| Objective: Adjuvant arthritis rats were used to explore the anti-RA effect of Anemarrhena saponins;the potential mechanism of anti-RA of Anemarrhena saponins was predicted by network pharmacology,and the absorption of Anemarrhena saponins after oral administration was explored through serum medicinal chemistry methods.The main components of blood provide research directions for vitro experiments;TNF-α-induced MH7 A human synovial fibroblasts are used as the carrier for in vitro experimental research,and cytopathic glycolysis is the research center to investigate the key to the glycolysis pathway of drugs.The influence of the target PKM2 reveals the mechanism of Anemarrhena saponins inhibiting the proliferation of RA synovium.Methods: 1.The effect of total saponins of Anemarrhena on the pathological characteristics of adjuvant arthritis rats.An adjuvant arthritis model was prepared by subcutaneously injecting 0.1 m L of complete Freund’s adjuvant(CFA)into the right back sole of SD rats.The rat foot swelling instrument was used to measure the rat foot swelling,the tape measure to measure the rat foot circumference,and the joints were measured according to the scoring rules.Clinical scoring,HE staining to observe the pathological morphology of rat synovium,to investigate the effect of total saponins of Anemarrhena on the symptoms and signs of AA rats.2.Network pharmacology and serum medicinal chemistry analysis.Use the Pharm Mapper server to predict and analyze the potential targets of Anemarrhena saponins,analyze the protein interaction network(PPI)through the String online database,and use the R language program to annotate and KEGG the functions of the selected target molecules Enrichment and analysis of the potential mechanism of Anemarrhena saponins actions in the treatment of RA.And use Cytoscape software to visualize the data,and build a drug component-protein target interaction network.And choose the pathway with the highest degree of drug enrichment and the strongest significance,and use computer-aided simulation screening(molecular docking technology)to explore the binding ability of Anemarrhena saponins compounds with the key targets of this pathway.After intragastric administration of the total saponins of Anemarrhena vulgaris to healthy SD rats,blood from abdominal aorta of rats was collected and plasma was separated.After sample preparation,UPLC/Q Exactive MS(Ultra High Performance Liquid Chromatography-Mass Spectrometry)TIC full scan sample was used Analyze the main components absorbed into the blood after oral administration of the total saponins of Anemarrhena,combined with the results of network pharmacology and molecular docking results,screening of the main active components of the total saponins of Anemarrhena with anti-RA effect.3.Effects of drugs on the proliferation and activation of MH7 A cells.Using TNF-α-induced MH7 A synovial fibroblasts as the research carrier,the MTT method and plate cloning method were used to explore the effects of drugs on the proliferation of MH7 A cells;Carry out scar healing experiments and cell Transwell shuttle experiments to explore the effects of drugs on the migration ability of MH7 A cells;use Transwell erosion experiments(With Matrigel)to explore the effects of drugs on cell invasion;ELISA kits to detect changes in cell IL-6 release;Flow cytometry was used to detect drug interference on cell growth cycle and cell apoptosis,and pyridine iodide(PI)was used on an inverted fluorescence microscope.Observe the effect of drugs on cell apoptosis.4.The effect of drugs on glycolysis pathway and related targets of MH7 A cells.TNF-α-induced MH7 A synovial fibroblasts were used as the research carrier,and the glycolytic function of the cells was the research center.The experiments were carried out with and without blocking PKM2.Use HPLC method to detect the effect of drugs on cell energy;use HK,PFK,PK activity kits to detect the effect of timosaponin on the activities of hexokinase,phosphofructokinase and pyruvate kinase in MH7 A cells;use glucose detection kit,pyruvate detection kit,and lactic acid detection kit to detect the effects of drugs on cell glucose uptake,pyruvate and lactate production;use RT-PCR and Western Blot detects the effects of drugs on the key targets of glycolysis PKM2 and downstream effect targets STAT3 and BCL-2 gene protein expression,and investigates the mechanism of drugs inhibiting synovial hyperplasia.Results: 1.The total saponins of Anemarrhena can effectively inhibit the pathological manifestations of adjuvant arthritis rats.In vivo experimental studies have found that the total saponins of Anemarrhena can significantly alleviate the clinical symptoms and foot swelling in adjuvant arthritis rats,and can inhibit synovial hyperplasia and immune cell infiltration in the synovial membrane.It has a significant joint protection effect,showing Good treatment effect.2.The total saponins of Anemarrhena may play a role through glycolysis pathway,and Rhizophora artemisiae saponin may be the most important active component Through network pharmacological enrichment analysis,it was found that the glycolysis/gluconeogenesis pathway may be the main mechanism of the total saponins of Anemarrhena to exert their efficacy,and it was found that sapogenin(Sarsasapogenin,SA)may be the most important active ingredient.Combined with the results of serum medicinal chemistry,it is suggested that SA may improve the clinical manifestations of rheumatoid arthritis through glycolysis/gluconeogenesis after entering the blood,which points out the direction for further research.3.Sarsasapogenin can inhibit the abnormal activation of MH7 A cells.In vitro experiments confirmed that Sarsasapogenin can effectively inhibit the proliferation and weaken the ability of migration and invasion of MH7 A cells;Observation of the mitotic cycle of MH7 A cells revealed that SA may mainly affect the G2/M phase of cell division;observations by flow cytometry and Hoechst staining showed that high doses of SA can effectively induce apoptosis of MH7 A cells.MH7A cells.It was found that SA decreased the production of ATP,glucose consumption,glycolytic rachetase activity,and the production of pyruvate and lactic acid in MH7 A cells.After blocking PKM2,it was found that SA no longer affected the process of MH7 A cells’ glycolysis,suggesting that SA may inhibit the pathologic glycolysis of MH7 A cells by inhibiting PKM2 tetramer activity.We also found that SA decreased the expression of Bcl-2 and the phosphorylation of STAT3 by inhibiting PKM2 phosphorylation at TYR105.After blocking PKM2,SA lost its ability to regulate these targets.These results suggest that SA may overinhibit PKM2(Tyr105)phosphorylation and block the transformation of PKM2 dimer to down-regulate STAT3 protein phosphorylation and Bcl-2 protein expression to inhibit the proliferation and activation of MH7 A cells and induce their apoptosis.4.Sarsasapogenin can regulate the glycolysis pathway and related targets ofConclusion: Anemarrhena saponins could effectively alleviate the inflammation and pathological characterization of rat arthroarthritis,and its main active component is Rhizopus arthrogenin,which could effectively inhibit the proliferation,migration,invasion and release of cytokines of FLS,interfere with the growth cycle of FLS and induce the apoptosis of FLS,and its mechanism of action is different from that of regulating the functions of different PKM2 subtypes.It also inhibits the pathological glycolysis of synovial fibroblasts. |
PDF Full Text Request