Study On The Mechanisms Of Wang-bi Capsule In Inhibiting Joint Destruction Of Rat With Collagen-induced Arthritis

ObjectiveRheumatoid arthritis(RA)is an autoimmune disease with high disability rate due to joint destruction.Although some agents have shown effect of articular protection to some extent,how to effectively block joint destruction is still a great challenge for clinical doctors because of lacking sufficient joint protection agents up to date.It is important to find more drugs with protective effects for joint.Wang-bi capsule(WB)has been used for RA treatment for many years and exhibited protection for joint.However,its molecular mechanisms are still obscured.In this study,we aimed to explore the molecular mechanisms that WB inhibited joint destruction in CIA rats based on transcriptomics and proteomics.Methods1.Therapeutic effect of WB on CIA ratsCIA rats were divided into five groups:normal group,model group,WB-T1 group,WB-T2 group,and WB-T3 group.WB-T1 group,WB-T2 group and WB-T3 group was intragastric administrated with WB(0.74g/kg/d,which was equal to that used in RA patients).Model group and normal group were given corresponding volume of distilled water.When treatment was over,the therapeutic effect of WB was evaluated based on arthritis index(AI),serum multi-factor test,serum bone metabolism markers test,H&E staining,safranin O green staining,TRAP staining and Micro-CT analysis.2.Tarsal bone transcriptomics-based analysis to explore the potential mechanisms that WB inhibited joint destructionThree tarsal bone samples from each model group,normal group and WB-T1 group were used to extract total RNA,and then which were reverse transcript to build the library for DNBSEQ sequencing.The different expression genes(DEGs)were analyzed to dissect significant enrich pathways associated with joint destruction.The networks were generated by Ingenuity Pathway Analysis(IPA).The key DEGs were validated by RT-qPCR.3.Synovium proteomics-based analysis to explore the mechanisms that WB inhibited joint destructionSynovium tissue samples from model group,normal group and WB-T1 group were used to extract protein.The protein from three groups was labeled by iTRAQ reagents,and then were analyzed by liquid chromatography and mass spectrometry(LC-MS).The different expression proteins(DEPs)were analyzed by IPA to dig the potential pathways and key proteins.In addition,the key proteins were validated by Western Blot test.Results1.Therapeutic effect of WB on CIA rats(1)Compared with model group,WB-T1 group could significantly decrease the AI score(P<0.01),the serum levels of inflammatory cytokines such as TNF-α,IL-6,IL-17α,IL-1α,IL-1β,IL-2,IL-12P70,IFN-γ,G-CSF and chemokines such as MIP-2,IP-10(P<0.01),the score of H&E staining(P<0.01),the score of safranin O green staining(P<0.01),and the score of TRAP staining(P<0.01).In addition,WB-T1 group could significantly inhibit the invasion of bone in micro-CT test,and increase BV/TV,Tb.N and BMD(P<0.01),but decrease the Tb.Sp and Tb.Pf(P<0.01).(2)Compared with model group,WB-T2 group could significantly decrease the AI score(P<0.01),the serum levels of inflammatory cytokines such as TNF-α,IL-17α,IL-1α,IL-1β,IL-2,IL-12P70,IFN-γ,G-CSF and chemokines IP-10(P<0.05),the score of H&E staining(P<0.01),the score of safranin O green staining(P<0.01),and the score of TRAP staining(P<0.05).In addition,WB-T2 group could significantly inhibit the invasion of bone in Micro-CT test,and increase BV/TV,Tb.N and BMD(P<0.01),but decrease the Tb.Sp and Tb.Pf(P<0.01).(3)Compared with model group,WB-T3 group could significantly decrease the serum levels of inflammatory cytokines such as IL-17α,IL-1β,G-CSF and chemokines MIP-2,IP-10(P<0.05).2.Tarsal bone transcriptomics-based analysis to investigate the potential mechanisms that WB inhibited joint destructionBy KEGG enrichment analysis,we found 12 significantly enrichment pathways associated with joint destruction,including "calcium signaling pathway","cell adhesion molecular(CAM)","cAMP signaling pathway","chemokine signaling pathway","Complement and coagulation cascades","MAPK signaling pathway","NF-kappa B signaling pathway","osteoclast differentiation","PI3K-Akt signaling pathway","Focal adhesion","Gap junction" and"Rap1 signaling pathway".Furthermore,three networks were generated by IPA,they were "resorption of bone","formation of bone" and"regulation of cartilage development".The key different expression genes(DEGs)of"resorption of bone" network included 116,Tnfsfl I,Ffar2,Plg,Tnfrsf11b,the key DEGs of "formation of bone" included Fgf4,Fprl,Siglecl,Vegfd,Cldnl,and the key DEGs of "regulation of cartilage development" network included Cxcl13,Chad,Arrb2,Fgf9,Egfr.RT-qPCR results showed the same tendency with transcriptomes.Compared with model group,WB group could down-regulate mRNA level of 116,Tnfsfl1(P<0.01),but up-regulate mRNA levels of Ffar2,Plg,Tnfrsf11b,Fgf4,Fpr1,Siglec1,Vegfd,Cldn1,Cxcl13,Chad,Arrb2,Fgf9,Egfr(P<0.01).3.Synovium proteomics-based analysis to explore the mechanisms that WB inhibited joint destruction539 DEPs were identified in WB-VS-Model,339 DEPs were down-regulated and 200 DEPs were up-regulated.1383 DEPs were identified in Model-VS-Normal,544 DEPs were down-regulated and 839 DEPs were up-regulated.We found several pathways associated with joint destruction,including "IL-1 signaling","IL-3 signaling","IL-8 signaling","inhibition of matrix metalloproteases","CXCR4 signaling","leukocyte extravasation signaling","integrin signaling".We found five key DEPs through the pathways and DEPs interaction network analysis,which were associated with cartilage degradation and bone resorption,including MMP3,MMP19,LBP,ARPC5 and IRAK4.Western Blot experiment was used to validate these DEPs,and we found that WB treatment could significantly inhibit the expression levels of MMP3,MMP19,LBP,ARPC5 and IRAK4 compared with rats with model group(P<0.01).Conclusion1.WB could inhibit the joint destruction of rats with CIA,and the therapeutic effect were associated with intervention time.2.Based-on tarsal bone transcriptomics analysis,we found that WB could inhibit bone resorption via regulation of 116,Tnfsf11,Ffar2,Plg,Tnfrsf11b,but promote bone formation via regulation of Fgf4,Fpr1,Siglec1,Vegfd,Cldn1,and promote cartilage development via regulation of Cxcl13,Chad,Arrb2,Fgf9,Egfr。3.Based-on synovium proteomics analysis,we found that WB could inhibit the expression levels of MMP3,MMP19,LBP,ARPC5 and IRAK4 proteins,which contributed to inhibiting joint destruction of rats with CIA.