| Background:Rheumatoid arthritis(RA)belongs to the "arthralgia" in the theory of traditional Chinese medicine,and cold syndrome and hot syndrome are two most common syndromes in the clinics.Wutou decoction(WTD)is a classical formula in the treatment of RA with cold syndrome,with the benefits of prominent therapeutic efficacy,long-term administration and low toxicity.However,its pharmacological material basis and acting mechanism remains unclear,which seriously limited the further wide application of WTD in clinics.Therefore,it is urgently demanded to identify the pharmacological material basis of WTD in alleviating the disease severity of RA with cold syndrome,and explore the acting mechanismAim:This study aims to screen for the key pharmacological material basis of WTD in alleviating the RA with cold syndrome,and to explore for the potential acting mechanism by the combination of microarray and differential data analysis,network pharmacology analysis,molecular docking,surface plasmon resonance(SPR),pharmacokinetics and in vivo and in vitro experimental verification.Methods:1.Detecting the therapeutic effects of WTD in alleviating the severity of AIA(Adjuvant-induced arthritis)with cold and hot syndromes,and its network regulation mechanism on the basis of AIA rats with cold and hot syndromes,as well as microarray and differential data analysis 1.1 Establishment of AIA rats models1.1.1 AIA rat model:Male Lewis rats(6~8 weeks)were injected intradermally at the base of the tail with 10mg/mL M tuberculosis H37 Ra(Difco,BD company,New Jersey,US)suspended in liquid paraffin.1.1.2 AIA rats with cold and hot syndromes models:From the day of primary immunization,male Lewis rats were kept in the model box(production license No:RXZ-380A)for 2 h daily with certain wind velocity(6m/s),temperature(cold:4?;hot:37?)and humidity(cold:60?;hot:90?)for a period of 15 d.1.2 Grouping and treatment36 rats were randomly divided into six groups(six per group):Control group,AIA model group,AIA-cold model group,AIA-hot model group,AIA-C-WTD group(7.5g/kg),AIA-H-WTD group(7.5g/kg).Meanwhile,the rats in control group and AIA model groups were intragastrically administrated with the same volume distilled water.All treatments were administrated orally from the day of primary immunization till the 26th day.1.3 The evaluation of the arthritis1.3.1 Evaluation of the severity of arthritis:the arthritis score,hind paw thickness,body weight and pain thresholds were measured every 2 to 3 d throughout the experiments.The arthritis score was the total of the scores for all 4 limbs(maximum possible arthritis score 80).Hind paw thickness was measured by vernier caliper.Body weight was recorded with the assistance of a balance.Pain thresholds include mechanical hyperalgesia,thermal radiation pain and acetone-induced pain(three times,5 min interval).The levels of various inflammatory cytokines,including interleukin-1 beta(IL-1?),tumor necrosis factor alpha(TNF-?),interleukin-6(IL-6)and interleukin-17(IL-17)in rat sera were detected by Elisa(Enzyme-linked immunosorbent assay)using Multiskan MK3 enzyme-labeled instrument(Thermo Fisher Scientific,Waltham,MA USA).The thymus and spleen indexes were also measured by dividing the weight of the organs by the body weight of rats.1.3.2 Characterization of energy metabolic changes in RA rats:the temperature of the articular surface,water consumption,energy metabolism indicators,including Na+-K+-ATPase,Ca2+-Mg2"-ATPase,succinate dehydrogenase(SDH),lactate dehydrogenase(LDH),as well as hormone levels,including thyroid hormone 3(T3),thyroid hormone 4(T4),thyrotropin releasing hormone(TRH)and thyroid stimulating hormone(TSH)were detected.The temperature of the articular surface,awarded to the left hind paw of male Lewis rats,was measured using an Infrared thermal imager(TESTO-875,Testo