Study On Dihydroartemisinin Inhibits AKT Phosphorylation By Up Regulating PTEN Gene Expression In T-cell Lymphoma

Objective:Exploration of the anti-T-cell lymphoma effect of dihydroartemisinin（DHA）by studying the effects of dihydroartemisinin on PTEN gene expression and AKT protein phosphorylation in human T-cell lymphoma cells（Jurkat,HuT 78）.Methods:Jurkat cells and HuT 78 cells of human T-cell lymphoma were cultured according to conventional cell culture methods.When the cells were in logarithmic growth phase,in good growth condition and without pollution,they were selected for follow-up experiments.After dihydroartemisinin was used to intervene Jurkat cells and HuT 78 cells respectively,（1）the cell proliferation rate was detected by CCK-8 method and the IC50 value was calculated;（2）The number and morphology of cells were observed under light microscope;（3）Apoptosis was detected by flow cytometry;（4）The expression of PTEN mRNA was detected by real-time fluorescent quantitative PCR.GAPDH was used as the internal reference,and the relative expression of PTEN mRNA was calculated by 2^-ΔΔCtmethod;（5）Western blot was used to detect the expression of PTEN,AKT and p-AKT proteins in the two kinds of cells.GAPDH was used as internal reference.Image J software was used to analyze the gray value,and the relative expression of PTEN,AKT and p-AKT proteins was calculated.Results:（1）The results of CCK-8 assay showed that the inhibitory effect of dihydroartemisinin on the proliferation of Jurkat cells and HuT 78 cells was positively correlated with its concentration.The IC50 value of dihydroartemisinin in Jurkat cells and HuT 78 cells for 48h was 34.19μM and 38.76μM,respectively,compared with the control group.The difference was statistically significant（P<0.05）.（2）After the treatment of Jurkat cells and HuT 78 cells with 40μM of dihydroartemisinin solution for 48h,the density of these two kinds of human T cell lymphoma cells was significantly decreased and the cell debris was significantly increased under low power microscope in the culture flask.At high magnification,the integrity of cell membrane was destroyed and the cell structure was irregular.（3）The apoptosis rate of human T cell lymphoma cells was increased by72.92%（Jurkat）and61.66%（HuT 78）after 48h treatment with 40μM dihydroartemisinin solution,and the difference was statistically significant compared with the control group（P<0.05）.（4）The PTEN mRNA expression in Jurkat and HuT 78 cells was increased by1.91 and 1.82 times after 48h treatment with 40μM and 80μM dihydroartemisinin,respectively,and the difference was statistically significant compared with the control group（P<0.05）.（5）The expression of PTEN protein in Jurkat cells was increased by 2.31 times and 4.15 times after treatment with 40μM and 80μM dihydroartemisinin for 48h,and the differences were statistically significant（P<0.05）.The expression of AKT protein was decreased by 4%and 8%,compared with the control group.There was no statistically significant difference（P>0.05）,the p-AKT protein expression decreased by 18%and37%,compared with the control group,the difference was statistically significant（P<0.05）.The expression of PTEN protein in HuT 78 cells was increased by 2.15-fold and 3.53-fold when treated with 40μM and 80μM dihydroartemisinin for 48h,and the differences were statistically significant（P<0.05）.The expression of AKT protein was decreased by 5%and 7%when compared with the control group.There was no statistically significant difference（P>0.05）,and the p-AKT protein expression decreased by 19%and 31%,compared with the control group,the difference was statistically significant（P<0.05）.Conclusion:Dihydroartemisinin（DHA）significantly inhibited cell proliferation and promoted apoptosis in human T-cell lymphoma cells Jurkat cells and HuT 78 cells;dihydroartemisinin increased PTEN protein expression at the transcriptional and translational levels by upregulating PTEN mRNA levels,resulting in the inhibition of phosphorylation of downstream AKT proteins;dihydroartemisinin exerts anti-T-cell lymphoma effects by regulating the PTEN/AKT signaling pathway.The experimental results obtained in this study provide a new research idea for the clinical treatment of T-cell lymphoma.