| Objective Rheumatoid arthritis(RA)is a chronic inflammatory joint disease that can lead to cartilage and bone damage as well as disability.The classic Chinese herbal formula Ganjiang Lingzhu decoction(GJLZD)is commonly used in the clinical treatment of RA due to its efficacy in dispelling cold and dampness,promoting blood circulation and relieving pain.However,there is limited research on the pharmacological basis and mechanism of action of Ganjiang Lingzhu decoction.In this study,mass spectrometry was used to explore the in vivo and in vitro chemical components of Ganjiang Lingzhu decoction,and network pharmacology was employed to further elucidate the target pathways and mechanisms of action of Ganjiang Lingzhu decoction in the treatment of RA.Animal experiments were conducted to verify the therapeutic mechanisms of Ganjiang Lingzhu decoction,providing a scientific basis for its clinical application as a classic Chinese herbal formula.Methods 1 Study of the in vitro and in vivo chemical components of Ganjiang Lingzhu decoction.This study identified and analyzed the in vitro chemical components and rat blood prototype components of Ganjiang Lingzhu decoction(GJLZD)using UPLC-QE-MS technology,in combination with the Chinese medicine chemistry database.First,SD rats were randomly divided into normal and treatment groups,with 6 rats in each group.The treatment group was given 36g/kg of GJLZD orally every day for 3 consecutive days.Blood samples were collected from the rat’s eye socket at 15 min,30min,1h,2h,4h,6h,8h,and 12 h after the last dose to prepare serum samples.Subsequently,the UPLC-QEMS technology was used to detect blank serum and drug-containing serum samples,and the Xcalibur software(Thermo Fisher Scientific)was used to process the data of blank and treatment group samples,subtracting the endogenous components in the blank serum.Based on the in vitro chemical component identification results,the retention time,massto-charge ratio,and secondary characteristic fragment ions of the ion peaks were compared,and the prototype components of GJLZD in rat blood were identified by combining with the database and literature information.2 A network pharmacology study of Ganjiang Lingzhu decoction on rheumatoid arthritis was conducted.Based on the identified prototype components in the blood,network pharmacology analysis was performed using Pharm Mapper and Swiss Target Prediction databases to search for the target proteins of the prototype components.Gene Cards and GEO databases were utilized to search for the target proteins of rheumatoid arthritis.The intersection of the target proteins of the prototype components and the disease was considered as potential targets of Ganjiang Lingzhu decoction for treating rheumatoid arthritis.A protein interaction network of the intersection targets was constructed using the String database,and key target genes were screened using Cytoscape 3.7.1 software.A network map of component-target-pathway was constructed,and GO functional enrichment and KEGG pathway enrichment analysis were performed using the DAVID database.The top3 key target genes and key components were validated by molecular docking using the Swiss Dock online website.3 The intervention effect of Ganjiang Lingzhu decoction on rheumatoid arthritis rats.We established an adjuvant-induced arthritis(AA)rat model using Freund’s complete adjuvant and administered Ganjiang Lingzhu decoction and methotrexate.We observed and recorded the body weight,arthritis index,and paw swelling degree of the rats.Histopathological examination of the knee joints of each group of rats was conducted using HE staining.Enzyme-linked immunosorbent assay was used to detect the levels of TNF-α and IL-1β inflammatory cytokines in the serum of rats in each group.RT-q PCR and Western blot experiments were performed to detect the expression levels of genes and proteins related to the PI3K/Akt signaling pathway in the synovial tissue of eachgroup of rats.Results1 Study of the in vitro and in vivo chemical components of Ganjiang Lingzhu decoction.A total of 104 compounds were identified in Ganjiang Lingzhu decoction using UPLC-QE-MS technology,including 41 flavonoids,18 phenolic acids,16 terpenoids,8 lignans,5 alkaloids,3 fatty acyls,5 amino acids,and 8 other compounds.By comparing the exogenous chemical components of Ganjiang Lingzhu decoction and the endogenous components in rat blank serum,38 prototype compounds were identified in the serum of rats orally administered with Ganjiang Lingzhu decoction,most of which were flavonoids.2 A network pharmacology study of Ganjiang Lingzhu decoction on rheumatoid arthritis was conducted.Using the 38 prototype components of Ganjiang Lingzhu decoction as the research object,network pharmacology analysis was conducted.Pharm Mapper and Swiss Target Prediction databases were used to obtain 957 targets for the prototype components in the bloodstream.From the GSE55457 dataset in the GEO database,665 disease genes were obtained,including 430 upregulated genes and 235 downregulated genes.The Genecards database provided 1276 targets,and after removing duplicate genes,1821 disease targets were obtained.Taking the intersection of the two databases resulted in 327 target genes.Using Cytoscape 3.7.1 software,PIK3R1,SRC,and MAPK1 were identified as potential key targets for Ganjiang Lingzhu decoction treating rheumatoid arthritis,and 6-gingerol,isoliquiritigenin,and glycyrol may be the key components for its efficacy.Molecular docking results showed that the top three core targets and key components had good docking activity.KEGG pathway enrichment analysis suggested that Ganjiang Lingzhu decoction may exert therapeutic effects on rheumatoid arthritis by affecting the PI3K/AKT signaling pathway,MAPK signaling pathway,Ras signaling pathway,and other pathways.3 The intervention effect of Ganjiang Lingzhu decoction on rheumatoid arthritis rats.Ganjiang Lingzhu decoction can improve synovial tissue proliferation and intraarticular inflammatory cell infiltration in AA rats,reduce the levels of IL-1β and TNF-α inflammatory cytokines in the serum of AA rats,and inhibit the expression of PI3 K,AKT m RNA and PI3 K,p-AKT protein in the synovial tissue of AA rats.Conclusion1 The identified blood-active components such as glycyrrhetinic acid N,glycyrrhetinic acid,liquiritigenin A,6-gingerol,osteogenic ester of Gleditsia sinensis,isoliquiritigenin,mangiferin,and genistein A,may be the main material basis for the treatment of rheumatoid arthritis with Ganjiang Lingzhu decoction.2 Network pharmacology has screened multiple targets and pathways for the treatment of arthritis with Ganjiang Lingzhu decoction,which are closely related to inflammatory response,revealing that the mechanism of Ganjiang Lingzhu decoction in treating rheumatoid arthritis may be related to intervening in the inflammatory response.3 Ganjiang Lingzhu decoction exerts its therapeutic effects on arthritis by inhibiting inflammatory cell infiltration,synovial hyperplasia,and cartilage destruction through the PI3K/AKT signaling pathway,and reducing the levels of inflammatory cytokines IL-1β and TNF-α in the serum. |
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