AG,Schwarzwald,Germany)once a day from the day when the first signs of inflammatory were observed.Three energy metabolism indicators(Na+-K--ATPase,Ca2+-Mg2+-ATPase,SDH)were measured by colorimetric method,and LDH were measured using microplate method.The hormone levels in rat sera were measured via Elisa.Water consumption were daily measured by graduated cylinder in a 12-hour cycle.1.4 Microarray and differential data analysis1.4.1 Collection of synovial membrane samples and chip processing:Synovial tissues were collected,immersed in Trizol(Invitrogen,CA,USA)and transferred immediately into liquid nitrogen for microarray detection.Agilent Whole Rat Genome Microarray 4×44K(CatNo.:014879,Santa Clara,California,USA.)were used.Generation of biotinylated complementary RNA and the following amplification,labeling,hybridization and washing were performed in accordance with the manufacturer's instructions.1.4.2 Data collection:the arrays were scanned using a laser scanner Agilent Microarray Scanner(Cat.#G2565CA,Agilent technologies,Santa Clara,CA,US)and analyzed by Affymetrix GeneChip Operating Software(GCOS;version 1.4)according to the manufacturer' s instructions.Genes with log2 ratio ? 1 or log2 ratio?-1 and P-value<0.05 were selected for data analysis.1.4.3 Data analysis:Hierarchical clustering was performed using Agilent GeneSpring GX software to identify DEGs according to the heat map package in R(version 1.0.2,R Core Team,Vienna,Austria),then followed by the Cluster analysis(Cluster 3.0)on the basis of Euclidean distance.Next,gene symbols were uploaded to the Gene Ontology(GO,http://www.geneontology.org/)for GO annotation and enrichment analysis.The DEGs were used for performing pathway analyses in the Kyoto Encyclopedia of Genes and Genomes(KEGG,http://www.genome.jp/kegg/)database.1.5 Network construction and analysis1.5.1 The RA-cold/hot-candidate-marker-gene—WTD-effective-gene networks:the gene-gene interaction data were collected from the public database STRING(Search Tool for Known and Predicted Protein-Protein Interactions,version 10.0,http://string-db.org/),and the interaction networks were all visualized by Navigator software(Version 2.2.1).1.5.2 Topological values of the network target:the topological importance of the nodes in the networks by calculating four topological features,including node' s degree,betweenness,closeness and k-coreness,and the nodes with the value of these indicators bigger than median value were selected for functional enrichment analysis.2.Screening for the key pharmacological material basis of WTD in alleviating the AIA with cold syndrome2.1 Molecular docking2.1.1 The collection of the key putative targets of WTD and the structure of active compoundsThe key putative targets of WTD in intervening with RA with cold syndrome:the three-dimensional(3D)structures of corresponding proteins and active pocket information were obtained from a PDB website(http://www.rcsb.org/pdb/home/home.do).If there exist complex crystal structures between the target and small molecules,the active pocket is directly defined according to the position of small molecules in the complex crystal structure.Otherwise,surface pocket analysis is conducted on the target,and the largest pocket is selected for docking analysis.The structure of the active compounds of WTD in alleviating the severity RA with cold syndrome:the active compounds were selected on the basis of the chemical compounds of WTD,and their ADME characteristics.Open Babel was used for transforming the two-dimensional(2D)structures of the active compounds into 3D.2.1.2 Molecular docking analysisThe key putative pharmacological material basis of WTD in alleviating the severity RA with cold syndrome were selected with the docking score higher than 4,which represents strong binding affinity between the ligand and