An Investigation Into The Network Modulation Mechanism Of Wutou Decoction Acting On Rheumatoid Arthritis With Cold Pattern

Aim:Under the guidance of treatment based on syndrome differentiation and holism conception,this study aimed to investigate the potential molecular mechanisms of wutou decoction(WTD)acting on rheumatoid arthritis(RA)with cold pattern by adopting the integrated method of network target prediction,network analysis and experimental validations.Methods:1.Data collection and organization1.1 WTD's compositive compounds:Composite compounds of each herb in WTD were obtained from TCM Database@Taiwan(http://tcm.cmu.edu.tw/,Updated in 2012-06-28);1.2 Known RA-related genes:Known RA-related targets were obtained from four existing resources,including DrugBank database,The Online Mendelian Inheritance in Man(OMIM)database,Genetic Association Database(GAD)and Kyoto Encyclopedia of Genes and Genomes(KEGG)database;1.3 Protein-protein interaction(PPI)data:PPI data were imported from eight existing PPI databases including Human Annotated and Predicted Protein Interaction Database(HAPPI)?Reactome?Online Predicted Human Interaction Database(OPHID)?InAct?Human Protein Reference Database(HPRD)?Molecular interaction Database(MINT)?Database of Interacting Proteins(DIP)and PDZBase.2.Putative target prediction for WTDThe putative targets of WTD's compositive compounds were predicted by drug-CIPHER-CS presented by Zhao and Li,on the basis of two hypotheses:(i)drugs with similar chemical structure usually bind functionally related proteins and(ii)functional relationship between the proteins can be measured by their distance in the protein interaction network,drugCIPHER-CS achieves good prediction performance and can infer drug targets in the genome-wide scale.3.Construction and analysis of WTD herbs-putative targets-RA-related genes network3.1 We first constructed an interaction network for known RA-related targets and putative drug targets of WTD based on their direct interaction data obtained from eight existing PPI databases;3.2 Four measures-the degree,node betweeness,closeness and k-coreness-were calculated to assess the topological importance of the nodes in the network.Key hubs are those hubs whose value is larger than corresponding medians.4.Pathway enrichment analysisWe used the Database for Annotation,Visualization and Integrated Discovery(DAVID,http://david.abcc.ncifcrf.gov/home.jsp,version 6.7)for pathway enrichment analysis based on pathway data obtained from Biocarta(http://www.biocarta.com/genes/index.asp).Only BioCarTa pathways(p<0.05)were included(Revised by Bonferron method).We next designed both in vitro and in vivo experimental validations,in view of our previous study of WTD's pharmacodynamics and urinary metabonomics,and related pathway functions.5.In vivo experimental validations5.1 Induction of CIA and CIA-cold/hot models5.1.1 Induction of Collagen-Induced Arthritis(CIA):Male SD rats were injected intradermally at the base of the tail with 200 ?g bovine type ? collagen(Chondrex,Redmond,WA,USA)in 0.05 M acetic acid emulsified in incomplete Freund's adjuvant(IFA,Chondrex,Redmond,WA,USA).On day 7,rats were boosted intraperitoneally with 100 mg type ? collagen in IFA;5.1.2 Induction of the CIA-cold/hot model:Induction of the CIA-cold/hot model was based on the sole induction of the CIA model.From the day of primary immunization,male SD rats were housed in the model box(production license no.:RXZ-380A)for two hours daily with specific wind velocity(6 m/s),temperature and humidity.Precisely,rats in the CIA-hot model group were retained in the box at 37? and 95%humidity,while rats in the CIA-cold model group were housed in the box at 6? and the 60%humidity.This process lasted for a period of 21 days.5.2 Groups and treatmentMale SD rats were divided into 10 groups with 10 rats per group,which were separately categorized into the normal control group,the CIA model group,the CIA-hot model group,the CIA-hot+WTD-low/middle/high groups,the CIA-cold model group and the CIA-cold+WTD-low/middle/high groups.Treatment of WTD was performed for 22 days via oral administration from the first day,when the animals were primary immunized and the dosage selection for the low-,middle-and high-WTD were nearly equivalent to 0.5,1 and 2 