Investigation On The Effect Of Callistemon Salignus Extract L72 On HEL Cell Apoptosis And Its Molecular Mechanism

Objective:In this study,it was found through activity screening that the extract of Callistemon salignus,the acylphloroglucinol compound L72,has a strong inhibitory effect on the proliferation of leukemia cells HEL,and the potential molecular mechanism was further explored through experiments,which made L72 become candidate lead compounds for the treatment of AML provide a theoretical basis.Methods:1.Use different concentrations of L72(20 μM,10 μM,5 μM,2.5 μM,1.25 μM)to act on leukemia cells HEL in the logarithmic growth phase,and use MTT colorimetric method to detect the effect of L72 on the proliferation and viability of HEL cells.At the same time,observe the cell number and morphology through an inverted optical microscope;2.Use Hoechst 33258 staining kit to observe the apoptosis of HEL cells treated with different concentrations of L72 for 24 h and 48 h;3.Double staining with Annexin V-FITC/PI after the kit was stained,flow cytometry was used to detect the apoptosis level of HEL cells treated with different concentrations of L72 for 24 h and 48 h;4.JC-1 staining kit was used to detect the loss of mitochondrial membrane potential in HEL cells when treated with different concentrations of L72 for 24 h;5.The expression of XIAP,FLT3,IDH2 and SOD2 genes in HEL cells treated with different concentrations of L72 was analyzed by q PCR;6.After staining by PI/RNase,use flow cytometry to detect the level of apoptosis of HEL cells treated with L72 for 24 h and 48 h.The proportion of each cycle of HEL cells after 48 h;analysis of the influence of L72 on the HEL cell cycle;7.Using Western Blot method to detect the effects of different concentrations of L72 on the expression levels of proteins related to apoptosis and cell cycle arrest in HEL cells.Results:1.MTT results show that L72 has a significant inhibitory effect on the proliferation of HEL cells,and it is time-and dose-dependent.It was observed under the microscope that the number of HEL cells treated with L72 was significantly reduced,and the cell fragmentation was serious;2.After Hoechst 33258 staining,the nucleus and chromatin of HEL cells were densely stained,and bright blue fluorescence appeared,indicating that L72 could be induce HEL cell apoptosis;3.Flow cytometry detection of cell apoptosis results further show that HEL cells have obvious early and late apoptosis after the action of L72,and the effect is time and dose-dependent;4.From the results of JC-1 staining,it can be seen that as the concentration of L72 increases,the proportion of mitochondrial membrane potential loss of HEL cells presents a dose dependent increase;observation under a fluorescence microscope shows that as the concentration of L72 increases,the red fluorescence becomes darker and the green fluorescence becomes brighter,indicating a significant decrease in mitochondrial membrane potential;5.q PCR results show that L72 can down-regulate XIAP,FLT3,IDH2 and SOD2 gene expression in HEL cells,thereby enhancing its anti-HEL cell proliferation;6.Flow cytometry detection of cells the cycle distribution results show that L72 can block the cell cycle of HEL cells in G2/M phase;7.Western Blot results further proved that L72 may inhibit the proliferation of HEL cells through the mitochondrial apoptosis pathway and cell cycle arrest.Conclusions:The results of this study indicate that L72 can inhibit the proliferation activity of HEL cells in a dose-and time-dependent manner;L72 may cause apoptosis of HEL cells through the mitochondrial apoptotic pathway,and at the same time block the cell cycle of HEL cells at G2/M stage;L72 can down-regulate the expression of STAT3,and at the same time down-regulate the expression of XIAP,FLT3,IDH2 and SOD2,enhancing its anti-HEL cell proliferation effect.These results indicate that the compound L72 extracted and isolated from Callistemon salignus can inhibit the proliferation of leukemia cells,block the cycle of leukemia cells,thus hinder the metastasis and spread of leukemia.It can be used as a candidate compound for anti-leukemia treatment,providing a theoretical basis for clinical leukemia treatment.