimental groups(n=12),which included DSS model group,sulfasalazine group(200mg/kg/d),artesunate low-dose group(75 mg/kg/d),artesunate medium-dose group(150mg/kg/d)and artesunate high-dose group(300 mg/kg/d).Experimental ulcerative colitis was established by drinking 3%DSS solution freely for 7 days in experimental groups.Drug interventions were conducted intragastrically 8 hours after initial supply of 3%DSS solution and last for 7 days.During the experimental period,general condition,body weight change,stool consistency and bleeding of mice were recorded,then disease activity index(DAI)was measured in accordance with a previously reported method.Mice were sacrificed on the 8th day of experiment.The efficacy and mechanistic action of artesunate were evaluated by means of body weight change,colon length,DAI and histopathological approach.4.Western-Blot and RT-qPCR approach was used to verify the expression of IL-6,TP53 and VEGFA in each group.5.Immunohistochemistry approach was used to detect and compare the expression of Bax,Bcl-2 and caspase-3 protein in colonic tissue,while RT-qPCR approach was used to verify the expression of TP53,Bax and Bcl-2 mRNA expression.Results1.154 drug targets and 2104 disease targets were screened by network pharmacology approach.Then 137 key targets of artesunate and 1423 key targets of ulcerative colitis were obtained from PPI network construction.After further intersecting key targets of drug and disease,22 potential targets of artesunate on ulcerative colitis,such as IL6,VEGFA,TP53,CCND1 and CTNNB1,were screened.The gene functions of these targets involved extracellular regulation,exocrine regulation,cell proliferation regulation,apoptosis regulation,small molecule metabolism,regulation of the immune system and so on.Pathways such as p53 signaling pathway,WNT signaling pathway,JAK/STAT signaling pathway are included.2.Docking results showed that artesunate had formed ligand-protein complexes with 20 of 22 key receptors,among which 19 targets except MS4A1 were docked with artesunate at the active pocket.After the corresponding 19 optimal conformations were re-docked,the results showed that the RMSD values of 19 receptor-ligand complexes were all less than 2.0.Among the 19 optimal complexes formed,first five target receptors with lowest binding energy were AKR1B1(-11.63 kcal/mol),GZMB(-11.52 kcal/mol),TP53(-11.27 kcal/mol),IL6(-10.74 kcal/mol)and LDHB(-10.68 kcal/mol)respectively.With the further interactions of target information from network pharmacology approach,we obtained the targets that both act as key roles in the drug-disease network and have a good combination to artesunate,of which the top five target protein receptors are IL6,TP53,VEGFA,AKR1B1 and GZMB respectively.Furthermore,we analyzed the 19 optimal conformations respectively.Results showed that there were hydrogen bonds and hydrophobic interactions in the binding sites of 19 ligand-receptor complexes.The oxygen atom on the terminal carboxyl group(-COOH)of artesunate is its main hydrogen bond donor,which forms hydrogen bonds with 18 of 19 targets' residues(TP53,IL6,VEGFA,GZMB,MMP2,CCND1,LYN,ANXA5,CTNNB1,ESR1,CD2,GALM,TF,RBKS,IFNGR1,PCNA,SRM,LDHB).IL6 and LDHB are the targets generated most hydrogen bonds with artesunate.3.In this study,experimental ulcerative colitis was successfully established by free drinking with 3%DSS(MW 36000?50000)solution.Significant differences were founded in body weight,colon length,DAI and pathological score between mice in DSS group and in control group(P<0.01).On the 8th day of the experiment,mice in sulfasalazine and low-dose artesunate groups had a notably higher body weight,longer colon length,lower DAI score than that in DSS group(P<0.01).Meanwhile,mice in medium-dose artesunate group also had a higher body weight,longer colon length,lower DAI score than that in DSS group(P<0.05).But there was no significant difference between DSS group and high-dose artesunate group in body weight,colon length and DAI score(P>0.05).Histopathological result showed that mice in sulfasalazine and low-dose artesunate group had a notably lower HI score than that in