The Research Of Mitochondrial Dysfunction And Cytotoxicity In Deguelin And 7-hydroxy-substituted Deguelin Derivative

Objevtive:Deguelin is a flavonoid extracted from rotena plant.It has broad spectrum antitumor activity by inhibiting cell invasion,migration,cell cycle arrest,antiangiogenesis and other mechanisms.Rotenine intervention led to a dramatic increase in oxidative stress levels in cells,and mitochondrial function was crucial in this process.As the ubiquinone I REDOX enzyme complex inhibitor,deguelin damage the neurons,heart and lung,obstruct the application of deguelin.In order to explore and released the mitochondrial damage caused by deguelin,our research group synthesized a large number of rotenine derivatives based on the structure of deguelin,and selected the 7-OH substituted deguelin derivatives with potential anti-tumor activity.This study mainly verified the anti-tumor activities of the two compounds,compared the degree of mitochondrial damage and apoptosis caused by the two compounds,and explored whether 7-OH substituted deguelin derivatives could achieve the effect of toxicity reduction.Methods:1.Verified the anti-tumor activity of deguelin and 7-OH substituted deguelin derivatives,observed the inhibitory effect of two compounds on the proliferation of five kinds of normal cells,and selected the two most obvious types of cells.2.By means of transmission electron microscopy,fluorescence inverted microscope,flow cytometry and western blot,the apoptotic damage of the two compounds on PC-12 cells and 16HBE cells was observed,and the regulatory effects of related apoptotic factors Bcl,2,Bax and Cytochrome C were studied.3.Explored the potential mechanisms of the two compounds which were causing toxic damage to mitochondria,including the effects on mitochondrial membrane potential,free radical scavenging ability,ATP level and mtDNA copy number.4.Study the regulatory effects of the two compounds on related proteins in the AMPK/PGC1-α pathway and the toxic mechanism of deguelin,and to observe whether the optimized 7-OH substituted deguelin derivatives can improve the mitochondrial damage caused by them.Results:1.CCK-8 assay showed that deguelin and 7-OH substituted deguelin derivatives could inhibit the proliferation of H1299 cells and MCF-7 cells in a time and concentration dependent manner.It was attractive to us that the inhibitory activity of 7-OH substituted deguelin derivatives on tumor cells is similar to deguelin,which can achieve anti-tumor activity at the level of micromole.We also found deguelin and 7-OH substituted deguelin derivatives affected five kinds of normal cell line(PC-12,16HBE,LO2,HEK293T,HUVEC)with different levels of inhibition of proliferation,found that deguelin affacted the PC-12 and 16 HBE with the strongest toxic effect.At the same time,the toxic effects of 7-OH substituted deguelin derivatives in that two kinds of cells was weaker than deguelin’s(P<0.05).2.Both compounds have obvious damage to the nucleus and mitochondria of cells to a certain extent.With the increase of drug concentration,cell nucleus showed obvious apoptosis,mitochondrial green fluorescence decreased gradually,and mitochondrial activity decreased significantly.At the same time,heterochromosomal aggregation was found in the nucleus,swelling of mitochondria and loss of cristae.After quantitative detection of the apoptosis-inducing effects of the two compounds on PC-12 and 16HBE cells,it was found that the drug concentration was positively correlated with the apoptosis rate.The damage of these two compounds to cells,resulting in apoptosis,may be related to the overflow of cytochrome C,the activation of pro-apoptotic factor Bax,and the inhibition of anti-apoptotic factor Bcl-2.It was worth noting that 7-OH substituted deguelin derivatives led to less apoptosis than deguelin(P<0.05).3.The two compounds under different degree of PC-12 and 16 HBE cells have damage,including the significant reduction of mitochondrial membrane potential and the decrease of SOD and GPx of antioxidant enzyme activity.Decreased the production of adenosine triphosphate(ATP)and the copy number of mitochondrial mtDNA,which may affect the biosynthesis of mitochondria and cause obvious mitochondrial damage.However,the mitochondrial damage caused by 7-OH substituted deguelin derivatives was weaker than deguelin(P<0.05).4.The two compounds could down-regulated phosphorylated AMPK in PC-12 cells and 16HBE cells,while reducing the protein expression levels of downstream CREB,PGC1-α,Nrf1,Nrf 2,and TFAM.However,7-OH substituted deguelin derivatives downregulated related proteins to a lesser than deguelin(P<0.05).