From The "inflammation-immunity" Network, Explore The Mechanism Of Baihu Jiaguizhi Decoction In Intervening Rheumatoid Arthritis

AimsBaihu-Guizhi decoction(BHGZD),a classic TCM-based herbal formula,was selected as the research object in current study.An integrative approach,including global gene expression profiling and network pharmacology analysis,was programmed as the route of "the establishment of BHGZD’s compositive compounds library→putative targets prediction→establishment and analysis of ’RA-related genes-BHGZD putative targets’network→key network targets screening and mechanism mining→experimental validation".This study aimed to identify the pharmacodynamic material basis,to reveal the underlying molecular mechanism,and to screen key putative targets of BHGZD acting on rheumatoid arthritis(RA)from "inflammation-immune" network.Methods1.The construction of Adjuvant-Induced Arthritis(AIA)rat model and pharmacodynamic evaluation of BHGZD acting on RA.1.1 Induction of AIA modelMale Lewis rats were immunized intradermally at the base of the tail with 0.1 mL Freund’s complete adjuvant(CFA)(1 mg of heat-killed Mycobacterium tuberculosis suspended in 0.1 mL paraffin oil;Difco).1.2 Animal groups,treatment and anatomyMale Lewis rats were divided into six groups:a normal control group(Con,n=9),an AIA model control group(AIA,n=9),BHGZD-low/middle/high groups(n=6 per group)and a 0.2mg·kg-1 Methotrexate(MTX)group(MTX,n=6).Five groups rats,including AIA model,BHGZD-low/middle/high and MTX groups,were induced AIA rat model.The Con group rats were injected with an equal volume of saline instead of CFA.BHGZD solution was prepared at a concentration of 2g·mL-1 and delivered by oral administration.The dosage selections for the low-,middle-and high-BHGZD groups were nearly equivalent to 0.5,1 and 2 times the daily RA patient dosage.The AIA rats were treated with 5.4g·(kg·day)-1 BHGZD(BHGZD-low),10.7g·(kg·day)-1 BHGZD(BHGZD-middle)and 21.4g·(kg·day)-1 BHGZD(BHGZD-high).The rats were administered with a volume of 1 mL/100g body weight from the day of immunization.The BHGZD intervention groups were administered for 6 days after an interval of a day,and MTX intervention group was administered for 3 days after an interval of a day.The Con group and the AIA model group were taken an equal volume of distilled water,and each group of animals was administered until the 25th day of immunization.On the day 26,the animals were sacrificed,and tissues such as joints,liver and kidney,and synovium were taken,disposed and reserved according to the operation protocol.1.3 Assessment of arthritisSeverity assessment,pain threshold,and histology of joint of AIA rats were determinated to clarify the treatment of BHGZD acting on RA.Sections of livers and kidneys from paraffin-embedded tissue(4 μm)were stained with hematoxylin and eosin(H&E)to assess pathologic changes.The sections were evaluated by two pathologists who were blinded with regard to liver and kidney pathologic changes in different groups.The liver and kidney index in a group were calculated as following formula:Liver or kidney tissue weight(mg)Body weight(g)2.An integrative approach combined global gene expression profiling and network pharmacology analysis2.1 Global gene expression profiling and the differentially expressed genes screeningRNA samples from synovial tissue were extracted using the Agilent Low Input Quick Amp Labeling Kit and the labeled cRNA was purified using the RNeasy mini kit.The limma algorithm package was used as the normalization algorithm for gene expression profiling(Agilent Whole Rat Genome Microarray 4×44K)and for the differentially expressed genes(DEGs)identification between AIA model group and Con group using Fold Change≤0.5 or Fold Change≥2,and T-test p-value<0.05 as the standard,which was regarded as AIA-related genes.2.2 The construction of BHGZD’s compositive compounds libraryThe chemical constitutes of five herbs contained in BHGZD were identified by the ultra-fast liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(UFLC-Q-TOF-MS/MS).The composite compounds of five herbs contained in BHGZD were obtained from TCM Database@Taiwan.2.3 Prediction of BHGZD putative targets Based on our previous studies,the putative targets of BHGZD were predicted.2.4 Network construction and analysis Based on the interactions between five herbs contained in BHGZD and putative targets,we constructed an interaction network for RA-related targets and putative targets of BHGZD according to the molecular interaction data,including known RA-related targets collected from existing databases and AIA-related genes obtained by global gene expression profiling.Three features,including "Degree","Betweenness Centrality" and "Closeness Centrality",were calculated so that assess the topological importance of the nodes in the network.Key hubs are those hubs whose value is larger than corresponding medians.RA-related pathways were screened by statistical calculation and pathway enrichment analysis.Then,experimental validation was performed.3.Investigation of the regulation of BHGZD on the predicted signal axisThe western blotting protocol and semi-quantitative analysis were performed to investigate the effect of BHGZD on the expression levels of four proteins in the inflamed joints of different AIA rats,including TLR4(Toll-like