Xuanhusuosan's Potential Pharmacodynamic Material Basis And Molecular Mechanism Of Anti-chondrocyte Apoptosis

Xuanhusuo Powder is composed of Rhizoma Corydalis,Fructus psoraleae,Angelica sinensis and Achyranthes bidentata,which has the effect of "tonifying kidney and strengthening bone,activating blood circulation and dredging collaterals".It is a classic compound prescription for clinical treatment of osteoarthritis.However,the material basis and mechanism of its efficacy are not completely clear.Osteoarthritis is a kind of chronic arthropathy characterized by degenerative lesions of articular cartilage and chondrocytes are the only cells in articular cartilage.Chondrocyte apoptosis is one of the important factors in the pathogenesis of osteoarthritis.In order to further study the pharmacodynamic material basis and mechanism of compound Xuanhusuo Powder in the treatment of osteoarthritis,we put forward the hypothesis of "target cell activity",and at the same time we studied the pharmacodynamic material basis and potential mechanism of Xuanhusuo Powder against chondrocyte apoptosis combining with the theory of serum pharmacochemistry,network pharmacology,molecular docking technology,molecular biology and other methods,so as to provide a basis for further development and utilization of Xuanhusuo Powder.The content of this study mainly includes the following parts:1 Analysis of prototype components of Xuanhusuo Powder based on UPLC-QTOF-MS and UPLC-QQQ-MSBecause the composition of traditional Chinese medicine is complex,we filtered out many non-blood ingredient by studying of blood ingredient,which was one of the important active chemical components.UPLC-Q-TOF-MS technology is widely used in the analysis of chemical components of traditional Chinese medicine because of its high speed,high accuracy and wide range of analysis.However,it is difficult to detect the prototype components of traditional Chinese medicine,due to the complex background of blood samples and low content of blood components.UPLC-QQQ-MS technology has the characteristics of high sensitivity,accuracy and good repeatability,which can avoid the interference of complex background.In this study,we used data processing software MassLynx,UNIFI and standard sample verification with the combination of UPLC-QTOF-MS and UPLC-QQQ-MS technology,and established an accurate and simple method for screening and identification of blood components in Xuanhusuo powder.Besides we analyzed the prototype components of Xuanhusuo powder in rat plasma quickly and accurately.After we further studied 20 prototype components in rat plasma,among them,Psoralen,Angelicin,Tetrahydropalmatine,Corydaline,Protopine,Berberine have high content in blood,which may be the key pharmacodynamic substance basis in the treatment of osteoarthritis of Xuanhusuo Powder.2 Analysis of potential Mechanism of Xuanhusuo Powder in the treatment of Osteoarthritis based on Network Pharmacology In order to further study the pharmacodynamic material basis and mechanism of Xuanhusuo Powder in the treatment of osteoarthritis,we used the network pharmacology technique to predict the action targets and pathways of 20 kinds of prototype components of Xuanhusuo Powder in the treatment of osteoarthritis.Finally there are 61 potential targets of Xuanhusuo Powder in the treatment of osteoarthritis were identified.And the results of KEGG pathway analysis showed that the main effects of 20 blood-entering prototype components of Xuanhusuo Powder in the treatment of osteoarthritis were anti-apoptosis and anti-inflammation,which was consistent with the hypothesis of the subject.Among them,PI3K-Akt and MAPK signal pathways are the main anti-apoptotic pathways of Xuanhusuo Powder.And network topology analysis showed that the potential targets of 20 prototype components of Xuanhusuo Powder in the treatment of osteoarthritis were closely related to AKT1,FN1,MMP9,MMP2 and HRAS.3 Study on the potential Pharmacodynamic material basis of Xuanhusuo Powder against Chondrocyte apoptosis based on Molecular docking technique In this study,we used the based on the "target cell activity hypothesis”and the results of network pharmacological study,combined with molecular docking technique to select apoptotic factors closely related to chondrocyte apoptosis.Target proteins were selected as follows:AKT1,FN1,MMP9 and HRAS were four key targets of network pharmacology study;PI3K,AKT1 and mTOR were three key targets of PI3K-AKT pathway;JUK?ERK?p38 were three key targets of MAPK pathway.The binding energy ?Gbind was sorted from low to high to select the compound with good binding ability as the potential pharmacodynamic component.The results showed that 20 prototype components had good binding activities with Caspase-3,Bax,Bcl-2,AKT1,PI3K,mTOR targets,among which Psoralen,Tetrahydropalmatine,Angelicin,Berberine and Dehydrocorybulbine were the top five compounds for binding energies of Caspase-3,Bax and Bcl-2.Psoralen,Angelicin,Berberine,Tetrahydropalmatine and Protopine are the top five compounds when binding with PI3K,AKT and mTOR.The mechanism of the action of Xuanhososan treating osteoarthritis may be related to the anti-apoptotic effect of Psoralen,Tetrahydropalmatine,Angelicin,Berberine,Protopine,Dehydrocorybulbine on chondrocytes,and the mechanism of anti-apoptotic effect of these components may related to PI3K-AKT pathway,which still needs to be further verified by cell experiments.4 Preliminary study on the effective substances of Xuanhusuo Powder against chondrocyte apoptosisChondrocytes are the only cells in articular cartilage.In this study,the potential pharmacodynamic components obtained from the experiments of serum pharmacochemistry,network pharmacology and molecular docking were preliminarily investigated at the cellular level toverify its anti-apoptotic activity.Chondrocytes were extracted by 0.2%II collagenase digestion.Apoptosis of chondrocytes was induced by 1.0 mmol/L sodium nitrate(SNP).The anti-apoptotic effects of different concentrations of Angelicin,Tetrahydropalmatine and Psoralen were studied by MTT method.The results showed that Angelicin,Psoralen 0.028?g/mL?0.14 ?g/mL had a certain protective effect on SNP-induced chondrocyte apoptosis;Tetrahydropalmatine 1 ?g/mL had a certain protective effect on SNP-induced chondrocyte apoptosis.