receptor using eHiTS(eHiTS 6.0,SimBioSysInc.eHiTS,Canada)software.2.2 SPR2.2.1 The preparation of key putative pharmacological material basis of WTD and targets:purchase or synthetic the putative pharmacological material basis which have strong binding affinity with the key putative targets,and corresponding key putative targets.2.2.2 SPR analysis:The tested proteins were fixed on CM5 chips in an amino coupling manner.Detection was applied on a Biacore 8K instrument(BIACORE 8K,GE,Sweden),and the results were analyzed using the Biacore 8K evaluation software 2.0(GE Healthcare)was used for calculating the related KD values.2.3 Pharmacokinetic detection2.3.1 Sample collection:Male Lewis rats(n=18,6-8 weeks old,200±20g in weight)were divided into three groups:Control group,Control-BAC group and Control-WTD(7.5g/kg).Blood samples(~0.25mL)were collected from the retinal venous plexus into heparinized 1.5mL polythene tubes before administration and 0,0.083,0.25,0.5,1,2,4,8,12 and 24h after administration of BAC and WTD,respectively2.3.2 Pharmacokinetic analysis:pharmacokinetic parameters were calculated via Ultra Performance Liquid Chromatography-Mass spectrum/Mass spectrum(UPLC-MS/MS)on an AB Sciex API 5500 Q Trap mass spectrometer(Toronto,Canada),interfaced with a Waters AcQuity UPLC separation module.3.Evaluating the therapeutic effects of the key pharmacological material basis of WTD in alleviating the severity of RA with cold syndrome,and exploring for the acting mechanism3.1 Establishment of AIA rats models(The same with 1.1)3.2 Grouping and treatment101 rats were randomly divided into fourteen groups:Control group(n=9),AIA-cold model group(n=9),AIA-hot model group(n=9),AIA-C-WTD group(n=9,7.5g/kg),AIA-C-BAC group(n=9),AIA-C-TLT group(n=5),AIA-C-PAE group(n=5),AIA-C-MTX group(n=9),AIA-H-WTD group(n=9,7.5g/kg),AIA-H-BAC group(n=9),AIA-H-TLT group(n=5),AIA-H-PAE group(n=5),AIA-H-MTX group(n=9).Meanwhile,the rats in control group and AIA model groups were intragastrically administrated with the same volume distilled water.All treatments were administrated orally from the day of primary immunization till the 26th day.3.3 The evaluation of the arthritis3.3.1 Evaluation of the severity of arthritis(The same with 1.3.1):In addition,assessing the pathological changes of the right hind limb of rats in different groups using hematoxylin and eosin(HE)and toluidine blue staining3.3.2 Characterization of energy metabolic changes in RA rats(The same with 1.3.2)3.3.3 Assessment of liver and kidney toxicity:liver/kidney indexes,biochemical indexes and pathological changes.3.4 Cell cultureHFLS-RA(Human fibroblast-like synoviocytes-RA)and 3T3-L1 preadipocytes were cultured in the medium containing 90%basis medium,10%fetal bovine serum(FBS)and 1%Penicillin-Streptomycin(PS),kept in the incubator with the temperature of 37? and 5%CO2.3.5 Induction and treatment of cells3.5.1 HFLS-RA:HFLS-RA cells were divided into the following groups:i)Control group:no stimulation and treatment;ii)Model group:cells were stimulated by 10 ng/mL of IL-1? for 24 hrs;iii):WTD treatment group:after stimulation,cells were treated with 0.4?g/mL WTD for 24 hrs;v):BAC treatment group:after stimulation,cells were treated with the BAC(1.17 ng/mL Paeoniflorin and 0.38 pg/mL Talatizidine,as the same content of that in 0.4 ?g/mL WTD formula)for 24 hrs;vi):MTX treatment group:after stimulation,cells were treated with 0.2 ?g/mL MTX for 24 hrs.The concentration of DMSO was less than 1‰of the solution in vitro.3.5.2 3T3-L1 preadipocytesCell viability:3T3-L1 preadipocytes seeded in 96-well plates were separately reacted with WTD and BAC for 24h at the following dosages:0.2,0.4,0.8,1.6,3.2,6.4,12.8 and 25.6?g/mL WTD and corresponding dosage BAC,and MTT assay were performed.Induction of 3T3-L1 preadipocytes and oil red oxygen staining:adipocyte