Mechanism Study On The Apoptosis Of NB4 Cells Induced By Total Astragalosides

Objective:To investigate the effect of Total Astragalosides(TA) on acute promyelocytic leukemia cell line NB4 cell growth inhibition and inducing apoptosis, and to explore the activity of NF-κB and Akt during the apoptosis by flow cytometry,try to find the mechanism of inducing apoptosis and to lay foundation for further relative research.Methods:The NB4 cells which grew in logarithmic phase were treated with TA of different concentration(200,400,600,800 mg/L) for 48 h in culture. Simultaneously, the blank control group was set with no TA.1. Using trypan blue staining to observe the growth of NB4 cells and effect of TA on NB4 cells.2. The changes of the NB4 cells shape after TA impacted were observed with inverted microscope.3. The effect of different concentrations of TA on NB4 cells proliferation was determined by CCK-8 assay.4. Flow cytometry was used to explore the cell apoptosis and the cell necrosis after the NB4 cells were treated with TA of different concentration for 48 h.5. Flow cytometry was used to explore the activity of NF-κB and Akt during the apoptosis after the NB4 cells were treated with TA of different concentration for 48 h.Results:1. After the NB4 cells were treated with TA of different concentration for 48 h, the apoptosis could be dicoverd with inverted microscope. The cells showed the volume of single cells reduced while the cell membrane was integrate and there were a lot of large and small droplet-shaped vacuoles in the cytoplasm. The cells also showed karyolemma disintegration, chromatin segmentation and apoptotic bodies, and other typical forms of apoptosis.2. TA of different concentration(200,400,600,800 mg/L) inhibited proliferation of NB4 cells in a dose-dependent manner.3. FCM showed that TA of different concentration(200,400,600,800 mg/L) could induce apoptosis of NB4 cells. At doses of 200,400 and 600 mg/L, the apoptotic rates of TA on NB4 cells increased in a dose-dependent manner. But there was no significant difference in apoptotic rates between the group of 800 mg/L and 600 mg/L. In the group of 800 mg/L,the necrotic cells elevated highly.4. After TA of different concentration (200,400,600μg/ml) treatment on NB4 cells the expression of NF-κB protein was decreased significantly compared with blank control,while Akt protein was not decreased obviously.Conclusion:TA can obviously inhibit the NB4 cell growth and proliferation. In some dose limit, TA can induce apoptosis of NB4 cells. TA can induce apoptosis of NB4 cells through an Akt-independent NF-κB signaling pathway. To inhibit the activity of NF-κB in NB4 cells may be one of the important mechanism of inducing apoptosis of NB4 cells by TA.