Study On The Activity And Mechanism Of Codonopsis Canescens Nannf. On Anti-rheumatoid Arthritis Based On TLRs/MAPKs/NF-κB Signal Pathways

Background and purpose of the studyRheumatoid arthritis(RA)is a symmetric,multiple,progressive and systemic autoimmune disease with synovial inflammation as the main clinical feature.RA is one of the ten chronic diseases.It not only seriously affects people’s quality of life but also brings a heavy economic burden to families and society.Traditional Chinese medicine has unique advantages in the prevention and treatment of RA.It can not only alleviate the toxicities and side effects of western medicine but also have the characteristics of multi-component,multi-target,and multi-channel regulation of diseases.Codonopsis.canescens Nannf.,a plant in the family Campanulaceae,has the effect of drying yellow water and reducing swelling,and is commonly used in the treatment of RA,eczema and leprosy.Toll-like receptors(TLRs)on the cell surface,especially TLR2 and TLR4,play an important role in the occurrence and development of RA.Nuclear transcription factor p65(NF-κB p65)and p38 mitogen-activated protein kinase(p38 MAPK)are the common downstream molecules of TLR2 and TLR4 activation,and also the key effector molecules.Therefore,these targets are closely related to the development of RA and are important targets of the TLRs/MAPKs/NF-κB signaling pathway.However,traditional Chinese medicine components can not only directly regulate these important targets of TLRs/MAPKs/NF-κB signaling pathway,but also exert anti-RA effects by regulating the upstream and downstream targets of these important targets,or by simultaneously regulating important targets and their upstream and downstream targets.In view of this,this study first used ultra-high performance liquid chromatography tandem timeof-flight mass spectrometry(UPLC-Q-TOF-MS)to identify the chemical constituents of the total extract of Codonopsis.canescens Nannf..Then,a rat model of collageninduced arthritis(CIA)was established.Based on the important targets of TLRs/MAPKs/NF-κB signaling pathway,the material basis and mechanism of Codonopsis.canescens Nannf.regulating anti-RA were revealed.Finally,based on molecular docking technique,we studied the active components of C.canescens Nannf.that directly and indirectly regulate TLR2,TLR4,NF-κB p65,p38 MAPK,which are important targets of anti-RA signaling pathway TLRs/MAPKs/NF-κB.In order to provide reference for clinical application and new drug development of C.canescens Nannf.against RA.Methods and results1.Study on the chemical constituents of the total extract of C.canescens Nannf.based on UPLC-Q-TOF-MS technologyUPLC-Q-TOF-MS was used to analyze the components of the extract of C.canescens Nannf.In the negative ion mode,the total ion flow diagram of the extract components was obtained.Based on the accurate molecular weight of the components,ion fragments and literature,34 main components of C.canescens Nannf.were identified,including 15 flavonoids,7 phenylpropanoids,4 terpenoids,4 organic acids,2 esters and 2 polyacetylenes.2.Study on the anti-RA material basis and mechanism of action of C.canescens Nannf.of regulating TLRs/MAPKs/NF-κB based on CIA model and important targetsThe rat model of collagen-induced arthritis(CIA)was induced by bovine type II collagen emulsion injection.The body weight and foot swelling of rats were measured,and the arthritis index and immune organ index were analyzed.The pathological changes of synovial tissue in each group were observed by HE staining.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in serum were determined by ELISA.The expression of TLR2,TLR4,NF-κB p65,p38 MAPK and p-p38 MAPK protein in rats was detected by Western blot.The results showed that,compared with the normal group,the body weight of the model group was significantly decreased(P<0.01);foot swelling was significantly increased(P<0.05,P<0.01);arthritis index was significantly increased(P<0.01);the immune organ index was significantly increased(P<0.01);synovial histopathological injury was obvious observed.Serum levels of inflammatory factors TNF-α,IL-1β and IL-6were significantly increased(P<0.01).The protein expression levels of TLR2,TLR4,NF-κB p65,p-p38 MAPK/p38 MAPK in synovial tissue were significantly increased(P<0.01).Compared with model group,body weight of ZDS dose groups was significantly increased(P<0.01);the swelling degree of toes was significantly decreased(P<0.01);arthritis index significantly decreased(P<0.01);the immune organ index was significantly decreased(P<0.01);the pathological changes of synovial tissue hyperplasia and inflammatory cell infiltration were alleviated.The levels of TNF-α,IL-1β and IL-6 in serum were significantly decreased(P<0.05,P<0.01).The protein expression levels of TLR2,TLR4,NF-κB p65,p-p38MAPK/p38 MAPK were significantly decreased(P<0.05,P<0.01).3.Study on the active components of C.canescens Nannf.direct and indirect regulating important targets of TLRs/MAPKs/NF-κB based on molecular docking technologyFirstly,the TCMSP database was used to screen the active ingredients of C.canescens Nannf.based on oral bioavailability(OB)and drug-likeness(DL)parameters.A total of 9 active compounds with DL≥0.18 and OB≥10 % were screened,which were luteolin,apigenin,astragalin,chrysoeriol,tricin,chlorogenic acid,esculin,lobetyolin and oleanolic acid.Then,Thirty-seven TLRs/MAPKs/NF-κB pathway targets were obtained through the KEGG database,and thirty-two human receptor target proteins were finally obtained through the PDB database.Lastly,Auto Dock Tools 1.5.6 software was used to perform molecular docking of active ingredients and pathway targets,and Pymol software was used to visualize the docking results.The docking results showed that oleanolic acid,luteolin,apigenin,chrysoeriol,tricin,astragalin and esculin could directly regulate the important targets TLR2,TLR4,NF-κB p65 and p38 MAPK to exert anti-RA activity;Chlorogenic acid can directly regulate the important targets TLR4,NF-κB p65 and p38 MAPK to exert anti-RA activity;lobetyolin can directly regulate the important targets TLR2 and p38 MAPK to exert anti-RA activity.This indicates that these active components can directly regulate important targets to exert anti-RA activity,but the direct regulation of different components on important targets is not exactly the same.In addition,oleanolic acid,luteolin,apigenin,chrysoeriol,tricin and esculin had direct regulatory effects on other targets of TLRs/MAPKs/NF-κB pathway.Astragalin,chlorogenic acid and lobetyolin also have direct regulatory effects on most other targets.However,the strength of direct regulatory activity varies.ConclusionThese results indicate that the active components of C.canescens Nannf.can not only directly regulate the important targets TLR2,TLR4,NF-κB p65 and p38 MAPK of TLRs/MAPKs/NF-κB,but also indirectly regulate these important targets by binding to other targets in the pathway to exert anti-RA effects.At the same time,luteolin,apigenin,astragalin,chrysoeriol,tricin,chlorogenic acid,esculin,lobetyolin and oleanolic acid of C.canescens Nannf.are potential anti-RA active substances.