Inhibition Of Proliferation Of Acute Lymphoblastic Leukemic Cells By Huaier Through P38MAPK Pathway

objective: In this study,two human acute lymphoblastic leukemia(ALL)cell lines Jurkat and Nalm-6 were used to investigate the effects of Huaier on the proliferation,apoptosis and cell cycle in vitro.To explore whether Huaier regulates the expression of HoxA5 gene through p38 MAPK signaling pathway,and provides some experimental evidence for the application of Huaier in the clinical treatment of leukemia.Methods: 1.Cell culture: Culture of Human ALL cell Jurkat and Nalm-6 through in vitro culture technology.2.Experimental grouping:(1)Each cell line was divided into four groups according to different concentration of Huaier(0,0.5,1,2 mg/ml).(2)According to the addition of Huaier and p38 MAPK signaling pathway inhibitor SB203580,Jurkat and Nalm-6 cells were divided into four groups: control group;Huaier group(1.0 mg/ml);SB203580 group(10 uM)and combined group(SB203580 10 uM and Huaier 1.0 mg/ml).3.CCK8(Cell Counting Kit-8): CCK-8 kit was used to detect the effect of different concentrations of Huaier(0,0.25,0.5,1,2,4,8mg/ml)on the proliferation of Jurkat and Nalm-6 cells at different time(24h,48 h and 72h).Screening for optimal drug concentration and optimal time of action for subsequent experiments.4.Cell apoptosis: Apoptosis of Jurkat and Nalm-6 cells treated with different concentrations of Huaier(0,0.5,1,2 mg/ml)and SB203580 for 48 h was detected by flow cytometry.5.Cell cycle: Cell cycle changes of Jurkat and Nalm-6 cells treated with different concentrations of Huaier(0,0.5,1,2 mg/ml)and SB203580 for 48 h were detected by flow cytometry.6.qRT-PCR(quantitative Real-time PCR): The mRNA relative expression levels of p38 and HoxA5 in Jurkat and Nalm-6 cells treated with different concentrations of Huaier and SB203580 for 48 h were detected.7.Western Blot:(1)The relative expression of p-p38 and HoxA5 protein in Jurkat and Nalm-6 cells were detected after 48 h of intervention with different concentrations of Huaier(0,0.5,1,2 mg/ml).(2)The expression of p-p38,p38 and HoxA5 protein in ALL cells were detected after pretreatment with SB203580 for 1h and intervention with 1 mg/ml Huaier for 48 h.Results: 1.Huaier significantly inhibited the proliferation of acute lymphoblastic leukemia cells.The growth of Jurkat cells was inhibited in a dose-and time-dependent manner,and Nalm-6 cells were inhibited in a dose-dependent manner,with the most obvious effect at 48 h.The IC50 of Huaier on Jurkat and Nalm-6 cells for 48 h were 2.247 mg/ml and 1.777 mg/ml,respectively.2.Huaier promoted apoptosis of ALL cells in a dose-dependent manner.SB203580 combined with Huaier partially counteracted the apoptotic effect of Huaier on Jurkat cells.3.Both Huaier and SB203580 could block ALL cell cycle at G2/M phase.4.quantitative Real-time PCR showed that Huaier promoted the mRNA expression of p38 and inhibited the expression of HoxA5.After adding SB203580,the expression of p38 decreased while the expression of HoxA5 increased.5.Western Blot results showed that Huaier could activate p38 MAPK signaling pathway,down-regulate the expression of HoxA5 protein and up-regulate the expression of p-p38 protein in ALL cells.One hour after SB203580 pretreatment,the p38 MAPK signaling pathway was inhibited,the expression of p-p38 was significantly decreased,the level of HoxA5 protein was increased,and the expression of total p38 protein was not significantly changed.Conclusion: 1.Huaier significantly inhibited the proliferation of Jurkat and Nalm-6 cells.2.Huaier promotes apoptosis of ALL cells and block cell cycle.3.Huaier down-regulates the relative expression of HoxA5 mRNA and protein,which may be related to the activation of p38 MAPK signaling pathway.