Neurotoxicity And Related Mechanisms Of Deguelin In Vitro

Deguelin,a natural flavonoid,has strong biological activity and has been proved to have strong inhibitory effect in a variety of tumor cells.Deguelin has strong anti-tumor effect,it can effectively inhibit the growth and reproduction of cancer cells,especially on lung cancer cells.It is a potential natural antitumor drug with good application prospect.However,experiments have shown that high doses of deguelin can cause damage to the heart,liver and kidney of organisms.In order to explore the mechanism of deguelin-induced toxicity and elucidate mechanism,we choose deguelin to do the pharmacological study.CCK-8 test,Hoechst stains,Transmission electron microscopy(SEM)experiment were used to evaluate the deguelin toxicity to cell proliferation and cell nuclear damage.AO-EB staining and Annexin V-FITC/PI double staining experiment were used to evaluate the effects of deguelin on cell apoptosis morphology and apoptosis rate.RT-qPCR and Western Blot experiments were used to detect the regulatory effects of deguelin on the Bax and Bcl-2 at the gene and protein level,respectively.Meanwhile,DCFH-DA staining,SOD Activity Assay Kit and LDH Quantification Kit were used to detect the effects of deguelin on oxidative stress in cells.Finally,Western Blot experiments were conducted to investigate the regulatory effect of deguelin on related proteins in the MAPK/ERK pathway.The conclusions obtained in this paper are as follows:(1)1.5625μM-100μM of deguelin had inhibitory effects on the proliferation of PC-12,SH-SY5 Y,16HBE,LO2 and HEK-293 T cells and the survival rate of cells was significantly decreased with the increase of deguelin concentration(P<0.05).Deguelin had the strongest inhibitory effect on the proliferation of PC-12 cells.(2)Hoechst stains and Transmission electron microscopy(SEM)experiment observed 12.5μM,25μM,50μM of deguelin can inhibited cells growth differentiation and cause nucleus damage of PC-12 cells and SH-SY5 Y cells.(3)AO-EB staining showed deguelin could enhance apoptosis morphology.Flow cytometry combined with Annexin V-FITC/PI double staining showed that 12.5μM,25μM,50μM deguelin could markedly induce apoptosis rate of PC-12 cells and SH-SY5 Y cells.RT-qPCR and Western Blot experiments demonstrated that deguelin could up-regulate the expression of pro-apoptotic molecule Bax and down-regulate the expression of anti-apoptotic molecule Bcl-2,respectively,from the gene and protein levels.(4)DCFH-DA staining flow cytometry detected 12.5μM,25μM,50μM could enhance the PC-12 and SH-SY5 Y cells ROS level.Meanwhile we detected the LDH lactate dehydrogenase release amount increased,the intracellular SOD dismutase(SOD)content decreased,MDA lipid oxidation markers malondialdehyde content increased,etc.The oxidative stress levels increased significantly in the cells.Deguelin induced oxidative stress on PC-12 and SH-SY5 Y cells,which weakened the ability to resist external damage for cells.(5)Western Blot experiment explored the regulatory effects of deguelin on MAPK/ERK pathway at 12.5μM,25μM,and 50μM.Deguelin significantly reduced the proteins expression of EGF,RAS,CREB,p-MEK,MEK,p-ERK/2,and ERK/2(P < 0.05).Deguelin impaired cell activity by down-regulating the expression of related proteins in the MAPK/ERK pathway,thus achieving the effect of cytotoxicity.