Study On The Material Basis Of Qianghuo Shengshi Decoction In The Treatment Of Rheumatoid Arthritis

Rheumatoid arthritis（RA）is a widespread,devastating autoimmune disease whose main clinical feature is progressive synovitis,which if left untreated can lead to destruction of joint bone and cartilage,joint deformity and loss of function,ultimately leading to disability.As a chronic disease,RA poses a serious risk to human health and a heavy burden to both individuals and society.Qianghuo Shengshi Decoction（QHSSD）is a common formula of traditional Chinese medicine（TCM）for the clinical treatment of RA,which derived from the Neiwaishang Bianhuolun created by Dongyuan Li of the Jin dynasty.It consists of seven herbs,including Notopterygii Rhizoma et Radix（Qiang Huo,QH）,Angelicae Pubescentis Radix（Du Huo,DH）,Saposhnikoviae Radix（Fang Feng,FF）,Ligustici Rhizoma et Radix（Gao Ben,GB）,Chuanxiong Rhizoma（Chuan Xiong,CX）,Viticis Fructus（Man Jing Zi,MJZ）and Glycyrrhizae Radix et Rhizoma（Gan Cao,GC）.QHSSD has ample clinical studies and has been proven to have good efficacy.Since QHSSD was included in the list of 100 classical formulas in 2018,its experimental research has gradually made some progress.However,there is still a lack of the systematic research on its material basis and pharmacological mechanism,resulting in the inability to clarify the pharmacodynamic material basis and explain its mechanism of action in treating diseases scientifically,thus limiting the further development of this classic formula.In this study,a new systematic research strategy was proposed to investigate the pharmacodynamic material basis and the mechanism of action of QHSSD in the treatment of RA by multi-level analysis which combining drug analysis,systems biology and in vitro experiment,providing a practical reference model for the study of pharmacodynamic material basis of TCM formula.The specific research works are listed as follows:1.Study on HPLC fingerprint of QHSSD24 batches of lyophilized powder of QHSSD were prepared from seven herbs of different origins by randomly combining.The HPLC fingerprint of QHSSD was established,and the"Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines"（2012 version）was used for similarity evaluation and common peak matching.The results showed that all the similarities were higher than 0.9,and 27 common peaks were matched.The methodological validation results showed that the developed HPLC fingerprint method was stable and reliable.The common peaks were identified by chromatographic behaviour and mass spectrometric information（23 of the27 common peaks were identified,of which 11 compounds were confirmed by standards）and attributed to the source of the herbs by chromatographic behaviour,which enriched the information of the fingerprint of QHSSD.It provides the method support for the quality control of QHSSD.2.Chemical profiling characterization of QHSSD and its in vivo metabolitesThe technology of UHPLC-ESI-Q-TOF-MS/MS was used to analyze QHSSD sample and the biological samples of rats after QHSSD gavage.By comparing the mass spectrometry information with literatures and databases,159 compounds and 29absorbable prototypes of QHSSD were identified by using Peak View 1.2 software.The chemical compounds in QHSSD includes 43 coumarins,23 flavones,7 chromones,26terpenoids,17 phenylpropionic acids,16 phthalides,12 organic acids and 15 others（nucleosides,amino acids,polysaccharides,anthraquinones,aromatic hydrocarbons）.The absorbable prototypes of QHSSD includes 9 coumarins,6 flavones,5 chromones,3terpenoids,3 phenylpropionic acids,1 phthalide,1 anthraquinone and 1 nucleoside,which may be the active ingredients in QHSSD,providing material basis for the subsequent systems biology studies.In addition,210 in vivo metabolites of the absorbable prototypes were detected using Metabolitepilot^TM 1.5 software.3.Systems biology studies of QHSSD in the treatment of RAA systems biology strategy was used to investigate the pharmacodynamic material basis and the mechanism of action of QHSSD in the treatment of RA.Firstly,it was found that the function of QHSSD in the treatment of inflammation mainly involves the regulation of purine,pyrimidine and amino acid metabolisms by cellular untargeted metabolomics study.Secondly,a network pharmacology study was conducted based on the absorbable prototypes of QHSSD,then integrated with metabolomics results to get a comprehensive analysis.The results showed that five active ingredients（notopterol,isoliquiritin,imperatorin,cimifugin,and glycyrrhizic acid）and eight key genes（STAT3,SRC,HSP90AA1,AKT1,EGFR,MAPK3,ADA and HPRT1）play crucial roles during the treatment of QHSSD,and the pathways mainly involved in the PI3K-Akt signaling pathway and MAPK signaling pathway.Finally,the great binding abilities of the potential active compounds to the key targets were validated by molecular docking techniques（all binding energies<-5.0 kcal/mol）.4.In vitro anti-inflammatory validation of QHSSD and active compoundsThe effects of QHSSD and selected active compounds on the viability of RAW 264.7cells were determined by CCK-8 method.LPS-induced RAW 264.7 cells were treated with different concentrations of QHSSD extract and selected active ingredients,the supernatants were collected and the levels of NO,TNF-αand IL-6 in the supernatants were measured by kits.The results showed that QHSSD and all the five selected active ingredients had great in vitro anti-inflammatory effects,preliminarily indicating that the five active ingredients screened may be the potent anti-RA substances in QHSSD,the systematic strategy developed in our study is feasible.