Study On Material Basis And Molecular Mechanism Of Bushen Huoxue Recipe In Treating Osteoarthritis

Osteoarthritis(OA),also called hypertrophic arthritis or degenerative arthritis,is frequently occurred and refractory.According to traditional Chinese medicine(TCM),it belongs to the category of "bone Bi syndrome".The main pathological symptom of osteoarthritis is lesion of cartilage tissue.Cytokine has the function of promoting catabolism of cartilage matrix,playing an important role in the pathogenesis of osteoarthritis.The thesis first summarized the development history of traditional Chinese medicine understanding and treating osteoarthritis and put forward that the reinforcing kidney and promoting blood therapy may be main principle for treating osteoarthritis and "deficiency of kidney and obstruction of collaterals due to blood stasis" may be the main pathogenesis of osteoarthritis.Combining with the pathological manifestation of osteoarthritis,this thesis analysed the key role of chondrocytes apoptosis in osteoarthritis which may be related to catabolism of matrix.In addtion,the thesis also made a systematic literature summary of chemical composition and pharmacological activity of each herb in the compound recipe.At last,the thesis summarized the application of LC-MS technology and computer simulation technology in medicine,especially the compound recipe.The experiment made a qualitative analysis on chemical components in water extract of compound medicine and Single medicine by HPLC-ESI-Q-TOF-MS.LC-MS/MS,C18(4.6mm×250 mm,5?m)column was adopted,with methanol-0.1%formic acid as the mobile phase for gradient elution;ESI ion source was used for mass spectra,and data were collected in positive and negative mode.88 compounds from water extract of medicine compound and Single medicine were identified by analyzing positive and negative ion mass spectra information and element composition and comparing controls with data from relevant literature.At the same time,we made a comparative analysis of the chemical composition of medicine compound and Single medicine.Through review of literatures,17 active substances with potential in treating osteoarthritis and inhibitting chondrocyte apoptosis were found,namely paeonol,paeoniflorin,catechin,ferulic acid,apigenin,quercetin,psoralen,isoimperatorin,osthole,polydatin,resveratrol,oleanolic acid,ecdysterone,icariin,icariside II,hyperoside,stachydrine.In order to compare content changes of chemical composition in the compound recipe and single recipe,the experiment further developed the LC-MS/MS analysis method which may simultaneously determinate the content of 12 kinds of monomer solution in Bushenhuoxue Recipe extraction.The content of catechin,paeoniflorin,ferulic acid,hyperoside,polydatin,quercetin,resveratrol,psoralen,isopsoralen,icariside II,osthole and isomperatorin in extract solution of Bushen Huoxue compound Recipe was namely 0.0422mg·g-1,4.2596mg·g-1,0.1211mg·g-1,0.0584mg·g-1,0.7594mg·g-1,0.1999mg·g-1,0.1570mg·g-1?0.8948mg·g-1,0.4634mg·g-1,0.0928mg·g-1,0.5364mg,g-1 and 0.1487 mg,g-1.Compared withQuercetin,isopsoralen,icariside II,osthole and isomperatorin in simple Taxilli Herba recipe,simple Angelicae Pubescentis Radix recipe,simple Polygoni Cuspidati Rhizoma Et Radix recipe and simple Epimedii Folium recipe,the content of each compositon in compound recipe increased remarkablely.Extract solution of Bushen Huoxue Recipe and monomeric compounds,such as resveratrol,paeoniflorin,osthole and polydatin,may protect cartilage by promoting the proliferation of chrondrocytes,GAG synthesis and expression of GlcAT-1 mRNA and inhibitting the expression of MMPs-1 and MMPs-3 mRNA,in order to prevent osteoarthritis.In the molecular mechanism experiment,by enzyme-linked immunosorbent assay(ELISA),the effect of 16 compounds such as oleanolic acid,as well as water extraction of the compound recipe,alcohol extraction of the compound recipe and polysaccharides of the water extract-alcohol extraction on IL-6,TNF-a and NO released by macrophage were detected.It was found that most of the reagents had some or all inhibitory effect on the above three cytolkines,which may demonstrate the possible cause and mechanism of compound recipe in treating osteoarthritis.Molecular mechanism experiment also investigated the effect of extract solution of Bushen Huoxue Recipe of different concentrations and 4 monomeric compounds,namely resveratrol,paeoniflorin,osthole and polydatin,on the rat IL-1?-induced arthritis chondrocytes in vitro and analyse its possible mechanism.The thesis initially clarified the extract solution of Bushen Huoxue Recipe and monomeric compounds,such as resveratrol,paeoniflorin,osthole and polydatin,may protect cartilage by promoting the proliferation of chrondrocytes,GAG synthesis and expression of G1cAT-1 mRNA and inhibitting the expression of MMPs-1 and MMPs-3 mRNA,in order to prevent osteoarthritis.In order to analyse multiple target effect of Bushen Huoxue Recipe in treating osteoarthritis,computation virtual screening technology were used to compare and analyze chemical space of compounds of Bushen Huoxue Recipe and inhibitors related to OA target enzymes searching from MDDR database.We selected some feature descriptors,and analysized the distributions decribed,and principal component analysis(PCA)were used to map these multiple descriptor chemical space into a 2D plane.The effective components of Bushen Huoxue Recipe have good diversity,and it has more same chemical space between inhibitors and compounds from Bushen Huoxue Recipe.This may be intuitive to reveal the comprehensive and multi-target effect of Bushen Huoxue Recipe on treating OA.Applying DS microsofe,the experiment studied chemical composition of receptor target enzymes,such as TNF-? and IL-1? in Bushen Huoxue Recipe and discuss the molecular mechanism of Bushen Huoxue Recipe in treating osteoarthritis and screened exsitting potential composition which may have therapeutic effect on osteoarthritis.The results showed that 11 or 5 compounds which were well connected with these target enzymes and had a higher score at the same time were respectively selected from Bushen Huoxue Recipe.This may have possible connection with activity of Bushen Huoxue Recipe for treating osteoarthritis.Therefor,it provides guideline for exploring the molecular mechanism of Bushen Huoxue Recipe in treating osteoarthritis.In the flexible docking experiment,apigenin and INOS protein were well docked,indicating that apigenin may be the potential inhibitor not only for TNF-? and IL-1?,but also for INOS.