differentiation was stimulated two d after reaching confluence(Day 0)with a transformation cocktail made up of insulin(lmg/mL;Sigma-Aldrich,St.Louis,MO),isobutyl-1-methylxanthine(500 mM;Sigma-Aldrich,St.Louis,MO)and dexamethasone(0.25 mM;Sigma-Aldrich,St.Louis,MO)until day 2(MDI mature medium).Then.preadipocytes were cultured in insulin-containing medium(1 ?g/mL)for two d(Day 4),followed by transferring into medium containing 10%FBS for an additional two d(Day 6),at which time more than 90%mature adipocytes appeared with obvious fat droplets accumulation.At the end of the incubation(Day 7),Oil red 0 staining was performed on 3T3-L1 adipocytes using oil red 0 dye solution kit(Solarbio,Beijing Solarbio Science&Technology Co.,Ltd,China).Effects of BAC on the adipogenesis:After the induction of cells,WTD and BAC(1.17 ng/mL PAE and 0.38 pg/mL TLT,as the same content of that in 0.4 ? g/mL WTD formula)were added into the differentiation medium at the same concentration with that in HFLS-RA cells for three d.Total proteins were prepared for western blot analysis to detect the expression of peroxisome proliferator activated receptor gamma(PPAR-?),PPAR-? coactivator lalpha(PGC-1?),uncoupling protein 1(UCP1)and PR domain containing 16(PRDM16).In addition,oil red 0 staining was performed on the 7th day after the induction.The acting mechanism of BAC on 3T3-L1 preadipocytes:the PPAR-?specific siRNA oligonucleotide(a pool of 3 target-specific 19-25nt siRNAs designed to known down gene expression,Lot:#D2517,Santa Cruz Biotechnology,CA,USA)and negative control siRNA(non-targeting 20-25nt siRNA designed as a negative control,Lot:23,Cell signaling technology,Boston,USA)was used in this study.3T3-L1 preadipocytes were seeded in six-well plates,and siRNAs transfection were performed 2 d after cells reaching confluence.Lipofectamine 3000(3.75 ?L)and 10 ?L siRNA(10 ?mol/L)were diluted and incubated in 125 ?L Opti-MEM medium(Invitrogen,Carlsbad,CA,USA)for 20min.Then,these solutions were mixed and incubated for 15min,followed by adding to the cells on the basis of Lipofectamine 3000 protocol provided by the manufacturer.The initial medium was removed and replaced with medium containing 90%DMEM and 10%FBS for 6h after transfection for a period of three d.Differentiation was induced in maturation medium with and without 0.4?g/mL WTD and BAC(The same content of PAE and TLT with WTD)for a further 3 d.Total proteins were prepared for western blot analysis 3.6 Detection of the expression levels of corresponding proteinsTo investigate the regulatory effects of WTD,BAC and MTX on the expression levels of the candidate therapeutics targets(ALOX15B,c-JUN,PPAR-y,FGF2,PTGS2,MMP13,IL-1?,TGF-?1)in both arthritic tissue samples of rats and HFLS-RA cells treated with the three drugs,as well as thermogenesis and metabolism-related proteins(PPAR-y,PGC 1?,UCP1,PRDM16)in 3T3-L1 cells,western blot analysis was performed as described previously.In addition,the changes of nuclear translocation for nuclear factors PPAR-? and c-JUN in the ankles of rats in different groups were detected using an immunohistochemical method.Results1.WTD has better therapeutic outcomes in AIA rats with cold syndrome than AIA rats with hot syndrome,and the acting mechanism differs from each other1.1 WTD is effective in alleviating the arthritis severity of AIA rats with cold syndrome,while not in AIA rats with hot syndrome1.1.1 WTD alleviates the arthritis severity of AIA rats with cold syndrome:The abilities to feed and function of the AIA model rats were markedly decreased due to severe redness or swelling in hind limbs.Especially,the ankles and knuckle joints were seriously affected with symmetrical arthrocele among rats.The administration of WTD(doses of 7.5g/kg)significantly alleviated the disease severity of rats in AIA-cold groups by