钩吻不同部位生物碱及非生物碱的抗肿瘤活性及机制研究OA

Objective:To compare and analyze the in vitro anti-tumor activities of alkaloids and non-alkaloids in the root,stem,and leaf parts of the Gelsemium elegans(Gardn.et Champ.)Benth,and explore their mechanisms of ac-tion.Methods:The inhibitory activity of alkaloids and non-alkaloids on cell growth in the roots,stems,and leaves of Gelsemium elegans(Gardn.et Champ.)Benth were tested by adopting the CCK-8 assay on seven human cancer cell lines,including human colon cancer cells(HCT116),liver cancer cells(HepG2)and human cervical cancer cells(HeLa).On this basis,high activity root alkaloids were selected for concentration dependent investigation of inhibiting tumor cell proliferation,and the effects of different concentrations and time treatments of root alkaloids on the morphological changes of tumor cells were observed under a microscope.Furthermore,molecular docking simulation was employed to investigate the binding inter-actions between the main active alkaloids(koumine,gelsevirine,gelsemine,gelsenicine,and koumidine)and the MDM2 pro-tein.By taking A549 cells as the research object,Western blot was used to verify the effect of Gelsemium alkaloids on the ex-pression of MDM2 and p53 proteins in cells in order to preliminarily explore its anti-tumor mechanism.Results:Alkaloids and non-alkaloids from the root,stem,and leaf parts of Gelsemium elegans have a certain inhibitory effect on the proliferation of the seven types of cells mentioned above,and the activity of alkaloids is generally higher than that of non-alkaloids,with root alkaloids being the strongest and leaf non-alkaloids being the weakest.Root alkaloids can inhibit the proliferation of seven types of cells in a concentration dependent manner,with the primary effective concentrations ranging from 50 to 100 μg/mL.Mor-phological observations showed that compared to the normal group,low concentrations of root alkaloids(below 50 μg/mL)had little effect on cell morphological changes.As the concentration increased,cell adhesion decreased,the skeleton disintegra-ted,and apoptotic bodies appeared,which were classic features of apoptosis.This suggests that it may inhibit tumor cell prolif-eration by inducing cell apoptosis.Through molecular docking,we found that the main active ingredients of Gelsemium elegans(Gardn.et Champ.)Benth could effectively bind to MDM2 protein through interactions with hydrogen bonding,carbon hydro-gen bonding,and van der waal forces.Western blot experiment also showed that there exists concentration dependent inhibition of MDM2 protein in A549 cells by root alkaloids and upregulation of p53 protein expression.Conclusion:The anti-tumor activ-ity of Chinese materia medica Gelsemium elegans(Gardn.et Champ.)Benth is primarily enriched in the roots,and the anti-tumor effect of alkaloid components is significantly better than that of non-alkaloid components.The mechanism of action may be related to the competitive binding of alkaloids to MDM2 protein,which could relieve the inhibition of p53 by MDM2,there-by releasing and activating the p53-mediated anti-tumor pathway.