Study On The Mechanism Of Pristimerin Anti-rheumatoid Arthritis Synovial Hyperplasia Based On Metabolomics Technology

Rheumatoid arthritis（RA）is an autoimmune disease characterized by synovial hyperplasia and inflammatory cell infiltration.Fibroblast-like synoviocyte（FLS）is the main cell type of synovial tissue,which will invade articular cartilage under pathological conditions,resulting in joint destruction,joint deformity and even disability in RA patients.At present,the commonly used therapeutic drugs for RA include nonsteroidal anti-inflammatory drugs,glucocorticoid and immunosuppressant clinically.However,these drugs are accompanied by serious side effects while treating diseases.Therefore,it is extremely urgent to find anti-RA drugs with better efficacy,less side effects and lower cost.Pristimerin is a natural quinone methyl triterpenoid isolated from the plants of the Celastraceae and the Hippocrateaceae families.Pristimerin possesses anti-cancer,antiinflammatory,antibacterial and antioxidant activities.At present,studies on its pharmacological effects mainly focus on its anti-proliferative activity against cancer cells from different sources,while there are relatively few studies on its anti-RA activity and mechanism.In this study,human rheumatoid arthritis synovial fibroblast（MH7A）,were used to study the effects of pristimerin on the proliferation,migration and cell cycle of MH7A cells by MTT assay,EdU proliferation assay,Wound healing assay,Transwell cell migration assay and cell cycle assay.The potential mechanism was explored by network pharmacology analysis and western blot assay.Then,the cell metabolomics method based on UPLC-LTQ-Orbitrap-MS was used to investigate the effect of pristimerin on endogenous metabolites in MH7A cells.Finally,an adjuvant-induced arthritis（AIA）rat model was established to investigate the therapeutic effect of pristimerin on AIA rats by arthritis score,enzyme-linked immunosorbent assay（ELISA）,histopathological analysis and immunohistochemistry.The thesis consists of the following three chapters:Chapter 1:Effects of pristimerin on MH7A cell proliferation,migration,cell cycle and related signaling pathwaysIn this chapter,human rheumatoid arthritis synovial fibroblast（MH7A）cells,MH7A was used as the research object.Firstly,MTT assay and EdU assay were used to detect the effects of pristimerin on the viability and proliferation of MH7A cells.Wound healing and Transwell assay were used to detect the effects of pristimerin on MH7A cell migration.Flow cytometry was used to detect the effect of pristimerin on MH7A cell cycle.The key protein targets of pristimerin in RA treatment were identified by network pharmacology analysis.Finally,Western blotting assay was used to detect the effect of pristimerin on cell cycle-related proteins such as p21,p27,cyclin E1,CDK2,Akt,p-Akt,Erk,and p-Erk.MTT assay showed that pristimerin significantly inhibited the survival rate of MH7A cells stimulated by TNF-α（20 ng/mL）with IC50 of 1.408 μM at 24 h.EdU experiment showed that pristimerin inhibited the proliferation of MH7A cells stimulated by TNF-α.Wound healing assay and Transwell assay showed that pristimerin significantly inhibited the migration of MH7A cells stimulated by TNF-α.Flow cytometry was used to detect the cell cycle of MH7A cells.Network pharmacology analysis identified 23 key core targets responsible for the anti-RA effect of pristimerin.Pathway enrichment analysis showed that these 23 key targets were mainly involved in the TNF signaling pathway and PI3K/Akt signaling pathway.Western blotting showed that the expression levels of cyclin E1,CDK2,p-Akt,and p-Erk decreased,while the expression levels of p21 and p27 increased after pristimerin intervention.Chapter 2:Metabolomics study of MH7A cells after pristimerin treatmentIn this study,we analyzed the changes of endogenous metabolites in human rheumatoid arthritis synovial fibroblasts（MH7A）stimulated by TNF-α after treatment with pristimerin by UPLC-LTQ-Orbitrap-MS.Twenty metabolites were identified after data preprocessing and chemometric analysis.Network enrichment analysis showed that these metabolites were mainly involved in phospholipid biosynthesis,fatty acid biosynthesis,glutathione metabolism and amino acid metabolism.Chapter 3:Therapeutic effects of pristimerin on AIA ratsIn this chapter,adjuvant-induced arthritis（AIA）rats were used.Results of paw volume,arthritis score,organ coefficient,serum TNF-α,nitric oxide（NO）content,histopathological analysis and immunohistochemical experiments in normal control group,AIA model group,positive control group,and pristimerin treatment group were compared.The results showed that pristimerin significantly reduced paw swelling,arthritis score as well as serum TNF-α and NO levels in AIA rats.Histopathological analysis showed that pristimerin could significantly reduce inflammatory cell infiltration and bone erosion in the ankle joint of AIA rats.Immunohistochemical experiments showed that the expression of p-Akt and p-Erk in the ankle of AIA rats decreased after treatment with pristimerin.In conclusion,this experiment employs rheumatoid arthritis synovial fibroblast（MH7A）cells and adjuvant-induced arthritis rat model,using a combination of cell metabolomics,network pharmacology,western blotting,histopathological analysis and immunohistochemistry techniques to explore the therapeutic effects of pristimerin on rheumatoid arthritis.Our study would give new insights into the development of new anti-RA drugs targeting synovial fibroblast cell.