The effect of reactive oxygen species regulation of expression of Bcl-2 and Bax in apoptosis of human umbilical vein endothelial cell induced by heat stress

Abstract

To observe the effect of heat stress-induced reactive oxygen species (ROS) burst on the regulation of expression of Bcl-2 and Bax in human umbilical vein endothelial cell (HUVEC) apoptosis induced by heat stress, and explore the pathogenesis of vascular endothelial damage caused by severe heat stroke. HUVEC heat stress model was reproduced. Cells of heat stress group were incubated at either 39, 41, or 43 centigrade for 2 hours, then all the cells were further incubated at 37 centigrade and 5% CO2 for 24 hours. Before heat stress, cells of 43 centigrade heat stress group were pretreated with 10 μmol/L MnTMPyP, which was a specific scavenger of ROS, for 1 hour. Cells of control group were incubated at 37 centigrade and 5% CO2. The amount of ROS was assayed with 2', 7'-dichlorofluorescin diacetate (DCFH-DA) and dihydroethidium (DHE) staining. Apoptosis was determined by using staining with Hoechst33258. The mRNA expressions of Bcl-2 and Bax were determined by reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of Bcl-2, Bax, caspase-3 were analyzed by Western Blot. In addition, the effect of MnTMPyP on heat stress-induced apoptosis was also studied. Compared with control group, there was no obvious change in cells after 39 centigrade heat stress. With the increase in heat stress temperature up to 41 centigrade and 43 centigrade, viability of cells showed a lowering trend, with a burst of ROS, and an increase of mRNA and protein of Bax, and the protein of caspase-3 was significantly increased, the mRNA and protein of Bcl-2 were significantly decreased in a temperature-dependent manner. These changes were marked in 43 centigrade heat stress group as compared with those of the control group [cell viability: (46.00±4.00)% vs. (96.33±1.53)%, t=20.164, P=0.001; ROS (fluorescence relative value): 400.67±12.10 vs. 99.33±4.04, t=32.909, P=0.001; Bax mRNA (A value): 3.03±0.15 vs. 1.00±0.00, t=23.056, P=0.001; Bax protein (gray value): 3.97±0.21 vs. 1.00±0.00, t=24.684, P=0.001; caspase-3 protein (gray value): 4.80±0.20 vs. 1.00±0.00, t=32.909, P=0.001; Bcl-2 mRNA(A value): 0.42±0.30 vs. 1.00±0.00, t=33.072, P=0.001; Bcl-2 protein (gray value): 0.39±0.25 vs. 1.00±0.00, t=42.212, P=0.001]. It was shown that pre-condition with the antioxidant MnTMPyP significantly decreased the heat stress-induced expression of Bax, caspase-3, and apoptosis, and the expression of Bcl-2 was elevated [Bax mRNA (A value): 2.00±0.20 vs. 3.33±0.25, t=7.184, P=0.002; Bax protein (gray value): 2.03±0.25 vs. 3.23±0.25, t=5.840, P=0.004; caspase-3 protein (gray value): 2.07±0.21 vs. 5.00±0.20, t=17.600, P=0.001; Bcl-2 mRNA(A value): 0.71±0.40 vs. 0.42±0.26, t=8.126, P=0.002; Bcl-2 protein (gray value): 0.57±0.31 vs. 0.40±0.06, t=5.091, P=0.007]. A burst in an increase of ROS plays an important role on heat stress-induced HUVEC apoptosis, and the mechanism is probably related to the expressions of Bcl-2 and Bax. The vascular endothelial cells apoptosis may be one of the pathogenetic factor in severe heat stroke.