Network Pharmacology And Metabolomics-based Study On The Mechanism Of The Extract From Celastrus Orbiculatus Thunb For The Treatment Of Rheumatoid Arthritis

Rheumatoid arthritis(RA)is a chronic autoimmune disease characterized by inflammation of synovial cells and destruction of articular cartilage and bone,which seriously endangers human health.The pathogenesis of RA is not clear,and it can occur at any age.At present,the goal of treatment is mainly to meliorate the symptoms of joint swelling and pain and delay the progress of the disease.Because of the long course of disease and high disability rate,it brings heavy psychological and economic burden to patients and their families.In recent years,although the rapid development of disease modifying anti-rheumatic drugs(DMARDs)has greatly improved the prognosis of patients with RA,especially the advent of drugs targeting key inflammatory factors or immune cells of RA,some drugs are relatively expensive and exhibited drug resistance or side effects.Thus,it’s necessary to discover antiRA drugs with better efficacy,less side effects and lower cost.Celastrus orbiculatus Thunb.belongs to the family Celastraceae and was first recorded in the Illustrated of Plants in Qing Dynasty.It has the effects of dispelling cold,activating blood circulation,relieving swelling and pain.The clinical experience for many years has shown that it can treat chronic rheumatic arthritis,rheumatoid arthritis,ankylosing spondylitis and other diseases either in sole or in combination.Celastrus orbiculatus Thunb.has been traditionally used in Asia countries such as China,Korea,Japan and other places due to its rich wild resources and low price.Compared with another widely used anti-RA traditional Chinese medicine Tripterygium Wilfordii of Celastraceae family,the toxicity of Celastrus orbiculatus Thunb.is relatively small,and has a wider range of safe application.At present,the research on its pharmacological activity is mainly focused on anti-tumor activity,especially antigastrointestinal tumor activity,while the research on its traditional efficacy against RA activity is relatively rare.In this paper,firstly,the potential active components and action mechanism of Celastrus orbiculatus extract against rheumatoid arthritis were explored by using network pharmacology and molecular docking technology.Then,the adjuvant-induced arthritis rat(AIA)model was established with complete Freund’s adjuvant(CFA).The therapeutic effect of Celastrus orbiculatus extract on AIA rat arthritis was confirmed by measuring indexes such as foot swelling degree,arthritis score,serum tumor necrosis factor-α(TNF-α)level,histopathological and immunohistochemical analysis,etc.Finally,non-targeted metabolomics based on UPLC-LTQ-Orbitrap-MS was used to find out the effects of Celastrus orbiculatus extract on endogenous small molecule metabolites.This paper is divided into the following three chapters:Chapter 1:Study on the mechanism of active components of Celastrus orbiculatus extract in the treatment of rheumatoid arthritis based on network pharmacology and molecular docking technologyIn this chapter,the potential targets of the active components of Celastrus orbiculatus extract(COE)were screened by Swiss Target Prediction and String databases,and RA-related target genes were screened by DisGeNET,GeneCards and OMIM databases,Venny maps were drawn for the ingredient targets of the active components of COE and RA targets,and protein network interaction map was obtained by String database.The network diagram of "active components of COE-core targets-RA" was constructed by using Cytoscape 3.7.1 software,and the Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were carried out on the intersecting targets through DAVID database and micro-information platform to predict its possible molecular mechanism.The experimental results show that there were 554 potential targets for the 29 active components from Celastrus orbiculatus extracts and 6174 potential targets of RA diseases,and 375 common targets and 10 core targets were obtained by taking the intersection;The network diagram of "active components of COE-core targets-RA" was constructed,and it was found that the multi-components of COE may play an important role in treating RA by regulating PI3K/Akt signaling pathway and MAPK signaling pathway.Chapter 2:Therapeutic effects of Celastrus orbiculatus extract on rheumatoid arthritisIn this chapter,Celastrus orbiculatus extract,(COE)was prepared according to the patented method in the laboratory(patent number:ZL200710025343.3),and then the adjuvant-induced arthritis rat was established by subcutaneous injection of CFA into the plantar region.The rats were divided into the normal control group(CON group),model group(AIA group),positive control group(MTX group),low dose group of Celastrus orbiculatus extract(COEL group),medium dose group of Celastrus orbiculatus extract(COE-M group)and high dose group of Celastrus orbiculatus extract(COE-H group).The successful establishment of AIA rat and the remission of COE on arthritis-related symptoms of AIA rats were determined by statistical analysis of body weight,paw volume,arthritis score.TNF-α and NO were quantified by corresponding enzyme-linked immunosorbent assay(ELISA)kits and NO assay kit according to the manufacturer’s instructions,which evaluate the effect of COE on the expression levels of inflammation-related factors in AIA rat serum.The anti-RA effects of COE was further evaluated by histopathological examination through H&E staining from the perspective of inflammation and bone erosion.The possible mechanism of COE on AIA rats was elucidated through immunohistochemical experimental analysis.The experimental results showed that COE could improve the body weight change and significantly alleviate paw swelling in AIA rats,decrease the paw volume and arthritis score,COE significantly reduced the expression levels of serum TNF-α and NO;and can significantly improved these histopathological changes.Reduce the expression of P-Akt,P-Erk and P-Stat3 proteins in the ankle.Chapter 3:Metabolomics study of Celastrus orbiculatus extract against rheumatoid arthritisIn this chapter,we used metabolomics techniques based on ultra performance liquid chromatography-mass spectrometry(UPLC-LTQ-Orbitrap-MS)to explore the effects of COE on endogenous small molecule metabolites in serum of AIA rats.First,serum samples were subjected to pretreatment.The LC-MS raw spectral data were first processed by Compound Discoverer 2.1(Thermo Fisher Scientific)for peak detection and alignment.Afterwards,the data were undergone background deduction,total area normalization.Finally,the data were analyzed by Principal Components Analysis(PCA),partial Least Square Discriminant Analysis(PLS-DA)and Orthogonal PLS-DA(OPLS-DA).Variable importance in projection(VIP)>1.0 values obtained from OPLS-DA models and p values(P<0.05)calculated from Mann-Whitney tests were used to screen differential metabolites,which were matched with the human metabolome database(HMDB)to identify the specific differential metabolites.The enrichment of KEGG pathway and GO function analysis were conducted to clarify the mechanism of COE in the treatment of RA.The experimental results showed that after CFAinduced arthritis in rats,the levels of 22 endogenous metabolites were found to be changed.After COE treatment,the content of 12 endogenous metabolites was increased,while that of 10 endogenous metabolites was decreased.Through GO function analysis and KEGG pathway enrichment analysis,the differential metabolites mentioned above were related to fatty acid metabolism,linoleic acid metabolism,glycerophospholipid metabolism,amino acid metabolism,tryptophan metabolism,and TCA cycle,indicating that COE could recover from metabolic disorders caused by RA by regulating energy metabolism and amino acid metabolism.In conclusion,this experiment employs adjuvant-induced arthritis rat model,using a combination of network pharmacology,molecular docking analysis,histopathological analysis and immunohistochemistry techniques to reveal the therapeutic effects of COE on RA.Our study would give new insights into the development of new anti-RA drugs and full exploitation of Celastrus orbiculatus Thunb.