receptor 4),c-Fos/APl(Fos proto-oncogene/AP-1 transcription factor subunit),IL2(interleukin 2)and TNF-α(tumor necrosis factor-alpha)in TLR4-c-Fos-IL2-TNF-α signaling axis.Results1.Successful establishment of AIA rat model and pharmacodynamic evaluation of BHGZD acting on RAThe macroscopic performance of arthritis,including erythema and swelling,was obviously observed in AIA model group,indicating that the AIA rat model was successfully established.BHGZD could significantly ameliorate the development and severity of arthritis,relieve the pain and improve the pathological changes of joints in Lewis rats in AIA groups in a dose-dependent manner(doses of 5.4 g·(kg·day)-1～21.4g·(kg·day)-1,p<0.05,p<0.01,or p<0.001).Liver and kidney damages caused by BHGZD administration had not observed using histopathological evaluation of liver and kidney in different groups.2.Molecular mechanism prediction of BHGZD in RA "inflammatory-immune"imbalance network2.1 Screening of RA-related geneA total of 26 differentially expressed genes(DEGs,AIA vs.Con)were obtained by the global gene expression profiling(fold change>2,p<0.05),9 of which were up-regulated and 17 were down-regulated.2.2 Collection of RA-related genesA total of 234 RA-related genes were obtained,including 208 known RA-related genes collected from the Drugbank and KEGG databases,and 26 AIA-related genes screened by global gene expression profiling.2.3 The construction of BHGZD’s compositive compounds libraryWith UFLC-Q-TOF-MS/MS system,a total of 41 chemical compositions were identified in the water extract of BHGZD based on reference standards,chromatographic elution behaviors,chemical composition,mass fragment patterns as well as mass spectral library.Among them,a total of 19 chemical compositions were authenticated by comparing the reference standards.A total of 123 composite compounds in BHGZD were obtained from TCM Database@Taiwan.2.4 The putative targets of BHGZD acting on RAA total of 1312 BHGZD putative targets were predicted based on the similarities in drug structure and function.The numbers of putative targets of BHGZD herbs,including Cinnamomi Ramulus,Glycyrrhizae Radix Et Rhizoma,Anemarrhenae Rhizoma,Gypsum Fibrosum,Round-shaped Rice were 545,701,197,424,266,respectively.Among them,53 known RA-related gene were included.2.5 Network construction and analysisWe constructed the network based on the direct interactions between herbs and putative targets of BHGZD.This network consisted of 1281 nodes.To screen the major nodes with topological importance,three topological features-"Degree","Betweenness Centrality" and "Closeness Centrality"-were calculated for each node in the network.The median values of "Degree","Betweenness Centrality" and "Closeness Centrality" were 28,0.00067746,and 0.39119497,respectively.As a result,623 major nodes-19 putative targets of BHGZD and 39 known RA-related targets-were identified.According to the pathway enrichment analysis,the major nodes of the RA-related genes-putative targets of BHGZD network were significantly associated with the following pathways:Toll-like receptor signaling pathway,B cell receptor signaling pathway,VEGF signaling pathway,T cell receptor signaling pathway,etc.Interestingly,the major nodes were significantly enriched in Fatty acid biosynthesis,Carbohydrate digestion and absorption,and Adipocytokine signaling pathway.Moreover,the four moleculars,including TLR4,c-Fos,IL2 and TNF-α remarkably enriched in Toll-like receptor signaling pathway and T cell receptor signaling pathway.3.BHGZD decreased the abnormally high expressions of TLR4-c-Fos-IL2-TNF-αsignaling axis related proteins of the "inflammatory-immune" imbalance network.Compared to Con group,the expression levels of four proteins in the arthritis joints of AIA rats were dramatically increased in the AIA model group(all p<0.001),while the expression levels of TLR4,c-Fos,IL2 and TNF-α were significantly decreased after the administration of BHGZD(p<0.05,p<0.01 or p<0.001).Conclusion1 In the current study,in vivo experimental validation offered strong evidence that BHGZD could markedly ameliorate the arthritis symptoms,including erythema and swelling,based on AIA rat model,which confirmed the effects of BHGZD acting on RA.2 An integrative approach based on global gene expression profiling and network pharmacology analysis was performed to screen the key putative targets enriched in"inflammatory-immune" imbalance network.Among them,the four major nodes,including TLR4,c-Fos,IL2 and TNF-a were significantly associated with Toll-like receptor signaling pathway and T cell receptor signaling pathway.3 Experimental validation offered convincing evidence that BHGZD may ameliorate partially RA by reversing inflammation-immune system imbalance and regulating the TLR4-c-Fos-IL2-TNF-a signaling axis4 Interestingly,the major nodes in the network were also significantly enriched in Fatty acid biosynthesis,Carbohydrate digestion and absorption,and Adipocytokine signaling pathway.Overall,the current study preliminarily revealed the underlying mechanism of action of BHGZD acting on RA from“inflammation-immunization" network,that provided the experimental basis for scientific connotation and rational application of this formula,and that played a crucial role in further "disease-syndromes-formulas" researches.