Role of mammalian target of rapamycin protein in oxidative stress-induced lung injury and pulmonary fibrosis in immature rats

Abstract

Objective To study the changes of transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF) and collagen 1 (COL1) in newborn rat's lung tissue and the expressions of 4EBP1 (eukaryotic promoter) and P70S6K (mammalian target of rapamyein pathway downstream target protein) after rapamycin and hyperoxia intervention, and to study the influence of mammalian target of rapamyein (mTOR) pathway on hyperoxic lung injury and the possible intervention methods. Method A total of 48 21-day-old neonatal rats were assigned into 8 groups (n=6), including air control group, 3 d group (3 days after hyperoxic exposure), 7 d group (7 days after hyperoxic exposure), 14 d group (14 days after hyperoxic exposure), air+ RAPA group (air+ rapamycin), 3 d+ RAPA group (3 days after hyperoxic exposure+ rapamycin), 7 d+ RAPA group (7 days after hyperoxic exposure+ rapamycin) and 14 d+ RAPA group (14 days after hyperoxic exposure + rapamycin). In the hyperoxic group, newborn rats were exposed to 90% oxygen for 3, 7, 14 days respectively. The rats in the hyperoxia+ rapamycin intervention groups received intraperitoneal injection of rapamycin and inhaled high concentrations of oxygen for 3, 7, 14 days respectively. Air+ rapamycin group received intraperitoneal injection of rapamycin for 3 days. To study the pathological changes of lung tissures after hyperoxia and rapamycin intervention, we used ELISA to detect the changes of TGF-β, CTGF and COL1 and Western blot to detect the variations of mTORC1, P70S6K and 4EBP1 expression. Result TGF-β, CTGF, COL1 levels at 3 days, 7 days and 14 days after hyperoxic exposure (TGF-β: 33.7±2.8 vs. 58.6±3.1 vs. 98.8±1.5 ng/mg, CTGF: 50.1±1.8 vs. 68.7±2.2 vs. 94.4±2.5 ng/mg, COL1: 471.9±5.7 vs. 529.7±7.0 vs. 556.4±8.5 ng/mg) were significantly higher than the air control group (TGF-β: 25.5±1.9 ng/mg, CTGF: 41.7±1.4 ng/mg, COL1: 414.4±8.9 ng/mg) (P<0.01). While the levels in rapamycin intervention group were significantly lower than all the hyperoxia+ rapamycin intervention groups (P<0.01). The lung tissue pathological grades in 3 d+ RAPA group and 7 d+ RAPAgroup were significantly lower than those in the 3 d group and 7 d group (3.5±0.8 vs. 6.3±2.3 and 9.7±2.0 vs. 14.0±2.4) (P<0.01). The mTORC1, P70S6K, 4EBP1 expressions in 3 d+ RAPA group were lower than 3 d group (mTORC1: 0.26±0.04 vs. 0.29±0.08, P70S6K: 0.29±0.01 vs. 0.31±0.08, 4EBP1: 0.31±0.06 vs. 0.33±0.06) (P<0.05), while the expressions in 7 d+ RAPA and 14 d+ RAPA groups were significantly lower than 3 d+ RAPAgroup (P<0.01). Conclusion mTOR signal pathway may be involved in the repairing process of hyperoxic-induced lung fibrosis. Rapamycin can reduce the levels of TGF-β, CTGF and COL1 and inhibit the expressions of mTOR pathway downstream target protein P70S6K and 4EBP1, thus reduce lung injury at early stage. Key words: Rapamycin; Lung injury; Oxidative stress; Pulmonary fibrosis; Rats