| Osteoarthritis(OA)is a common degenerative disease of cartilage.With the aging of population,the incidence of OA is increasing,which seriously affects the quality of life of patients.Bushen Jiangu Fang has been used in clinic for many years,and it has a definite curative effect on OA,but its functional components and mechanism still need to be further explored.Objective: The purpose of this study is to screen the best extraction process of effective components of Bushen Jiangu Fang based on the clinical medication mode of decoction.To clarify the protective effect of Bushen Jiangu Fang on cartilage at animal and cell level,and to screen its effective components.On this basis,the effective components were further modified to improve their action time and effect,and their action targets and biological mechanisms were deeply explored,providing theoretical basis for the treatment of cartilage injury diseases such as osteoarthritis.Method:1.Different extracts of Bushen Jiangu Fang were obtained by adjusting the material-liquid ratio,decocting time and decocting times.The best extraction method of Bushen Jiangu Fang was determined by calculating the yield rate and comparing and evaluating the effects of each group of extracts at the cellular level.2.The model of OA rats was established,and Bushen Jiangu Fang was given by gavage.The protective effect of Bushen Jiangu Fang on knee cartilage in OA rats was confirmed by Micro-CT and saffron O-solid green staining.Primary chondrocytes were isolated,and the primary chondrocytes induced by IL-1β were used as OA cell model.CCK-8,q RT-PCR,Alcian blue staining and immunofluorescence were used to explore the regulatory effect of Bushen Jiangu Fang on chondrocyte proliferation and matrix metabolism.ROS,SOD,GSSG,ATP and mitochondrial membrane potential were used as indicators to detect the protective effect of Bushen Jiangu Fang on oxidative stress of OA chondrocytes.3.Based on RNA-Seq,explore the differentially expressed genes between Bushen Jiangu Fang treatment group and control group,make functional annotation and pathway enrichment analysis of differentially expressed genes by bioinformatics method,and verify the expression level of key pathway genes by Western blot method,so as to explore the mechanism of Bushen Jiangu Fang for cartilage protection.4.The components of Bushen Jiangu Fang were identified by liquid chromatography-mass spectrometry(LC-MS),and the structure of the compound and the relative content information of each component were obtained by comparing with the database of known compounds.Taking chondrocyte proliferation and matrix metabolism as evaluation indexes,CCK-8 and q RT-PCR methods were used to screen the key monomer components that play a role in cartilage protection,and Alcian blue staining,immunofluorescence and flow cytometry were used to evaluate the protective effects of monomer components on chondrocytes.5.The degradable mesoporous silica(bMSN)was synthesized and the monomer components in Bushen Jiangu Fang were modified.The structure,particle size and potential of bMSN and monomer-bMSN were characterized and analyzed by transmission electron microscope(TEM),Fourier transform infrared spectroscopy(FTIR),dynamic light scattering(DLS)and Zeta potential.High performance liquid chromatography and in vivo imaging were used to evaluate its absorption and degradation in OA rat joints and chondrocytes.6.OA rat model(left hind limb: control group,right hind limb: OA group)was established,and monomer and monomer-bMSN were injected into joint cavity.The protective effects of monomer and monomer-bMSN on OA rats were compared and evaluated by behavioral analysis,Micro-CT,H&E staining,saffron O-fast green staining,ELISA and other methods.7.Drug affinity responsive target stability(DARTS),cellular thermal shift assay(CETSA),isothermal titration calorimetry(ITC),molecular docking,molecular dynamics and so on were used to screen the target of the monomer components of Bushen Jiangu Fang,thus clarifying its mechanism.Results:1.Comparison of the yield rate and efficacy of Bushen Jiangu Fang with different extraction methods: Based on the yield rate and the protective effect on chondrocytes,the best extraction process of Bushen Jiangu Fang was as follows: the material-liquid ratio was1:10,the decocting