Effects Of Ethanol On Apoptosis In Endothelial Cells And Its Mechanism Of Action Establishment Of HPLC Method For Determining Neferine Concentration In Rats

Part one Effects of ethanol on apoptosis in endothelial cells and its mechanism of actionSection one Effects of ethanol on apoptosis in endothelial cells Objective: Human endothelial cell line EA.hy926 was incubated in vitro, to investigate the effect of ethanol on cell proliferation and apoptosis..Methods: Human endothelial cell line EA.hy926 was cultured in vitro. Cells were incubated with 0.3%,0.6%,1.0% (v/v) ethanol for 6,9,12 h respectively. Inhibition of cell proliferation was determined by MTT assay. Cell survival rate was evaluated by Trypan blue exclusion method. Fluorescent dye Hoechst 33258 was used for displaying the morphological alteration of apoptosis. Cells were divided into different groups: negative control group, ethanol group ( 0.6% ), ethanol combined with NAC group (0.6%+10 mmol/L NAC), and NAC group (10 mmol/L NAC, N-acetyl-L-cysteine ). EA.hy926 of either group was cultured and incubated with corresponding drugs for the same 12 h. Apoptosis rate was determined by flow cytometry with sub-G1 method.Results: Ethanol(0.3%,0.6%,1.0%) inhibited the proliferation of EA.hy926. After being incubated with ethanol for 6h, growth inhibition rate was (29.38±1.69)%,(35.98±2.23)%, (40.75±0.55)% and cell survival rate was (78.09±9.81)%, (73.60±8.34)%, (69.03±11.56)% with increase of ethanol concentration. After being incubated with ethanol for 9h, growth inhibition rate was( 12.58±1.44)%, (18.39±3.26)%,( 28.62±1.13)% and cell survival rate was (85.42±10.97)%, (77.58±5.01)%, (71.85±14.06)% with increase of ethanol concentration. After being incubated with ethanol for 12h, growth inhibition rate was (7.57±1.17)%,(10.96±1.28)%,(13.81±0.83)% and cell survival rate was (94.72±5.65)%, (89.46±3.67)%, (84.31±9.77)% with increase of ethanol concentration. EA.hy926 exhibited typical morphological alteration of apoptosis following incubation of ethanol. Compared with control group incubation of EA.hy926 with 0.6% ethanol for 12 h caused a significant increase on apoptosis rate from ( 0.49±0.16 ) % to ( 5.31±0.54 ) % (P﹤0.01). Antioxidant agent NAC attenuated the apoptosis effect induced by ethanol, and the apoptosis rate decreased from ( 5.31±0.54 ) % to (1.28±0.24)% (P﹤0.01).Conclusion: Ethanol(0.3%,0.6%,1.0%) inhibited the proliferation of EA.hy926, under the same incubation time, growth inhibitory rate increased and cell survival rate decreased with the increase of ethanol concentration. 0.6% Ethanol can induce apoptosis of endothelial cells. Antioxidant agent NAC can attenuate ethanol-induced apoptosis.Section two Mechanism of ethanol-induced apoptosisObjective: Human endothelial cell line EA.hy926 was incubated with 0.6% ethanol for 12 h, to investigate the effect of ethanol on SOD activity, MDA level, intracellular ROS and intracellular free calcium concentration ([Ca2+]i).Methods: Cells were divided into different groups: negative control group, ethanol group (0.6%), ethanol combined with NAC group (0.6% ethanol+10 mmol/L NAC), and NAC group (10 mmol/L NAC). Cells of either group were cultured and incubated with corresponding drugs for 12 h. According to the kits'directions, SOD activity and MDA level were detected respectively. Intracellular ROS was determined by means of an oxidative-sensitive fluorescent probe DCFH-DA. Intracellular free calcium concentration was measured by fluorescent probe Furo-2/AM.Results: Compared with control group incubation with 0.6% ethanol for 12 h led to a significant increase in the level of MDA from 46.5±3.8 nmol/mg protein to 89.5±5.6 nmol/mg protein and the generation of ROS from 93.69±7.58 to 162.30±18.85, but a remarkable decrease in SOD activity from 40.8±2.9 U/mg protein to 29.2±4.0 U/mg protein. The level of MDA deceased to 45.4±3.3 nmol/mg protein and ROS to114.35±9.63, while SOD activity added up to 37.9±3.0 U/mg protein(P﹤0.01) in combination group. Compared with control group incubation of EA.hy926 with 0.6% ethanol for 12 h caused a remarkable increase in [Ca2+]i from 149.0±18.0 nmol/L to 183.8±19.9 nmol/L(P﹤0.05). Combination of NAC can reduce the intracellular calcium concentration to 158.2±8.6 nmol/L. Conclusion: Exposure of EA.hy926 cells to 0.6% ethanol can promote the level of MDA and degrade SOD activity significantly, and ethanol can result in the augment of ROS fluorescence intensity and the intracellular free calcium concentration. Antioxidant agent NAC can attenuate the effect caused by ethanol.Part two Establishment of HPLC method for determining neferine concentration in ratsObjective: To develop an HPLC method for the determination of neferine (NEF) in rat plasma and liver homogenate.Methods: The samples were separated on a reversed phase column (Hypersil BDS C18, particle size 5μm, 4.0 mm×250 mm) after extracting by ether. The mobile phase was consisted of methanol∶0.2 M phosphate buffered saline∶0.2 M NaOH∶triethylamine ( 71∶17∶12∶0.002 ), the flow rate was 0.8 ml/min , the sample size was 20μL, and the UV detection wavelength was 282 nm.Results: The linear range was 0.03125～2.00μg/mL in plasma, and 0.0625～16.0μg/mL in liver homogenate. The absolute recovery rate was between 80.4% and 89.2 % in plasma, and between 82.5% and 88.4% in liver homogenate. It showed good reproducibility that the precision was within 8.0% in plasma, and within 3.0% in liver homogenate.Conclusion: This method accorded with the standard of drug analysis in biological specimen and was successfully applied to determining the concentration of NEF in rat plasma and liver homogenate.