Study On The Pharmacodynamic Mechanism Of Total Flavonoids Extracted From Moghania Philippinensis Against Rheumatoid Arthritis

ObjectiveRheumatoid arthritis(RA)is a chronic systemic autoimmune disease characterized by synovial inflammation and joint destruction that ultimately leads to disability.The ethnic drug-Moghania philippinensis has a good clinical effect on RA,However,its quality control research is not comprehensive and the treatment mechanism is not clear,which is very detrimental to the development and utilization of this drug.In this project,HPLC-DAD-QTOF-MS echnology was used to study Moghania philippinensis fingerprint,and the fingerprint data were analyzed by combining with hierarchical cluster analysis(HCA),orthogonal partial least squares discriminant analysis(OPLS-DA),similarity evaluation and other stoichiometric methods,so as to improve the existing Moghania philippinensis quality control system.In addition,MPTF was used as the experimental drug,and human fibroblast-like synoviocyte-rheumatoid arthritis(HFLS-RA)of human rheumatoid arthritis core effector cells were used as the experimental object.Through the establishment of cell inflammation model,the efficacy of MPTF was evaluated and the treatment mechanism was further explored,so as to provide a basis for the treatment of RA.Methods1.The study of Moghania philippinensis of ingerprintThe extraction solvent and chromatographic conditions were optimized.HPLC-DAD was used to determine 36 batches of Moghania philippinensis and establish the control fingerprint of Moghania philippinensis.Chemical pattern recognition was used to evaluate 36 batches of Moghania philippinensis.HPLC-QTOF-MS was used to identify chemical constituents and characterize fingerprint peaks as well.2.Effect of MPTF on inflammation of HFLS-RAFirst,we monitored the growth characteristics of cells.Then,MPTF concentrations that had no effect on cell viability and were in good condition were screened.In addition,IL-6 and MMP-3 were used as indicators to construct TNF-α induced inflammatory model of HFLS-RA.Finally,we selected IL-1,IL-6,IL-8,MMP-3 and MMP-9 as indicators to determine the effect of MPTF on inflammation in HFLS-RA.3.Study on inflammatory mechanism of MPTF against HFLS-RAWith Beclin 1,p62,LCⅡ as detection index and the introduction of autophagy inhibitor 3-MA.determination of MPTF affect autophagy.Autophagy inhibitor 3-MA and autophagy agonist Rapa were introduced to study the intervention of autophagy on inflammatory indicators MMP-3 and MMP-9,and to investigate the effect of autophagy on inflammation.IκBα、p-p 65 as testing indexes,the determination of the MPTF affect NF-κB signaling pathway.NF-κB signaling pathway inhibitors PDTC and NF-κB signaling pathway agonist PMA were introduced to determine the indicators of NF-κB signaling pathway while focusing on autophagy indicators,to investigate whether NF-κB signaling pathway regulates autophagy.A preliminary study was conducted to investigate whether there is a series regulatory effect between NF-κB signaling pathway,autophagy and inflammation.Results1.The study of the Moghania philippinensis fingerprintOptimal chromatographic conditions:acetonitrile-0.1%formic acid water as mobile phase,254 nm detection wavelength.By optimizing the elution gradient of the chromatogram,the HPLC-DAD fingerprint of was Moghania philippinensis established.A total of 30 common peaks were identified.A total of 47 components(32 flavonoids)and 21 common peaks(15 flavonoids)were identified by HPLC-QTOF-MS.HCA was collected into two groups in 36 batches of Moghania philippinensis.Based on similarity evaluation of 36 batchs Moghania philippinensis,found that the similarity of 30 batches of tendrils were all over 0.820,and the HCA and OPLS-DA were basically consistent with the similarity analysis results,suggesting that the quality of Moghania philippinensis of tendrils of commercially available is stable.2.Effect of MPTF on inflammation of HFLS-RAThe experiment determined that MPTF lower than 32.5 μg/mL was a safe concentration range for HFLS-RA,so 7.5 μg/mL,15 μg/mL and 30 μg/mL(low,medium and high dose)were selected as the dose concentrations of MPTF.It was found that TNF-αinduced by 10 ng/mL and above significantly increased the expression of inflammatory mediators in HFLS-RA for 6 h,thus confirming that TNF-α induced by 10 ng/mL induced by 6 h was the model condition.MPTF decreased the expression of inflammatory cytokines and inflammatory proteins in a dose-dependent manner,and it was concluded that MPTF inhibited inflammation in HFLS-RA.3.Study on inflammatory mechanism of MPTF against HFLS-RAWe found that the autophagy level increased with the increase of MPTF concentration.When autophagy inhibitor 3-MA was added,the autophagy level was called back,indicating that MPTF activated autophagy.Compared with the MPTF alone treatment group,the inflammatory degree in the MPTF and 3-MA co-treatment group was significantly increased,and the inflammatory degree in the MPTF and Rapa co-treatment group was significantly reduced,indicating that autophagy activated by MPTF can inhibit the inflammatory degree.MPTF was also found to inhibit the NF-κB signaling pathway in HFLS-RA.Compared with the MPTF alone treatment group,autophagy level in the MPTF and PDTC co-treatment group was significantly enhanced,while autophagy level in the MPTF and PMA co-treatment group was significantly weakened,indicating that inhibition of NF-κB signaling pathway could activate autophagy.Conclusions1.In this study,HPLC-DAD fingerprint,HCA,OPLS-DA,similarity evaluation and other pattern recognition methods were used to evaluate the quality of Moghania philippinensis.For the first time,HPLC-DAD combined with HPLC-QTOF-MS method was used to qualitatively analyze the chemical components of Moghania philippinensis and characterize the common peaks of some fingerprints.2.This study demonstrated for the first time that MPTF can inhibit inflammatory response in vitro.Autophagy and NF-κB signaling pathways were first proposed to investigate the mechanism of MPTF inhibiting the inflammatory response of HFLS-RA.Conclusion the mechanism of MPTF against HFLS-RA inflammation may be:inhibition of NF-κB signaling pathway to activate autophagy,thus achieving anti-inflammatory efficacy.The research results of the pharmacodynamic mechanism of MPTF against HFLS-RA inflammation provide a more sufficient basis for the resistance to HFLS-RA.