HL-60 Cells Apoptosis Induced By Etomidate And Inhibited By Preconditioning Of Etomidate

Objectives: Apoptosis is an active form of cell death observed in various physiological and pathological scenarios and may play a role in determining clinical outcome. As an intravenous anesthetic, etomidate seldom affects hemodynamics, and can protect nervous system. However, the clinical use of etomidate has been restricted because of its side effects. Experimental studies revealed that etomidate directly inhibited the activity of 11-β-hydroxylase, an enzyme necessary for corticosteroids synthesis. An anesthesia-induction dose of etomidate (0.3mg/kg) is sufficient to suppress adrenocortical function for 5-8 hours. Decrease in serum cortisol will affect the hypothalamus-pituitary-adrenal axle, suppress the ability to respond to stress, and indirectly inhibit the immune function. In addition, some reports indicated that etomidate directly affect the immune function by inhibiting T-lymphocyte proliferation and neutrophil function.Therefore, there are clinical and pharmacological significances to study the immune cells apoptosis induced by etomidate. Human promyelocytic leukemia cells (HL-60) are the standard target cell line for apoptosis research, concerning which many biochemical reactions have been recognized and established. These cells also maintain some properties of neutrophils and monocytes that constitute the first line of immune protection. The study about the effect of etomidate on HL-60 cells may decipher the effect of etomidate on immune cells in vitro and its mechanisms. In addition, this study will observe whether precondition at low concentration of etomidate may inhibit apoptosis induced by etomidateitself. The object is to find the new method to decrease the immune inhibition of Etomidate.Methods: HL-60 cells were cultured at a concentration of 1.5×105/ml. Three groups were processed in accordance to different methods. The control group was blank (0μM etomidate for 24h); the preconditioning group was preconditioned with 1μM etomidate for 1h, washed etomidate out and allowed to recover for 4h, and then treated with 500μM for 24h; the injury group was treated with 500μM etomidate for 24h. Methyl thiazolyl tetrazolium (MTT) assay measured the cell viability and proliferation. Using Annexin V and propidium iodide (PI) staining, we examined apoptosis with flow cytometry. Western blot analysis was used to quantify the caspase-3, 8, 9 activities.Results: 1. In MTT assay, etomidate inhibited HL-60 viability in preconditioning and injury groups, but the percentage of active cells increased 35.35% in preconditioning group compared with that in the injury group. 2. In flow cytometry analysis by Annexin V and PI, we found the percentages of the early apoptotic as well as the dead cells decreased 27.48% and 20.23% respectively in preconditioning group compared with those in the injury group, although the percentages of the early apoptotic and the dead cells both increased compared with those in the control group. 3. The results of Western Blot displayed that etomidate induced procaspase-3 decrease and caspase-8, 9 subunits increased compared with the control group, but the preconditioning inhibited the tendency compared with the injury group. All the differences were significant (P< 0.01).Conclusions: 1. Etomidate can inhibit the viability of HL-60 cell. 2. Etomidate may be the inducer of caspase-dependent apoptosis in HL-60, and through both the intrinsic and extrinsic pathways. 3. Precondition at low concentration of etomidate can inhibit HL-60 apoptosis, and the protection may be independent of the pathways of apoptosis.