Study On The Activity Of Three Anthraquinone Compounds Of Rubia Cordifolia L.to Treat Rheumatoid Arthritis

According to previous result,the alcohol extract of Rubia cordifolia L.and total anthraquinone has showed great treatment to RA,and the madder,purpurin and rubiadin were identified in the Rubia cordifolia L.alcohol extraction into the blood components.In order to further study the role of active compounds of Rubia cordifolia L.to RA,we used network pharmacology,mouse macrophage RAW264.7 inflammation model induced by LPS and adjuvant rheumatoid arthritis rat model,to discusse the treatment and possible mechanism of above three anthraquinone compounds to RA.ObjectiveNetwork pharmacology and molecular docking were used to study the anti-inflammation and the treatment of madder,purpurin and rubiadin,and LPS-induced mouse macrophage RAW264.7 inflammation model and adjuvant rheumatoid arthritis rat model were used to verification.Methods1.Network pharmacology and molecular docking predicted the potential of treatment madder,purpurin and rubiadin to RAPharmapper online platform was used to collect chemical compound prediction targets,and Genecard database was used to collect RA Target:String database was used to construct target protein interaction(PPI),David database and Kobas3.0 database for KEGG enrichment and biological function analysis of active pathway.Cytoscape3.5.1 software was used to construct "compound-target" network and "compound-target-pathway-disease" network.The molecular docking was used to analyze the interaction of P65 protein and COX-2 protein between compounds.2.LPS-induced mouse macrophage RAW264.7 inflammatory model was used to evaluate the anti-inflammatory effect of the three compoundsThe mouse macrophagic RAW264.7 inflammatory model induced by LPS,the three compounds were evaluated to screen for better anti-inflammatory effect by the determination of IL-6,IL-1β,TNF-α nd IL-10.3.Evaluate the therapeutic effect of purpurin in the rat model of adjuvant arthritisThe rat model of arthritis induced by freund’ s complete adjuvant was established,the CD4+/CD8+ values and other indicators,as well as pathological and clinical biochemical indicators were used to evaluate the anti-inflammatory effect of purpurin.Results:1.Network pharmacology and molecular docking predicted the potential of treatment madder,purpurin and rubiadin to RAThe network of madder,purpurin and rubiadin acting on RA was analyzed,and there were 288 targets of madder,250 targets of purpurin,and 296 targets of rubiadin,and the common targets of above three compounds were 152.KEGG pathway enrichment was carried out on the targets of each compound,and a total of 128 related pathways of madder,166 of purpurin and 182 of rubiadin were obtained.The related pathways were analyzed,including PI3K-Akt signaling pathways,NF-κB signaling pathways,ect,the role of the three compounds pathways are similar,but the correlation with pathway were different.The molecular docking showed that the binding energies of madder,purpurin and rubiadin to P65 protein and COX-2 protein are all negative,indicating that the three compounds have the potential to interact with P65 protein and COX-2 protein.Combined the result of extract,the total anthraquinone of Rubia cordifolia L.inhibits IL-6,IL-1β,and TNF-α.This study further investigated the NF-κB pathway.2.LPS-induced mouse macrophage RAW264.7 inflammatory model was used to evaluate the anti-inflammatory effect of the compoundMadder,purpurin and rubiadin at different concentrations and proportions showed inhibition to IL-6,IL-1β and TNF-α(P<0.05),and increased the concentration of IL-10.Among the three compounds,purpurin showed greater inhibitory effect of IL-6,IL-1β and TNF-α.The expression of P65 protein decreased most significantly when low concentration of purpurin was given(P<0.01).Low and medium concentrations of purpurin inhibited COX-2 expression(P<0.01,P<0.01),while high concentrations of purpurin did not inhibit cox-2 expression.3.Evaluate the therapeutic effect of purpurin in the rat model of adjuvant arthritisCompared with the model group,the foot swelling of rats in the treatment group was significantly improved(P<0.001).The spleen index and liver index of the medium dose group were significantly different(P<0.05,P<0.05),but there was no significant difference among other groups.Purpurin significantly improve the structure and necrosis of AIA rats hepatocytes and injury of spleen,besides,purpurin increase the level of Foxp3 protein,and CD4+/CD8+values decreased in all groups.When serum inflammatory factors were detected,medium dose of hydroxymadarin significantly reduced the content of IL-6(P<0.01).Low,medium and high doses groups significantly decreased the content of IL-1β,respectively(P<0.05,P<0.05 and P<0.01).Low and medium doses of purpurin reduced IL-6 and MMP3 content in ankle of AIA rats.The low,medium and high doses group had significant inhibitory effect on the MPO content in the liver of AIA rats(P<0.01,P<0.001,P<0.001).Low dose of purpurin also reduced MDA in the liver of AIA rats,while medium and high dose of purpurin significantly reduced MDA(P<0.01,P<0.01).Conclusion:The result of network pharmacology showed Madder,purpurin and rubiadin showed potential effect on PI3K-Akt signaling pathways,NF-κB signaling pathways,ect;Madder,Purprine and Rubiadin can reduce the inflammatory factors with degree,and purpurin inhibited the expression of IL-6,IL-1β and TNF-α,as well as P65 protein and COX-2 in NF-κB pathway.Purpurin showed some treatment to AIA rats.