decreasing arthritis score and swelling degree,reducing the levels of inflammatory cytokines(IL-1?,INF-?,IL-6 and IL-17),and thymus and spleen indexes,as well as elevating the weight and the pain thresholds.However,WTD didn't affect these parameters of rats in AIA-hot groups1.1.2 WTD improves the abnormal energy metabolic changes of AIA rats with cold syndrome:the temperature of articular surface,water consumption,various energy metabolism parameters(Na+-K+-ATPase,Ca2+-Mg2+-ATPase,SDH and LDH),and hormones(T3,T4,TRH and TSH)were lower in AIA-cold groups,while higher in AIA-hot groups,compared with control and AIA groups.WTD significantly improves the abnormal energy metabolic changes of AIA rats with cold syndrome,not on AIA rats with hot syndrome.1.2 The acting mechanism of WTD in alleviating the severity RA with cold syndrome is related to its regulation on thermogenesis and energy metabolism,as well as inf lammation-immune system-related pathwaysThe interaction networks of AIA-cold-related genes were constructed using AIA-cold related genes,AIA related genes and known RA related genes obtained from the microarray-based expression profiling.This network includes 422 nodes(225 AIA-cold related genes,33 AIA related genes,170 known RA related genes)and 1939 pairs of interaction.Especially,141 nodes were identified as key nodes according to their topological importance in the network,which were mainly involved in the modulation of thermogenesis,and inflammation-immune system.The "AIA-cold candidate marker-WTD therapeutic effect-related gene" interaction network was constructed using the links among the AIA-cold candidate markers and the WTD therapeutic effect-related genes,which contains 179 nodes(57AIA-cold related genes,40 WTD therapeutic effect-related gene and 82 known RA related genes)and 1095 pairs of interaction.Especially,63 nodes were identified as key WTD therapeutic effect nodes according to their topological importance in the network,which were most enrichment in the modulation of thermogenesis,and then the inflammation-immune system.Among them,ALOX15B and FGF2 expression was upregulated in AIA-cold rats compared to AIA rats,but downregulated by the treatment of WTD based on the microarray data.The subnetwork of ALOX15B,FGF2 and genes that interacts directly with them were constructed,the key putative nodes of which were significantly associated with the biological function of energy metabolism and hormone response.Therefore,ALOX15B-PPAR-?-PTGS2-FGF2-IL-1?-c-JUN-MMP13-TGF-?1 signal axis,involved into thermogenesis and energy metabolism,as well as maintaining the balance of inflammation-immune system,were identified as the candidate target of WTD in alleviating the severity RA.1.3 The acting mechanism of WTD in alleviating the severity of RA with hot syndrome is related to its regulation on inflammation-immune system-related pathwaysThe interaction networks of AIA-cold-related genes were constructed using AIA-hot related genes,AIA related genes and known RA related genes obtained from the microarray-based expression profiling.This network includes 236 nodes(48 AIA-hot related genes,21 AIA related genes,165 known RA related genes)and 1203 pairs of interaction.Especially,82 nodes were identified as key nodes according to their topological importance in the network,which were mainly involved in the inflammation-immune system-related pathways.The "AIA-hot candidate marker-WTD therapeutic effect-related gene"interaction network was constructed using the links among the AIA-hot candidate markers and the WTD therapeutic effect-related genes,which contains 88 nodes(15 AIA-cold related genes,8 WTD therapeutic effect-related gene and 65 known RA related genes)and 566 pairs of interaction Especially,32 nodes were identified as key WTD therapeutic effect nodes according to their