times of the daily dosage of RA patients,which were 1.88 g/kg,3.75 g/kg and 7.5 g/kg,respectively.Animals in control and model groups were administrated with the same volume of distilled water,and all animals were sacrificed on the 23th day of the experiment.5.3 Severity assessment of arthritis5.3.1 Arthritis severity was evaluated by arthritis score,percentage of arthritic limbs and the time of arthritis first appeared which were performed by two independent,blinded observers.The arthritis score was the total of the scores for all 4 limbs(maximum possible arthritis score 80).Arthritis incidence values are the number positive/total number in group.In addition,the number of arthritic limbs of individual rats were counted and added to represent the number of arthritic limbs in a group.Moreover,the time of arthritis first appeared referred to the first day of the onset of the clinical symptoms of arthritis observed.The expression of IgG and IgM in serum were measured by Elisa;5.3.2 Indexes of the combination of disease and syndrome:The temperature of the articular surface,awarded to the left hind paw of male SD rats,was measured using an Infrared thermal imager(TESTO-875,Testo instruments of international trade co.,Ltd.,Schwarzwald,Germany)once a day from the day when the first signs of inflammatory were observed.And the blood flow volume,awarded to the left hind paw of male SD rats,was measured using laser Doppler flowmetry(Moor VMS-LDF,The Moore equipment company,London,UK)on day 22 after the first immunization.6.In vitro experimental validations6.1 Cell culture:HFLS-RA(Cell Applications,San Diego,CA,USA)was used for in vitro experimental validation.The cells were cultured in sterile synoviocyte growth medium(Cell Applications,USA)containing 100 U/mL 1 penicillin,80 U/mL 1 streptomycin,and 2 mM Gln-glutamine in a humidified atmosphere at 37? in the presence of 5%CO2.6.2 Induction of cell models and WTD treatment:For in vitro experimental validation,HFLS-RA cells were used at passage numbers between 4 to 8 and were incubated with 10 ng/mL of IL-1? and treated with various concentrations of WTD(0.1,0.2 and 0.4 ?g/mL)for 24 h.7.Western blotting analysis:The western blotting protocol and semi-quantitative analysis were performed to investigate the effect of WTD on the expression levels of six proteins—NCOA1(Nuclear receptor coactivator 1),NCOA2(Nuclear receptor coactivator 2),RXR-a(Retinoid X Receptor-a),PPAR-y(Peroxisome proliferator-activated receptor-y),CBP(cyclic AMP response element binding protein),and MED1(Mediator complex subunit 1)—both in the inflamed joints of different CIA rats and HFLS-RA cells treated with various concentrations of WTD.Results:1.Key putative targets of WTD acting on RA with the TCM cold pattern1.1 We constructed the network based on the direct interactions among herbs,putative targets of WTD and known RA-related targets(herbs-putative targets-known RA targets network).This network consisted of 254 nodes:5 herbs contained in WTD,218 putative targets of this formula and 31 known RA-related targets;1.2 To screen the major nodes with topological importance,4 topological features—"Degree,""Node betweenness," "Closeness" and "K value"—were calculated for each node in the network.The median values of "Degree," "Node betweenness," "Closeness" and "K value"were 5.00,0.30,8.13,and 4.00,respectively.As a result,74 major nodes—61 putative targets of WTD and 13 known RA-related targets—were identified.2.Pathway enrichment analysisAccording to the pathway enrichment analysis based on the Biocarta pathway database,the major nodes of the herbs-putative targets-known RA targets network were significantly associated with three pathways:the "Role of PPAR-y Coactivators in Obesity and Thermogenesis","Nuclear Receptors in Lipid Metabolism and Toxicity" and "NF-?B activation".According to WTD's pharmacodynamics and urinary metabonomics,and related pathway functions,we speculated that PPAR-y coactivators may be the potential targets of WTD acting on RA with cold pattern.3.In vivo experimental validations3.1 Alleviation of the severity of arthritis of CIA rats by WTD:The macroscopic performance of arthritis,including erythema and swelling,was obviously observed in CIA,CIA-cold/-hot model groups.WTD could significantly ameliorate