Study Of Active Ingredient Of Indigo Naturalis On The Proliferation, Apoptosis And Differentiation Of HEL Cell Line

Objective:It is to study the effect of active ingredient of indigo naturalis on the proliferation, apoptosis and differentiation of human erythroleukemia (HEL) cell line in vitro.Methods:1.Prepared of the active ingredient of indigo naturalis:The active ingredient of indigo naturalis was prepared by ethanol extraction and was dissolved into DMSO at 10mg per mililiter. 2.Cell culture:HEL cells were cultured in RPMI 1640 supplemented with 10% calf serum at 37℃with 5% CO2.3.MTT reduction assay:when HEL cells were grown to a density 0.5×106～1×106/Ml, cultured with different concentration of the extraction to detect the inhibitory effect on HEL cells.4. The morphological changes of HEL cells before and after the treatment were observed under light microscope.5.Detection of apoptosis:the apoptosis induction of the extract on HEL cells was demonstrated with Annexin V-FITC/PI double label method by flow cytometry (FCM).6.Detection of differentiation of HEL cells:The monocytic differentiation of HEL cells was evaluated by changes of the expression of CD11b and CD 14 detected by FCM..Results:1. The proliferation inhibition rates of 16.01%,39.12%,50.06% and 59.28% were recorded with the concentrations of 25,50,75 and 100μg/mL respectively (P<0.01).2. The morphological changes of HEL cells after the treatment of extraction were characterized by certain morphologic features of apoptosis.3. The induction of apoptosis was also illustrated as 5.60%,13.7%,17.8% and 18.4% with four different concentrations, which was significantly higher than the spontaneous apoptosis rate (2.70%) of the control cells.4. Compared with control, monocytic differentiation of HEL cells was presented with the treatment of the active ingredient of indigo naturalis at concentrations of 25,50,75 and 100μg/mL, with the 100μg/mL the most obvious.Conclusion:The active ingredient of indigo naturalis tends to inhibit proliferation, induce monocytic differentiation and promote apoptosis of HEL cells in vitro.