topological importance in the network,which were most enrichment in the inflammation-immune system-related pathways.Among them,CD3G and THBS1 belong to two redundant genes.The subnetwork of CD3G,THBS1 and genes that interacts directly with them were constructed,the key putative nodes of which were significantly associated with the biological function of the regulation of inflammation-immune system.2.PAE and TLT are putative key pharmacological material basis of WTD formula in alleviating the severity AIA with cold syndrome2.1 PAE and TLT has strong binding affinity with corresponding proteins in ALOX15B-PPAR-?-PTGS2-FGF2-IL-1?-c-JUN-MMP13-TGF-?1 pathwayActive compounds of WTD were screened by ADME assessment and its chemical component profiling.Molecular docking simulation was performed to evaluate the binding affinities of the candidate BACs of WTD to corresponding proteins in ALOX15B-PPAR-?-PTGS2-FGF2-IL-1?-c-JUN-MMP13-TGF-?1 signal axis.As a result,22 chemical compounds were found to bind with these proteins with the eHits score more than 2.0.Especially,PAE from Radix Paeoniae Alba and TLT were demonstrated to be able to bind with PPAR-y,c-JUN,MMP13 and TGF-? 1most efficiently with the eHits score more than 4.0.Then,SPR assay was applied to confirm the direct binding efficacy of PAE and TLT to PPAR-y,c-JUN,MMP13 and TGF-?1.PAE was strongly bound to the c-JUN and MMP13 with a KD value of 30.3 ?M and 6.57 ?M,respectively,and TLT was strongly bound to the TGF-?1 and MMP13 with a KD value of 89.1 ?M and 2.63 ?M,respectively.2.2 PAE and TLT contained is highly accumulated in Lewis rats after the continuous administration of WTD and BACAfter continuous oral administration of WTD and BAC(2201.32 ?g/mL PAE and 0.72 ?g/mL TLT)for 12 d at the dosage of 0.75 g/mL each day in Lewis rats,the time to reach the maximum plasma concentration(Tmax)for PAE and TLT in rats receiving two-BAC-combination were 0.63h and 0.80h,respectively,less than that(1h and 1.6h,respectively)in rats receiving WTD.The maximum plasma concentrations(Cmax)of PAE(775ng/mL)and TLT(34.0ng/mL)in rats receiving two-BAC-combination were respectively about 3.51-fold and 14.0-fold higher than that in rats receiving WTD(221ng/mL,2.43ng/mL),as well as the corresponding area under the plasma concentration time curve(AUCINF)of PAE and TLT in rats administrated by BAC larger than that in WTD(1.29-fold and 5.62-fold,respectively),indicating higher exposure of the compounds in rats receiving BAC than WTD.Therefore,PAE and TLT were speculated to be the key pharmacological material basis of WTD in alleviating the severity RA with cold syndrome.3.BAC is effective in alleviating the arthritis severity of AIA rats with cold syndrome,and the mechanism is related to its regulation on ALOX15B-PPAR-?-PTGS2-FGF2-IL-1?-C-JUN-MMP13-TGF-?1 pathway3.1 BAC is effective in alleviating the arthritis severity of AIA rats with cold syndrome,while not in AIA rats with hot syndrome3.1.1 BAC alleviates the arthritis severity of AIA rats with cold syndrome:the abilities to feed and function of the AIA model rats were markedly decreased due to severe redness or swelling in hind limbs.Especially,the ankles and knuckle joints were seriously affected with symmetrical arthrocele among rats.The administration of BAC significantly alleviated the disease severity of rats in AIA-cold groups by decreasing arthritis score and swelling degree,reducing the levels of inflammatory cytokines(IL-1?,TNF-?,IL-6 and IL-17),and thymus and spleen indexes,elevating the weight and the pain thresholds,and improving the pathological changes of rats in AIA model groups,the effects of which were not significantly different than that of WTD and MTX.However,WTD didn't affect these parameters of rats in AIA-hot groups.3.1.2 BAC improves the abnormal energy metabolic changes of AIA rats