the development and severity of arthritis in SD rats in CIA-cold groups,including lowering the mean arthritis score,arthritis incidence,percentage of arthritic limbs and delaying the time when arthritis first appeared.In contrast to the CIA-hot groups,all doses of WTD could ameliorate the severity of arthritis.However,the therapeutic effects were not as prominent as those in WTD-treated CIA-cold groups and did not show any differences with statistical significance among the CIA-hot andCIA-hot-WTD treated groups.WTD cold also lower the expression of IgG and IgM in CIA-cold groups,and the expression of IgG in CIA-hot groups;3.2 Improvement of indexes of the combination of disease and syndrome:Compared to the control groups,the temperature of the articular surface and blood flow volume in the joints were markedly up-regulated in the CIA,CIA-cold/-hot model groups.WTD could efficiently lower the temperature of the articular surface,and slightly up-regulated the blood flow volume in both CIA-cold/-hot model groups.3.3 Regulatory effects of WTD on major components in the pathway "PPAR-?coactivators in thermogenesis":Compared to the control groups,the expression levels of six proteins in the arthritis joints of CIA rats were dramatically decreased in the CIA,CIA-cold/hot model groups,while the changes in the CIA-hot model group were less obvious than those in the CIA-cold model group.After the administration of WTD,the expression levels of PPAR-?,RXR-?,MED1 and NCOA2 proteins were significantly increased in a dose-dependent manner in the CIA-cold groups.Doses of 3.75 and 7.5 g/(kg·day)WTD could enhance the expression of NCOA2 and CBP proteins in the CIA-cold groups.With regard to the CIA-hot groups,the regulatory effects of WTD on the six proteins were all weaker than those in the CIA-cold groups.4.In vitro experimental validations:Western blotting analysis showed that compared to control group,the expression of NCOA1,NCOA2,RXR-?,CBP,PPAR-? and MED1 in IL-1?-induced HFLS-RA were reduced,which were significantly reversed by WTD treatment.Summary:This study successfully established CIA model,as well as CIA hot/cold models in rats.On the basis of treating cold syndrome with hot medicine,we compared the therapeutic effect of wutou decoction acting on RA hot/cold syndrome,and detected the potential molecular mechanism.The related research conclusion is as follows:(1)Under the basis of our previous established drug target prediction method,this study constructed an interaction network for known RA-related targets and putative drug targets of WTD,and as a result,74 major nodes—61 putative targets of WTD and 13 known RA-related targets—were identified.Pathway enrichment analysis showed that these major nodes were significantly associated with three pathways:the "Role of PPAR-y Coactivators in Obesity and Thermogenesis","Nuclear Receptors in Lipid Metabolism and Toxicity" and "NF-?B activation";(2)By the combination of WTD's pharmacodynamics and urinary metabonomics,and the pathway functions of PPAR-? coactivators,we speculated that the therapeutic effects of WTD on RA with cold pattern were significantly associated with its modulation on six major nodes(PPAR-?,NCOA1,NCOA2,MED1,RXRA and CREBBP)of this pathway;(3)Experiments showed that WTD could improve the severity of RA with cold pattern more obviously than that in hot pattern in a dose-dependent manner(1.88g/kg-7.5g/kg).In particular,WTD could lower the mean arthritis score,arthritis incidence,percentage of arthritic limbs and delay the time when arthritis first appeared.WTD significantly decreased the level of IgG and IgM in CIA cold group,as well as IgG in CIA hot group.It also decreased the temperature of the articular surface and increased blood flow volume in the joints in CIA cold group;(4)Both animal and cell experiments showed that WTD improved the expression of NCOA1,NCOA2,RXR-?,CBP,PPAR-? and MED1 in CIA cold group,while had much lighter effects in CIA hot group.Conclusions:This study revealed that WTD might attenuate RA with the cold pattern partially by modulating the role of PPAR-? coactivators,which opened a new avenue for the individual treatment of RA patients,and further benefited the mechanisms investigations of traditional Chinese medicine formulae and new drugs development.