The Therapeutic Effect And Toxicity Of The Alcoholic Extract Of Euphorbia Pekinensis On Rats With Adjuvant Arthritis Model Therapeutic Effect And Toxicity Study

Objective:In this study,the alcoholic extracts of Euphorbia Pekinensis were used as example drugs.To investigate the efficacy and toxicity of Euphorbia Pekinensis on adjuvant arthritis model rats and normal rats by using adjuvant arthritis rats and normal rats as study subjects;Prediction of the targets and signaling pathways of the anti-rheumatoid arthritis effect of 95% ethanolic extract of Euphorbia Pekinensis using a network pharmacology approach,and validate the core targets related to rheumatoid arthritis through animal experiments;analyze the effect of Euphorbia Pekinensis on the change of intestinal flora in adjuvant arthritis model animals using 16 Sr DNA high-throughput sequencing.To provide a scientific basis for its safe and rational clinical application and further research and development.Methods: 1.Using a network pharmacology approach,we collected 95% of the active ingredients of the alcoholic extract of Euphorbia Pekinensis and screened the corresponding action targets to search for targets related to RA disease.Constructing a "drug-active ingredient-target" network,constructing a network between disease related targets based on PPI database,and screening key targets based on the 2-fold median of nodes.The Metascape platform was used to analyze the target-associated major GOs and KEGG,respectively,for pathway enrichment analysis,and the top 10disease-related core targets were molecularly docked to the corresponding top 10 chemical components using molecular docking software.2.54 healthy SD male rats were divided into 6 groups according to the random number method,which were blank group,model group(adjuvant-induced arthritis,AA),positive rehmannia polysaccharide tablet group(9 mg/kg),model administration high dose group(360.4 mg/kg),model administration medium dose group(180.2mg/kg),model administration low dose group(90.1 mg/kg),and model administration(90.1mg/kg).Adaptive feeding for seven days,replicate the rat adjuvant arthritis model,continuous treatment administration for 4 weeks,observe the model administration of each group of rats foot and plantar swelling,ELISA to detect the effect of the relevant indicators in the serum,and histopathological examination of the synovial membrane of the toes.3.81 SD rats,divided into 9 groups,respectively,normal group,model group,positive group(9mg/kg),model administration high,medium and low dose group(360.4mg/kg,180.2mg/kg,90.1mg/kg),normal administration high,medium and low dose group(360.4mg/kg,180.2mg/kg,90.1mg/kg).In addition to the normal and model groups were given the corresponding drugs to observe and compare the toxicity characteristics and toxic target organs after 4 weeks of administration of Kyodarabine to the model administration group and the normal administration group.4.intestinal flora analysis: rat feces were collected at the end of the experiment and sequenced with 16 S r RNA 341F-806 R region.To investigate the effect of Euphorbia Pekinensis on the intestinal flora of rheumatoid arthritis(AA)rats.Results:1.The results of network pharmacology experiments show that the 25active ingredients obtained from the screening of Euphorbia Pekinensis,and then get 376 ingredient targets,disease database search to 5017 rheumatoid arthritis disease targets,229 after the intersection of ingredient-disease targets.Compound-crossing target topology analysis revealed that 7,4’a dihydroxydihydroflavone(degree=73),apigenin(degree=71),kaempferol(degree=66),rhamnetin(degree=66),quercetin(degree=65),dibutyl phthalate(degree=61),7-hydroxycoumarin(degree=46),ellagic acid(degree=41),daidzeinol(degree=39),and glycyrrhizin(degree=38)dominated the network.PPI network topology analysis showed that 43 target proteins such as TNF,AKT1,SRC,EGFR,MMP9 and ERBB2 were the more core proteins.The intersecting targets involved1944 biological processes and 200 KEGG pathways(p<0.01),and Kyodoji mainly regulates cancer pathways,PI3K-Akt signaling pathway,MAPK signaling pathway,TNF signaling pathway,T cell receptor and other signaling pathways to achieve therapeutic RA effect.2.The results of the study on the therapeutic effect of Euphorbia Pekinensis Alcoholic extracts on AA rats showed that:(1)Swelling of toes: Compared with the normal group,the swelling of feet in the rat modeling group was significantly higher(p<0.01),and each treatment group showed different degrees of swelling reduction compared with the model group(p<0.05),among which the high-dose group had the best swelling reduction effect,and the effect was dose-related.