with cold syndrome:the temperature of articular surface,water consumption,various energy metabolism parameters(Na+-K+-ATPase,Ca2+-Mg2+-ATPase,SDH and LDH),and hormones(T3,T4,TRH and TSH)were lower in AIA-cold groups,while higher in AIA-hot groups,compared with control and AIA groups.BAC significantly improves the abnormal energy metabolic changes of AIA rats-with cold syndrome,the effects of which were not significantly different than that of WTD and MTX.However,WTD and BAC didn't affect these parameters of rats in AIA-hot groups.3.1.3 BAC has no toxicity on the liver and kidney of rats:the liver/kidney indexes,biochemical indicators and pathological changes were not obviously altered in both AIA model groups and WTD/BAC treatment groups.3.2 BAC significantly regulated the expression levels of proteins in ALOX15B-PPAR-?-PTGS2-FGF2-IL-1?-c-JUN-MMP13-TGF-?1 pathway of rats in AIA-cold group,while not in AIA-hot groupWestern blot analysis indicates that compared to the control rats,ALOX15B,PTGS2,FGF2,TGF-?1,IL-1?,c-JUN and MMP13 protein expression levels were significantly increased,while the expression levels of PPAR-? protein were markedly decreased in the inflamed joints of AIA-cold rats.Similar to WTD,BAC significantly reversed the dysregulation of the ALOX15B-PPAR-?-PTGS2-FGF2-IL-1?-c-JUN-MMP13-TGF-?1 signal axis in AIA-cold rats,while not in AIA-hot rats.Consistently,the immunohistochemistry indicated the weaker(for PPAR-?)/stronger(for c-JUN)nuclear positive staining of the nuclear factors in AIA-cold ankle tissues than that in control groups,which were effectively recovered by the treatments of WTD,BAC and MTX Statistically,the expression levels of PPAR-? and c-JUN proteins in the cellular nucleus of the AIA-cold ankle tissues were markedly lower and higher than those in normal control groups,respectively.The treatments of WTD,BAC and MTX significantly reversed their abnormal nuclear expression levels.3.3 BAC significantly regulated the expression levels of proteins in ALOX15B-PPAR-?-PTGS2-FGF2-IL-1?-c-JUN-MMP13-TGF-?1 pathway on the basis of HFLS-RA induced by IL-1?Consistently with in vivo study,BAC significantly regulated the abnormal expression levels of proteins in ALOX15B-PPAR-?-PTGS2-FGF2-IL-1?-c-JUN-MMP13-TGF-?1 pathway,which were similar to that of WTD and MTX.3.4 BAC at the dosage of 0.4 ?g/mL exerted no toxicity on 3T3-L1 preadipocytesThe IC50 of the BAC and WTD on 3T3-L1 adipocytes were respectively 5.9 ? g/mL and 5.626 ?g/mL,which was much higher than the treatment concentration(both 0.4 ?g/mL).Therefore,WTD and BAC exerted no toxicity on 3T3-L1 preadipocytes at the dosage of 0.4 ?g/mL.3.5 BAC promoted the adipogenesis of 3T3-L1 preadipocytesOil Red 0 staining indicated that both of BAC promoted the biological process of adipocyte differentiation by increasing the account of lipid droplets in 3T3-L1 cells.In addition,the western blot data demonstrated that BAC can significantly enhance the expression of PPAR-?,PGC-?(PPAR-? coactivator 1 alpha),uncoupling protein 1(UCP1)and PR domain containing 16(PRDM16),which was similar to that of WTD3.6 BAC promoted the adipogenesis and lipogenesis via regulating PPAR-?-induced lipogenic proteinThe transfection of PPAR-y siRNA significantly downregulated the expression of PPAR-? in 3T3-L1 cells when compared with that in control and model groups,indicating the knockdown of PPAR-y siRNA in 3T3-L1 cells.Moreover,the expression of PPAR-y,PGC-1 ?,PRDM16 and UCP1 proteins were significantly upregulated by BAC in 3T3-L1 cells transfected with control siRNA,while no obvious up-regulation trend was observed in 3T3-L1 cells transfected with PPAR-y siRNA,which was similar to that of WTD.ConclusionThis study adopts a comprehensive strategy,that combined microarray and differential data analysis,network pharmacology analysis,molecular docking,SPR,pharmacokinetics and in vivo and in vitro experimental verification,to screen for the key pharmacological material basis of WTD,and explore for the potential acting mechanism.