On Mechanism Of DTYMD In Treating Rheumatoid Arthritis Through Targeting MAP3K2 Based On Network Pharmacology And Molecular Docking Approaches

BackgroundRheumatoid arthritis(RA)is a chronic disease characterized by synovial inflammation.The main goals of treatment are to control pain and inflammation,reduce joint injury and disability,and maintain or improve physical function and quality of life.Treat-to-target has been established as a guiding principle for the treatment of rheumatoid arthritis?However,more and more therapeutic strategies tend to be used in combination of medicines.Thetraditional Chinese medicine has a long history for treating rheumatoid arthritis and has a remarkable effect.Under the guidance of the concept of "treating according to local conditions" and "treatment based on syndrome differentiation",we put forward theDutengyimu decoction(DTYMD).In previous studies,componds like Leonurine,kaempferol and triptolide have been investigated for therapeutic effect.However,monomeric compound hardly can represent the overall efficacy of compound formula,thus it is of significance to study the mechanism of DTYMD based on network pharmacology.Network pharmacology has been widely used for drug design and discovery,and the use of this method is based on the hypothesis that many of the effective drugs function by modulating a variety of proteins rather than a single target.Emerging evidences in systems biology suggest that highly selective compounds,compared with multitarget drugs,may exhibit lower clinical efficacy.Thus,this new paradigm holds a promising future of drug discovery and design.Along with the rapid developments in informational and computers,Molecule docking has become a powerful tool and an essential technique to predict the affinity of a ligand with receptor when it is bound to a protein receptor or enzyme.In general,we can choose the best 'binding affmity,to be the potent ligand or receptor forfurther biochemistry experiments.MEKK2 belongs to the mitogen-activated protein kinase(MAPK)kinase kinase gene family,which is involved in regulating multiple MAPK signaling pathways.MAPK signaling pathways plays an important role in regulating downstream inflammatory response.It is reported that in rheumatoid arthritis and osteoarthritis synovial tissues,MEKK2 were detected highly expressed.In our previous study,the results suggested that DTYMD has a regrulatory effect on MAPK signal pathway.However,whether DTYMD plays a regrulatory role in MAP3K2 remains unknown.Part 1 on Mechanism of the Effect of DTYMD on Rheumatoid Arthritis Based on Network Pharmacology ApproachesObjectives1.Virtual filter of the components of DTYMD.2.Target prediction of DTYMD.3.Rheumatoid arthritis targets collection.4.Construction of component-target-pathway network.MethodsThe traditional Chinese medicine herbs contained in DTYMD were searched in TCMSP,TCMID and CNKI database.All the ingredients in the formula were collected and screened according to the ADME parameters(absorption,distribution,metabolism,excretion).The results are the potential active components of DTYMD.The selected compounds were searched in TCMSP and HIT database for potential targets.Combining the results of two databases and removing duplicate targets,the remains are potential targets of DTYMD."rheumatoid arthritis" was inputted into Drugbank database and TTD database as key words to retrieve the targets related to rheumatoid arthritis.Combining the results of two databases,the duplicates were removed,thus the targets related to rheumatoid arthritis were obtained.After mapping the targets of DTYMD and rheumatoid arthritis,the potent targets of treating rheumatoid arthritis were obtained.Signal pathway network,protein-protein interaction network(PPI),component-protein-signal pathway network were constructed for visualized analysis.ResultsThe results suggest that 14 components,including tetrahydroberberine,quercetin,kaempferol and so on,had a good ADME properties.14 components from DTYMD could target 42 protiens related to rheumatoid arthritis.These 42 targets are involved in multiple signaling pathways,including IL-17 signal pathway,TNF signal pathway,Thl7 cell differentiation signal pathway,NF-?B signal pathway,osteoclast differentiation etc.ConclusionsThere are many components in DTYMD,which may play a therapeutic role in RA through multicomponents,multitargets,multipathways.The targets of DTYMD seems mainly concerning inflammation and apoptosis.The involved signaling pathways mainly related to inflammation and bone metabolism.Therefore,DTYMD may play an important role in anti-inflammatory activity in rheumatoid arthritis.Part 2 On Mechanism of DTYMD Targeting to MAP3K2 based on Molecular Docking ApproachesObjectives1.Screening of potent Ligand in DTYMD and preparation of 3D structure.2.Evaluation of binding ability between DTYMD and MAP3K2 receptor.MethodsBased on previous study of network pharmacology,14 components of DTYMD which may play a role in the treatment of rheumatoid arthritis were selected as ligands in study of molecule docking.14 ligands MOL2 format files were downloaded from the TCMSP database and converted to PDB