DSS group(5.42±1.83 vs 8.42±1.08,P<0.01),mice in medium-dose artesunate group also had a lower HI score than that in DSS group(P<0.05),while no significant difference was found between DSS group and high-dose artesunate group in HI score.4.Compared with the control group,western blotting results demonstrated that the expression of TP53 and VEGFA in the colon of mice in DSS group were significantly decreased(P<0.05),and the expression of IL6 mRNA was significantly increased(P<0.05).After oral administration of different drugs,mice in sulfasalazine,medium-dose and low-dose artesunate group turned to have a notably higher expression of TP53 and VEGFA,and a notably lower expression of IL6 than that in DSS group(P<0.05),while no significant difference was found between DSS group and high-dose artesunate group in TP53,VEGFA and IL6 mRNA expression.Real-time qPCR analysis revealed that the expression of TP53 and VEGFA mRNA in the colon of mice in DSS group were significantly decreased(P<0.01),and the expression of IL6 mRNA was significantly increased(P<0.01).After oral administration of different drugs,mice in sulfasalazine and low-dose artesunate group turned to have a notably higher expression of TP53 and VEGFA mRNA,and a notably lower expression of IL6 mRNA than that in DSS group(P<0.01),mice in medium-dose artesunate group also had a higher expression of TP53 and VEGFA mRNA,and a lower expression of IL6 mRNA than that in DSS group(P<0.05),while no significant difference was found between DSS group and high-dose artesunate group in TP53,VEGFA and IL6 mRNA expression.5.In comparison of key targets'expression in P53 signaling pathway,results of this study showed that the expression of Bax mRNA decreased significantly and the expression of Bcl-2 mRNA increased significantly in colon of mice in DSS group compared with that in control group(P<0.01).After oral administration of different drugs,mice in sulfasalazine and low-dose artesunate group turned to have a notably higher expression of Bax mRNA,and a notably lower expression of Bcl-2 mRNA than that in DSS group(P<0.01),mice in medium-dose artesunate group also had a higher expression of Bax mRNA,and a lower expression of Bcl-2 mRNA than that in DSS group(P<0.05),while no significant difference was found between DSS group and high-dose artesunate group in Bax,Bcl-2 mRNA expression.Further detection of Caspase-3 showed that its expression in colonic mucosa of 3%DSS model group was significantly lower than that of normal rats.After treatment with sulfasalazine,low dose artesunate and middle dose artesunate,expression of Caspase-3 in colonic mucosa of mice was significantly higher than that of the model group(P<0.01),while there was no significant difference between the artesunate high dose group and the model group(P>0.05).Conclusion1.Target prediction outcomes of network pharmacology approach suggest the multi-targets and multi-pathways pharmacologic properties of artesunate.The outcomes also reveal that artesunate might be a potential alternative medial approach for treating ulcerative colitis via targets such as IL6,VEGFA,TP53,CCND1,CTNNB1 and mechanisms such as extracellular regulation,exocrine regulation,cell proliferation regulation,apoptosis regulation,small molecule metabolism and immune system regulation.2.Result of molecular docking approach shows that artesunate could form stable ligand-receptor complexes with targets such as AKR1B1,GZMB,TP53,IL6 and LDHB,which further indicates the multi-target pharmacologi c property of artesunate.Combined with the results of network pharmacolo gical research,it might suggests the potential pharmacologic properties of artesunate on ulcerative colitis through key targets such as IL6,TP53,VEGFA and mechanisms related to immune and inflammatory,apoptosis and m ucosal protection.3.Results of animal experiments shows that both low and middle dose s of artesunate could effectively alleviate the inflammation and damage b rought by experimental colitis.Its mechanism might be related to the inc rease in apoptosis of inflammatory cells which is associated with the upregulation of p53 signal pathway.