(1)AIA-cold/hot-related gene networks were constructed respectively on the basis of the microarray and differential data analysis of synovial tissues of rats in control and different AIA model groups.After the topological calculation of nodes in the network and pathway enrichment,it has been revealed that AIA-cold related genes were significantly enriched in the pathways that were related with energy metabolism,hormone regulation and hemodynamics.AIA-hot related genes were significantly enriched in the pathways that were related with inflammation-immune system,both of which were involved in extracellular matrix regulation,cell adhesion and nervous system regulation-related pathways.(2)After the construction and analysis of the "AIA-cold/hot candidate marker-WTD therapeutic effect-related gene" interaction network,it has been revealed that WTD exerted different intervention in AIA-cold and AIA-hot networks.The key therapeutic effect nodes in AIA-cold network are mostly enriched in the modulation of thermogenesis,and then the inflammation-immune system.Further network analysis indicated that ALOX15B-PPAR-?-PTGS2-FGF2-IL-1?-c-JUN-MMP13-TGF-?1 signal axis,involved into thermogenesis and energy metabolism,as well as maintaining the balance of inflammation-immune system,were identified as the candidate target of WTD in alleviating the severity of RA.However,the key therapeutic effect nodes in AIA-hot network are mostly enriched in the inflammation-immune system.(3)PAE and TLT has strong binding affinity with the corresponding proteins in ALOX15B-PPAR-?-PTGS2-FGF2-IL-1?-c-JUN-MMP13-TGF-?1 signaling pathway,and are highly accumulated in the plasma of rats,analyzed by the combination adoption of chemical profiling of WTD,ADME,molecular docking,SPR and pharmacokinetic detection.Therefore,PAE and TLT were speculated to be the key pharmacological material basis of WTD in alleviating the severity RA with cold syndrome.(4)In vivo experiments on the basis of AIA-cold/hot rats showed that both BAC is effective in alleviating the arthritis severity of AIA rats with cold syndrome,the mechanism of which was related to their regulation on ALOX15B-PPAR-?-PTGS2-FGF2-IL-1?-c-JUN-MMP13-TGF-?1 signaling pathway,which was similar to that of WTD.(5)In vitro experiments that on the basis of HFLS-RA and 3T3-L1 preadipocytes showed that BAC,on one hand,alleviated the severity of the disease via regulating inflammatory-immune related pathways,on the other hand,promoted the adipogenesis and lipogenesis via regulating PPAR-?-induced lipogenic protein(UCP1,PGC-1?,PRDM16),which was similar to that of WTDAbove all,this study adopted a comprehensive strategy that combines microarray and differential data analysis,network pharmacology analysis,molecular docking,SPR,pharmacokinetics,and in vivo and in vitro experimental verifications,to find out that WTD is effective in alleviating the severity of RA with cold syndrome by regulating ALOX15B-PPAR-y-PTGS2-FGF2-IL-1?-c-JUN-MMP13-TGF-?1 signal axis,which were significantly associated with the thermogenesis and energy metabolism,as well as maintaining the balance of inflammation-immune system.Meaningfully,PAE originated from Radix Aconiti and TLT originated from Radix Paeoniae Alba were identified to be the key pharmacological material basis of WTD in exerting corresponding therapeutic effects.This study preliminarily revealed the regulation mechanism of "hot medicine cures cold syndrome",and its relation with the therapeutic effects,which provided the references and experimental evidence for the further individualized treatment of RA. |
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