| Objective:(1)The aim of this study was to investigate the effect of ilexonin A on proliferation inhibition of human erythroleukemia K562 cells and whether it can induce autophagy and apoptosis of K562 cells.(2)LC3 pathway was used as the entry point to explore the possible mechanism of the inhibition of the proliferation,autophagy and apoptosis of K562 cells by ileocin a.So as to determine the target and mechanism of the anti-leukemia effect of ilex lanceolata a,and provide the laboratory basis for clarifying the anti-leukemia effect mechanism of traditional Chinese medicine and the treatment of leukemia by traditional Chinese medicine.Methods:M T T method was used to detect the effect of ilexonin A on the proliferation of K562 cells.The morphology of K562 cells and the intracellular autophagosome structure of K562 cells were observed by transmission microscope.Flow cytometry was used to detect the apoptosis rate of K562 cells.Western Blot method was used to detect the changes in the expression of autophagy related protein l C 3 after the action of ilexonin A.Results: 1.MTT assay showed that the inhibition rate of K562 cell proliferation was significantly higher in the experimental group than in the blank control group(P < 0.05).There were significant differences in proliferation inhibition between different concentration groups(P < 0.05).The proliferation inhibition rate of K562 cells in the experimental group increased significantly with the extension of culture time(P < 0.05).The inhibitory effect of ilexonin A on the proliferation of K562 cells was significant,and the inhibitory effect was positively correlated with time,showing a dose-dependent effect.2.Cell morphology was observed by transmission microscope: after 48 h of intervention by ilexonin A,partial cell membrane of K562 cells in the experimental group was observed under electron microscope to be incomplete,chromatin condensation and density increase,and part of chromatin was broken and dispersed in the cytoplasm,and then condensed into lumps with uneven staining.The number of apoptosis of K562 cells increased with the increase of drug solubility.The morphology of K562 cells in the control group was normal.3.The fluorescence microscopic observation after MDC staining showed that the cells in the experimental group showed more fluorescence particles and stronger fluorescence than those in the control group.Cells containing green fluorescent particles were called MDC positive cells.Compared with the MDC positive rate of the experimental group and the control group,the positive rate of MDC in the experimental group increased significantly,with significant difference(P < 0.05).There was no significant difference in the positive rate of M,D and C between the two groups(P > 0.05).4.The apoptosis rate and LC3 expression of K562 cells were determined by flow cytometry: the apoptosis rate of K562 cells after 48 hours of the action of iodidin(0,20,40μg/ml)was4.52±1.98%,14.98±2.63%,38.45±1.64%,respectively.Compared with the control group,the apoptosis rate of K562 cells in the experimental group was significantly increased(P < 0.05).Compared with the groups with different concentrations,the apoptosis rate of cells in the high concentration group was significantly higher than that in the low concentration group(P < 0.05),and the effect of ilexonin A on promoting the apoptosis of K562 cells was significantly dose-dependent.The positive expression rates of LC3 were 4.52±1.98%,14.98±2.63%,38.45±1.64%,respectively.The positive expression rate of LC3 in the experimental group was significantly higher than that in the control group(P < 0.05).The positive expression rate of LC3 in the high concentration group was significantly higher than that in the low concentration group(P < 0.05).It was suggested that the expression of LC3 in K562 cells was up-regulated by cea,and the positive expression rate was positively correlated with the drug concentration.5.Western Blott method to detect pubescent Holly root shell element LC3 protein expression of K562 cells: the influence of using different concentration pubescent Holly root element intervention in K562 cells after 48 h,with the increase of drug concentration of autophagy related protein l C 3 Ⅰ/Ⅱ expression gradually increases.These results suggested that histolisin a could effectively induce autophagy and apoptosis in K562 cells by upregulation of L C 3 expression,and showed a dose-dependent effect.Compared with control group,experimental group L C 3 Ⅰ/Ⅱ expression were significantly increased(P < 0.05).Compared with low concentration group,pubescent Holly root element L C 3 Ⅰ/Ⅱ express high concentration group was obviously raised,significant difference(P < 0.05).Pubescent Holly root element of a high concentration of K562 cells in the group L C 3-Ⅰ and LC3-Ⅱ expression has no obvious difference(P > 0.05).Conclusion: 1.lexonin A can affect the proliferation inhibition of human leukemia K562 cells.2.lexonin A can affeccan induce autophagy and apoptosis in human leukemia K562 cells.3.The autolysis of K562 cells may be induced by up-regulating the expression of autophagy related protein L C 3,there by inhibiting the proliferation activity of K562 cells.4.The inhibition of K562 cell proliferation effect and the induction of autophagy were time-dependent and dose-dependent. |
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