Systematic Study on Molecular Mechanism of Aconitum Alkaloid in Treatment of Rheumatoid Arthritis / 中国实验方剂学杂志

ABSTRACT

Objective:To explore the effect of chemical compound of aconitum alkaloid on the lipopolysaccharide （LPS）-induced inflammatory response of RAW264.7 macrophages and investigate its mechanism. Method:The chemical compounds of Aconitum Kusnezoffii Reichb were collected from TCMSP database with consideration of oral bioavailability （OB）≥30% and drug-likeness （DL）≥0.18. The potential targets of each chemical component were predicted with use of Pubchem database and Swiss Target Prediction database. Rheumatoid arthritis （RA） targets were collected from GeneCards database and selected by intersection screening. Gene ontology （GO） classification enrichment and Pathway enrichment analysis were carried out with use of DAVID database. Cytoscape was used to construct "Chemical Compound-Potential Targets-Pathway-Disease" network. Protein-protein interaction （PPI） network was constructed by using STRING database and Cytoscape software. RAW264.7 macrophages were stimulated by LPS to establish macrophage inflammation model <italic>in vitro</italic>. Western blot was used to detect the effects of chemical compounds on the expression of tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） and cyclooxygenase-2 （COX-2） in RAW264.7 cells induced by LPS， as well as on the expression of JAK kinase and nuclear transcription factor- kappa B （NF-<italic>κ</italic>B） signal pathway. Result:A total of 27 chemical compounds were obtained by searching TCMSP database and consulting literature （OB≥30%， DL≥0.18）. 12 chemical compounds were obtained after screening. 177 potential targets were obtained after database prediction and screening， and 97 targets were obtained as potential targets for the treatment of RA after intersection between 177 potential targets and 4 329 RA targets. A total of 32 biological processes （BP）， 5 cellular components （CC）， and 12 molecular functions （MF） were enriched by DAVID database. The construction of network topology map showed that different chemical compounds can act on the same target and the same chemical compound can also act on different targets in the treatment of RA. Aconitum alkaloid can be connected with the same pathway through different targets or with different pathways through the same target， indicating that different targets may have synergistic effect， which fully reflected the complex multi-compound， multi-targets and multi-pathways mechanism. Different concentrations of LPS in stimulation （0-200 μg·L^-1） can significantly up-regulate the expression of COX-2 protein in RAW264.7 macrophages （<italic>P</italic><0.05）， indicating that the inflammatory model was successful. Compared with the normal group， the expression of TNF-<italic>α</italic> and COX-2 protein in the inflammatory model of RAW264.7 cells increased significantly（<italic>P</italic><0.05）， while the expression of TNF-<italic>α</italic> and COX-2 protein in bulleyaconitine A（BLA）， songorine， yunaconitine and karacoline groups decreased in varying degrees compared with the model group （<italic>P</italic><0.05）. Compared with the normal group， the expression of IRAK4， NF-<italic>κ</italic>B， JAK1 and STAT3 in the inflammatory model of RAW264.7 cells were significantly increased （<italic>P</italic><0.05）， while such levels in BulleyaconitineA， songorine， yunaconitine and Karacoline groups were significantly lower than those in the model group（<italic>P</italic><0.05）. Conclusion:Based on systematic pharmacology and <italic>in vitro</italic> experiments， the related targets and signal pathways were analyzed to provide new insights into the pathogenesis of RA， reveal the molecular mechanism of aconitum alkaloid in the treatment of RA， and provide new ideas for the application of Mongolian medicine in modern medicine.