(2)Enzyme-linked immunoassay results: RF,TNF-α,CD4,CD25,IL-2,MMP-9,and IFN-γ were significantly increased(p<0.05 or p<0.01)and CD8,IL-10,and TGF-β were decreased(p<0.05 or p<0.01)in the model group compared with the normal group.Compared with the model group,RF,TNF-α,CD4,CD25,MMP-9,IFN-γ were decreased(p<0.05 or p<0.01),IL-10,TGF-β were increased(p<0.05),IL-2 was decreased(p<0.05)and CD8 was increased(p<0.05)in the high,medium,low and dose groups in each treatment group.(3)HE synovial pathology results: synovial cells in the normal group were arranged in a single layer without proliferation and no inflammatory cells were found;in the model group,synovial tissue in the ankle joint was hyperplastic,with a large number of inflammatory cells infiltrating and inflammatory exudation in the joint cavity and serious bone loss;after treatment,the value-added of synovial cells was significantly better than that in the model group,and lesions such as inflammatory cell infiltration,cartilage erosion,synovial proliferation and formation of vascular opacities were all improved to different degrees.3.The results of the study on the toxic effects of Kyocera alcohol extract showed that:(1)the effect on biochemical indicators: compared with the normal group,model administration of high and normal administration of each dose group AST elevated(p<0.01 or p<0.05);normal administration of each dose group ALT elevated(p<0.01 or p<0.05);normal administration of medium dose group UREAL elevated(p<0.05);model administration of high dose group CREP elevated(p<0.05).CREP was elevated in the high dose group(p<0.05).(2)Effects on organ indices: compared with the normal group,the heart and liver indices were increased in the normal administration groups(p<0.01 or p<0.05);the lung indices were increased in the positive group(p<0.05);the kidney indices were increased in the positive group,the model administration low dose group and the normal administration groups(p<0.01 or p<0.05);the spleen indices were decreased in the model group(p<0.05),and compared with the model group Compared with the model administration medium and low dose groups,the spleen index was increased(p<0.01 or p<0.05).4.16 sr DNA high-throughput sequencing results: compared with the normal group,the model group differential bacteria were 13 differential bacteria,such as Bacillales,Actinomycetales,Jeotgalicoccus,Staphylococcaceae,Pasteurellales,Pasteurellaceae,Akkermansia,Verrucomicrobiales,Verrucomicrobiae,Verrucomicrobia,Verrucomicrobiaceae,Atopobium,Butyricimonas,Haemophilus.And the difference bacteria in the administration group were 14 difference bacteria such as Turicibacterales,Turicibacter,Turicibacteraceae,Corynebacterium,Corynebacteriaceae,Aerococcaceae,Facklamia,Neisseriaceae,Neisseria,Neisseriales,Oribacterium,Planococcaceae,Sporosarcina,Lachnoanaerobaculum.Conclusion: The alcoholic extract of Euphorbia Pekinensis can significantly improve the swelling degree of toe and synovial pathological changes in AA rats,and regulate the levels of cytokines such as TNF-a,RF,IL-2,MMP-9,CD4,CD8,CD25,TGF-β,IFN-γ and IL-10 in the serum of AA rats,so Euphorbia Pekinensis has obvious therapeutic effects on adjuvant arthritis rats;The mechanism of action may be through 25 active components such as 7,4’-dihydroxydihydroflavone,apigenin,kaempferol,rhamnetin,quercetin,dibutyl phthalate,7-hydroxycoumarin,ellagic acid,macrogallol,glycyrrhizin,etc.acting on 43 target proteins such as TNF,AKT1,SRC,EGFR,MMP9,ERBB2,etc.through cancer pathways,MAPK,PI3K-Akt,T-cell receptor,TNF and other core pathways to act directly or mechanism of action may be Turicibacterales,Turicibacter,Turicibacteraceae,Corynebacterium,Corynebacteriaceae,Aerococcaceae,Facklamia,Neisseriaceae,Neisseria,Neisseriales,Oribacterium,Planococcaceae,Sporosarcina,Lachnoanaerobaculum and other 14 differential bacteria to modulate back intestinal bacterial activity to modulate rheumatoid factor RF in AA rats.The content of inflammatory cytokines TNF-a,IL-2,T-cell associated factors CD4,CD8,CD25,B-cell associated factors TGF-β,IFN-γ,IL-10,etc.thus exerted anti-RA effect and relieved the swelling degree of rat toe and synovial membrane pathological changes.The toxicity target organs of Kyodarabine are liver and kidney,and the toxicity of administration under normal condition is greater than that of administration under disease condition.