Objective: To explore the effects of heme oxygenase-1 (HO-1) on the apoptosis, expression of caspase-3 and cytochrome-C (Cyt-C) on hydrogen peroxide (H(2)O(2)) oxidative damage in primary cultured rat's type II alveolar epithelium cell (AECII).
Methods: AECII from male Sprague-Dawley (SD) rats were separated and cultured. The cells were randomly divided into four groups: control group (A group, the cells were cultured with normal saline), H(2)O(2) group (B group, cultured with 0.5 mmol/L H(2)O(2)), HO-1 pretreatment group (C group, pretreatment with 0.2 μmol/L HO-1 for 2 hours followed by 0.5 mmol/L H(2)O(2)), HO-1 inhibition group [D group, pretreatment with 10 μmol/L zinc protoporphyrin IX (ZnppIX) for 2 hours followed by 0.5 mmol/L H(2)O(2)]. The cells in each group were cultured after different treatment. The cell apoptosis rate was determined by flow cytometry at 2, 6, 12 hours after the intervention. The protein expression of caspase-3 and Cyt-C were determined by Western Blot.
Results: The cell apoptosis rate in each group was gradually increased with prolonged time of H(2)O(2) treatment. The apoptosis rate at different time points after treatment in B group was significantly higher than that in A group, while the rate in C group was significantly lower than that in B group [2 hours: (11.46 ± 1.47)% vs. (20.83 ± 1.55)%, 6 hours: (12.30 ± 1.37)% vs. (27.14 ± 1.53)%, 12 hours: (12.62 ± 1.39)% vs. (35.66 ± 0.74)%, all P<0.05]. There were no significant differences in apoptotic rate at 2, 6, 12 hours between D group [(24.33 ± 1.36)%, (31.67 ± 1.24)%, (36.93 ± 2.40)%] and B group. The protein expression of caspase-3 and Cyt-C was gradually increased with prolonged time of H(2)O(2) treatment. The protein expression of caspase-3 and Cyt-C at different time points in B group was significantly higher than that in A group. The protein expression of caspase-3 (gray scale) in C group was significantly lower than that in B group (2 hours: 0.250 ± 0.039 vs. 0.650 ± 0.072, 6 hours: 0.470 ± 0.080 vs. 0.960 ± 0.118, 12 hours: 0.680 ± 0.099 vs. 1.830 ± 0.220, all P<0.05), and the Cyt-C protein expression (gray scale) was also significantly lowered in C group (2 hours: 0.250 ± 0.074 vs. 0.390 ± 0.069, 6 hours: 0.340 ± 0.043 vs. 0.670 ± 0.120, 12 hours: 0.470 ± 0.072 vs. 1.360 ± 0.112, all P<0.05). There were no significant differences in protein expression of caspase-3 and Cyt-C between D group (caspase-3: 0.720 ± 0.052, 1.060 ± 0.109, 1.880 ± 0.159; Cyt-C: 0.500 ± 0.110, 0.860 ± 0.050, 1.480 ± 0.140) and B group.
Conclusions: HO-1 preconditioning reduced the apoptotic rate of the AECII oxidative damaged by H(2)O(2) at different time points (2-12 hours) and decreased the expression of caspase-3 and Cyt-C. The mitochondrial apoptosis pathway participated in the protection mechanism of HO-1 in oxidative damage AECII.