time was 1h and the decocting times were 2 times.2.Evaluation of therapeutic effect of Bushen Jiangu Fang on OA: Bushen Jiangu Fang can significantly reduce the formation of osteophyte in OA rats,improve the microstructure of subchondral bone,alleviate cartilage degeneration and delay the progress of OA.In addition,Bushen Jiangu Fang can promote the proliferation of primary chondrocytes and the synthesis of extracellular matrix,and reduce the level of ROS induced by IL-1β in chondrocytes.The above results suggest that Bushen Jiangu Fang can effectively treat OA.3.Study on the mechanism of Bushen Jiangu Fang in treating OA: Bushen Jiangu Fang regulates the genes related to matrix metabolism,inflammation and oxidative stress in chondrocytes,and inhibits the expression of NF-κB(P65)in OA cells in a dose-dependent manner.In addition,Bushen Jiangu Fang can increase the expression level of Sox9 and Col2a1 in the downstream of NF-κB.These results suggest that Bushen Jiangu Fang may treat OA by regulating NF-κB pathway.4.Identification of active components of Bushen Jiangu Fang: A total of 619 monomer compounds were identified in Bushen Jiangu Fang,among which catalpol content was higher.Compared with other compounds,catalpol can significantly promote chondrocyte proliferation,up-regulate the level of chondrocyte anabolism(Col2a1,Acan,Comp)and inhibit the level of catabolism(Mmp9,Mmp13,Adamts4).Catalpol can inhibit the production of ROS in chondrocytes,improve the antioxidant capacity and mitochondrial membrane potential of chondrocytes,and inhibit the apoptosis mediated by IL-1β.The results suggest that catalpol may be one of the key components in Bushen Jiangu Fang.5.bMSN modification and characterization analysis of catalpol: bMSN with good dispersion and uniform particle size was successfully synthesized,and the Si-O-Si structure was detected by FTIR.The particle size of bMSN was about 200 nm and the potential was-12.42.Catalpol-bMSN has a particle size of about 450 nm and a potential of 31.3.In addition,bMSN and catalpol-bMSN cannot be removed by lysosomes,and can be kept for at least 7 days after intra-articular injection,and can be degraded and removed.These results suggest that catalpol-bMSN has been successfully synthesized and can be used to treat OA.6.Comparison of therapeutic effects of catalpol and catalpol-bMSN on OA: The differences of behavioral characteristics such as bearing area,weight and swing time of the left and right hind limbs of OA rats treated with catalpol and catalpol-bMSN are significantly reduced,and the therapeutic effect of catalpol-bMSN is better than catalpol.At the same time,catalpol and catalpol-bMSN treatment inhibited the abnormal remodeling of subchondral bone of knee joint in OA rats,improved cartilage tissue morphology,increased cartilage thickness,and alleviated the inflammatory reaction of synovial fluid,and catalpol-bMSN treatment was more effective.These results suggest that catalpol modified by bMSN shows greater potential in the treatment of OA.7.Exploring the mechanism of catalpol improving OA: catalpol binds to Hsp90βprotein in chondrocytes(KD=1.83e-6 433e-9m),and amino acids ASP88,THR179,ASP49 and ASN46 at the N-terminal of Hsp90β protein may be the main sites of catalpol binding,thus inhibiting the activity of Hsp90β and further regulating NF-κB signaling pathway to inhibit cartilage.These results suggest that Hsp90β is the target of catalpol,and catalpol can treat OA by regulating Hsp90β/NF-κB signaling pathway.Conclusion: The Bushen Jiangu Fang extract that obtained by the best extraction process(material-liquid ratio of 1:10,decocting time of 1h,decocting times of 2 times)have a good therapeutic effect on OA cells and OA animal models,which can effectively improve OA cartilage injury by regulating NF-κB signal pathway.Further screening of the effective components in Bushen Jiangu Fang showed that catalpol may be the key component.Compared with catalpol alone,catalpol modified by bMSN has a longer action time and better therapeutic effect.The mechanism of catalpol was deeply explored,and the results showed that Hsp90β was the target of catalpol in chondrocytes,and it could prevent cartilage injury by regulating Hsp90β/NF-κB signaling pathway. |
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