| Objective To study the effect of radiosensitivity of Dihydroartemisinin(DHA)on Raji lymphoma cells and to explore its mechanism.Methods Raji cells were cultured in vitro and inoculated in logarithmic growth phase,the cells were divided into four control groups: DHA group(5 μM DHA),radiation group(IR,4 Gy γ ray)and IR+DHA group(4 Gy γ ray+5 μM DHA).After 24 hours of culture,the cells were treated accordingly,and then the relevant tests were carried out at each time point.Cell growth suppression of each group was analyzed by CCK8,Flow cytometry(FCM)was used to detect the effect of apoptosis,cell cycle,mitochondrial membrane potential and intracellular ROS of each group,Western Blot was used to detect the expression of AKT,p-AKT,Bcl-2,Bax and Cleaved-Caspase-3.Results(1)Radiation promotes the apoptosis of Raji lymphoma cells in a dose-dependent manner,When the radiation dose was 4 Gy,the apoptosis rate was 12.21 ± 0.80%.(2)Raji cells were treated with Different concentrations of DHA for 24 h and 48 h.when the concentrations of DHA was 2.5 μM,there was no significant difference compared with the control group(P> 0.05),when the concentration of was greater than or equal to 5 μM,DHA inhibited cell proliferation in a concentration-dependent manner.(3)Flow cytometry showed that DHA combined with IR significantly inhibited the viability of Raji cells.Compared with the control group,the apoptotic rate of Raji cells in IR+ DHA group was significantly increased(45.15 ± 2.67%,P <0.01),Mitochondrial membrane potential(0.127 ± 0.01,P <0.01)was significantly decreased,the intracellular ROS content(7097 ± 287.51,P <0.01)increased significantly.Radiation led to G2/M phase arrest(P <0.01),DHA could inhibit the G2/M block by radiation.(4)Western Blot showed that there was no significant difference in the expression of AKT protein in Raji cells(P> 0.05)compared with the control group,However,the phosphorylation of AKT was inhibited(P <0.01),and the IR+DHA group was the most obvious,the expression of Bcl-2 protein was decreased,and the IR+DHA group was the most significant,Bax protein and Cleaved-Caspase-3 protein were up-regulated,and the up-regulation degree of IR+DHA group was higher than that of IR group and DHA group.Conclusion(1)We confirmed that the radiosensitization effect of Dihydroartemisinin on Raji cell line in vitro by FCM and fluorescence staining.(2)It was demonstrated that DHA increased the sensitivity of Raji cells to radiation through mitochondrial apoptosis by detecting mitochondrial membrane potential,ROS,and apoptosis.(3)Western blot showed that DHA could induce the Caspase cascade through the BCL-2 family of proteins,and thus increase the sensitivity of Raji cells to radiation,which may inhibit the phosphoinositide 3-kinase(PI3K-Akt)signaling pathway. |
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