The endoplasmic reticulum stress pathway involving protein kinase R-like ER kinase-activating transcription factor 4-CCAAT/enhancer binding protein homologous protein implicated in apoptosis in lungs of rats with bronchopulmonary dysplasis

Abstract

Objective To investigate the role of endoplasmic reticulum stress (ERS) pathway involving protein kinase R-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-CCAAT/enhancer binding protein homologous protein (CHOP) in apoptosis in lungs of rats with bronchopulmonary dysplasis (BPD). Methods Forty-eight premature SD rats were divided into BPD group and control group according to random number table.Rats in BPD group were continually exposed to O2 with volumetric concentration factor of 850 mL/L, while rats in control group were exposed to air.Lung tissues in each group were obtained in 7, 14 and 21 days respectively.The apoptosis in lung cells was evaluated by terminal dexynucleotifyl transferase-mediated dUTP nick end labeling (TUNEL) assay.The mRNA levels of glucose regulated protein 78 (GRP78), PERK, ATF4 and CHOP were detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of GRP78, phosphorylated PERK (pho-PERK), ATF4 and CHOP were detected by using Western blot. Results Compared with control group, the lung cells of the rats in BPD group developed more serious apoptosis.Furthermore, the apoptosis index (AI) in lung cells of the rats increased rapidly with the hyperoxia exposure time.This had been statistically verified by comparison with the control group at different timing(7 d: 15.50±0.58 vs 1.25±0.50, 14 d: 27.75±1.71 vs 3.25±0.96, 21 d: 50.50±3.70 vs 4.00±1.15; t=57.00, 20.58, 25.16, all P<0.01). The mRNA levels of GRP78, PERK, ATF4 and CHOP in BPD group increased significantly compared to the control group [GRP78: 7 d (33.88±3.73 )vs(11.65±1.00), 14 d (54.50±2.18)vs(12.84±1.41), 21 d (95.34±7.61)vs(12.43±0.59); PERK: 7 d (5.23±0.92)vs(1.45±0.46), 14 d (7.60±1.56)vs(2.18±0.97), 21 d (16.55±0.50)vs(2.90±1.18); ATF4: 7 d (23.04±2.45)vs(12.56±2.81), 14 d (28.66±2.66)vs(15.18±2.92), 21 d (36.63±2.99)vs(15.14±2.09); CHOP: 7 d (2.21±0.19)vs(0.81±0.02), 14 d (4.19±0.17)vs(0.90±0.08), 21 d (6.08±0.38)vs(0.88±0.10); all P<0.05]. The protein levels of GRP78, pho-PERK, ATF4 and CHOP in BPD group increased significantly as well [GRP78: 7 d (1.33±0.03 )vs(0.85±0.04), 14 d (1.31±0.02)vs(0.92±0.01), 21 d (1.82±0.28)vs(0.87±0.01); pho-PERK: 7 d (0.68±0.02)vs(0.54±0.01), 14 d (1.04±0.01)vs(0.65±0.01), 21 d (1.29±0.02)vs(0.73±0.01); ATF4: 7 d (1.26±0.01)vs(0.83±0.01), 14 d (1.39±0.02)vs(0.87±0.02), 21 d (1.67±0.02)vs(0.94±0.02); CHOP: 7 d (1.37±0.01)vs(0.47±0.06), 14 d (1.50±0.04)vs(0.74±0.05), 21 d (1.61±0.03)vs(0.55±0.02); all P<0.05]. Positive correlation was demonstrated between the expression levels of CHOP protein and AI, PERK, ATF4 in the BPD group (r=0.87, 0, 92, 0.93 respectively, all P<0.05). Conclusion PERK-ATF4-CHOP mediated ERS may participate in and contribute to the apoptosis mechanism in lungs of rats with BPD. Key words: Endoplasmic reticulum stress; Protein kinase R-like ER kinase; CCAAT/enhancer binding protein homologous protein; Bronchopulmonary dysplasis; Apoptosis