Application Of Label-free Quantitative Proteomics In The Study Of The Efficacy And Mechanism Of Traditional Chinese Medicine In The Treatment Of Osteoarthritis

Research BackgroundOsteoarthritis(OA)is one of the most common types of arthritis.The main pathological features are spinal destruction,synovial damage,osteophyte formation and subchondral bone remodeling.The clinical manifestations are joint swelling and pain,limited movement,deformity and dysfunction,which seriously affect the health and quality of life in patients.The pathogenesis of osteoarthritis caused by movement is intricate and related to multiple factors but has not yet been completely understood.Nowadays,clinical treatments mainly focus on relieving pain and reducing local inflammation.Due to the limitations of the lack of treatment methods to prevent disease and delay the progression of disease,it is necessary to develop more effective drugs and treatment strategies to prevent the occurrence of osteoarthritis and delay the development of the disease.In Chinese medicine,osteoarthritis is belonging to the category of "Chinese arthralgia syndrome".It is believed that the pathogenesis is related to many factors such as old age,physical failure,long-term strain,and invasion of foreign pathogens.JointAlive(?),as a new supplement to protect the spine and joints,has been approved and registered for the first time by the U.S.Food and Drug Administration(FDA)as the new insulin(NDI).JointAlive is composed of three Chinese medicine extracts of Epimedium,Dioscorea nipponica and Salvia miltiorrhiza(EDS).Based on the theory of traditional chinese medicine,it has the effect of tonifying liver and kidney,dispelling wind,dispersing cold and dehumidifying,promoting blood circulation and removing blood stasis,warming meridians and dredging collaterals.Clinical trials have proved that it can prevent joint swelling,relieve pain,and improve joint flexibility,However,its pharmacodynamic substance basis and underlying effect mechanism have not been reported.Research purposesEstablishing and evaluating the rat model of knee osteoarthritis induced by papain.Checking the protective effect of EDS on the tibia tissue and mobility of model rats at multiple levels;Using biological information based on unlabeled quantitative proteomics analyze the difference protein and signal transmission between the blank group,model group,positive drug group,and EDS group by scientific methods,and further verify the material basis and molecular mechanism of EDS.Exploring the proteomics technology to study complex as a new paradigm for evaluating the efficacy of Traditional Chinese Medicine ingredients.Research method(1)To prepare EDS compound methanol extract and use LC-MS/MS technology to separate the main components.Searching from of the TCMSP database,the m/z ratio of the main compounds in EDS is retrieved to determine the attribution of each mass spectrum peak.(2)To establish a rat knee osteoarthritis model,and to carry out traditional Chinese medicine intervention.To detect of joint reflex threshold and joint curvature between different experimental groups,and to detect the serum inflammatory factor levels,pathological tissue slices and other methods for EDS efficacy evaluation.(3)Label-free quantitative proteomics is used to screen differential proteins and delivery of EDS anti-arthritis rat models.Then using molecular biology methods to verify the key proteins and potential drug targets screened out.Experimental results(1)A total of 12 compounds were identified in EDS,including:5,6,7,4’-tetramethyl ether baicalein(5,6,7,4’-tetramethyl ether yellow,element),Epimedium Icariin(icariin),icariin C(epimedin C),8-pentenylpyridin 4’-methyl ether 3-rhamnoside(8-isopentene quercetin 4’methyl Ether 3-rhamnoside),ferulic acid(ferulic acid),1,2-dihydrotanshinone(1,2dihydrotanshinone),diosgenin acetate(acetate base),cryptotanshinone(Cryptotanshinone),tanshinone Ⅰ(tanshinone Ⅰ),tanshinone ⅡA(tanshinone ⅡA),δ-3,5-deoxytigogenin(δ3,5deoxytigogenin),tanshinone(salvirecognone).(2)The rat knee osteoarthritis model was successfully established.Through the intervention of high and low doses in the rat model,it can be observed that the joint reflex threshold of the EDS group was significantly increased,the joint curvature was significantly restored,and the serum inflammatory factor TNF-α,IL-1β,and IL-6 levels decreased significantly.(3)A total of 13199 peptides were identified,and a total of 1408 quantified proteins(specific peptides≥ 2,coverage>33.3%).The relative variance is distributed between 0.4 and 0.5,and the trends are similar between groups.Among them,the model group and the blank group list 62 up-regulated proteins and 208 down-regulated proteins.(4)Most of the down-regulated proteins in the model group were up-regulated in the EDSL group.The recovery of this part of down-regulated protein levels may lead to the recovery of knee motor function restriction caused by osteoarthritis.Some proteins down-regulated in the model group were up-regulated in the EDS group,especially the up-regulation in the EDSH group.The differential proteins recovered in the distribution group included proteins related to the complement cascade,leukocyte-mediated immune response,plasma lipoprotein particle level regulation,muscle contraction,and myofilament sliding.Conclusion:(1)EDS can improve the action ability of closed osteoarthritis model rats,restore cartilage function to a certain extent,and reduce the level of serum inflammatory factors.(2)Complement activation immune response Immune response may play a central role in the pathogenesis of osteoarthritis.(3)EDS may rebuild retinal pathways and regulate humoral and cellular immune responses to capillary protection.(4)The main compound components in EDS are related to the efficacy of treating osteoarthritis.(5)The label-free proteomics method can achieve high coverage of protein identification,providing new ideas and methods for the research of complex drug mechanisms.