format in PyMoL software.The ligand PDB format files were loaded in the AutoDockl.5.6 program and were detected for rotatable roots,then saved as PDBQT format for the construction of three-dimensional crystal grid.The above is the preparation of the ligand.The MEKK2(MAP3K2)three-dimensional crystal structure file was searched in PDB database and saved in PDB format.The receptor was pretreated with PyMOL software to remove water and some small molecules such as glycerin,zinc,magnesium and so on.The above is the preparation of the receptor file.The three-dimensional crystal structure of PDB receptor was uploaded in POCASA system.The receptor MEKK2 was docked with all the rotatable bonds of the ligand by semi-flexible docking method.According to the AutoDock Vina score system,the top 10 free binding energy was calculated,thus ligand-receptor complexes were evaluated.ResultsMAP3K2 receptor has three active pockets.Except quercetin,sylvestroside and isorhamnetin did not bind to the active pocket of MAP3K2 directly,other 11 ligands could bind to the active pocket of MAP3K2 in whole or in large part.ConlusionsThe results suggest that 11 of 14 ligands in DTYMD could directly bind to the active pocket of receptor,while 3 ligands could not directly bind to or merely contact with the amino acid residues of active pockets.DTYMD may work through the direct binding of 11 and ligand to MAP3K2.Part 3 Effect of DTYMD on the Expression of MAP3K2 in the RA FLSsObjectives1.Study the effect of DTYMD on MAP3K2 in RA FLSs.2.Study the effect of DTYMD on the expression of inflammatory factors.MethodsSynovial tissues were collected from RA patients after knee arthroplasty which meet the diagnostic criteria of ACR 1987.RA FLSs were isolated and cultured from synovial tissues.The survival rate of RA FLSs cells was detected by MTT assay,and the apoptosis of RA FLSs cells was detected by flow cytometry after pretreatment with different concentrations of DTYMD.The expression of MAP3K2 protein was detected by Western blot;The expression of MAP3K2 mRNA was measured by RT-qPCR;The expression of inflammatory factor such as IL-6,TNF?,MMP-1 by ELISA.ResultsThe synovium tissue was cultured for 5-7 days,and the elongated shuttle-shaped cells moved from the edge of the tissue and distributed in a radial pattern.The survival rate of RA FLSs was inhibited in a concentration-dependent manner by DTYMD,and the difference was significant(P<0.05 or P<0.01).The results of RT-qPCR suggestted that,under the pretreat of IL-1,the level of MAP3K2 mRNA was significantly increased,and the expression of MAP3K2 mRNA was significantly downregrulated compared to model group(P<0.05 or P<0.01).Western blot analysis showed that,compared with the model group,the expression of MAP3K2 protein was inhibited and the difference was statistically significant(P<0.05).The results of ELISA showed that the expression of MMP-1,IL-6 and TNF-1 decreased significantly(P<0.001).ConclusionsDTYMD might down-regulate the expression of MAP3K2 protein and nRNA in fibroblast-like synovial cells of rheumatoid arthritis,so as to inflammatory factor MMP-1,IL-6,TNF-?.Part 4 The therapeutic effect of DTYMD on arthritis in CIA mice modelObjectivsTo observe the therapeutic effect of DTYMD on CIA mice modle.Methods 42 SPF DBA/1 male nice,8 weeks old were divided into six groups:model group,DTYMD high/middle/low dose group,methotrexate group(MTX).Normal mice were taken as normal group and MTX as positive control group.The sample size of each group was 7.The collagen-induced arthritis model,CIA nouse model,was used.At the second immunization of the CIA mice model,the experimental groups were given the corresponding treatment on the 21st day of the establishment of the model.The normal and model groups were injected intraperitoneally with 0.2 ml saline.The gastric doses in the low/middle/high dose groups were 6.25 g9kg,12.5 g/kg and 25 g/kg per day,respectively.The dose of MTX was 2 mg/kg,Once a week.The mice were observed and tested every 2 days,weighed and scored for arthritis.The spleen index of mice was calculated and the pathological changes of ankle joints were observed by HE staining.ResultsCompared with the normal group,the body weight of other groups decreased significantly(P<0.05 or P<0.01).The body weight of high dose group was higher than that in other groups except normal group,but there was no statistical significance(P>0.05).The spleen index of the model group was higher than that of the normal group(P<0.01).Compared with the model group,the spleen index of the low-dose group showed a decreasing trend,but there was no statistical difference(P>0.05).3-5 days after the first immunization,the ankle joints of some mice began to appear redness and swelling,and most of the mice developed the disease after bostered immunization,and some mice suffered from joint movement restriction.But in the high-dose group,middle-dose group and MTX group,the ankle redness and swelling were not obvious,and the arthritis score was lower than that of the model group(P<0.05).ConclusionsDTYMD may reduce the swelling degree of arthritis in CIA mice,and maintain the body weight and spleen index of mice.