| Background:Bronchopulmonary dysplasia is a common complication of mechanical ventilation-induced hyperoxia in newborn. However, its exact mechanism is not yet clear. Endoplasmic reticulum stress is closely related to the occurrence and development of many diseases. Recently, the relationship between ERS and hyperoxia-induced lung injury has generated a great attention as possibly mechanism for hyperoxic lung injury. Several studies revealed that peroxidation of endoplasmic reticulum can influence the correct folding of the protein, which in turn activates the unfolded protein response (UPR). UPR react through the membrane PERK, IRE1, ATF6 and other three transmembrane protein stress signal transduction. However, the role of UPR in BPD and its mechanism is unclear. IGF-1 is an important growth factor in the body which plays a pivotal role in the regulation of the tissue’s cell proliferation, differentiation, apoptosis, body’s growth, and development. The mechanism of action is to promote cell DNA synthesis and metabolism, prompting cells to enter the G1 phase, which is conducive to cell proliferation. At the same time interactions through PI3K/Akt and MAPK signaling pathways inhibit apoptosis in various cells. Therefore, in current study, we made hyperoxic lung injury model using neonatal rats to explore the role and targets of the ERS and UPR signaling pathway in the BPD; on this basis, to further clarify the protection role of exogenous IGF-1 on ERS reactive signal in hyperoxia-induced lung injury. This will effectively reveal the role of ERS in hyperoxic BPD and its molecular mechanisms, In addition, it will provide scientific and theoretical basis for the clinical applications of IGF-1 in prevention and treatment of BPD.Part oneThe role of ERS in hyperoxia-induced lung injuryObjective:To investigate the changes of ERS signaling pathways in hyperoxia-induced lung injury and its molecular mechanism.Methods:Full-term newborn Wistar rats were prepared to make hyperoxia-induced lung injury model,after modeling,lung tissues wet and dry ratio was measured on the the lst,3rd,7th nd 14th day; taken the the BALF to do Diff-quik staining, Detected white blood cell count，observed lung alveolar development and degree of injury with HE staining. Changes in the ultramicrostructure of1. High concentrations of oxygen can induce pulmonary edema, inflammatory response, leading to widening of the alveolar septa, increase the number of lamellar bodies in alveolar type Ⅱ epithelial cells and increase vacuoles.2. ERS involved in hyperoxia-induced lung cell apoptosis.Part twoProtective role of IGF-1 in hyperoxia induced lung injury and its effects on the target of ERS signalObjective:To observe protective effect of IGF-1 in hyperoxia-induced lung injury, and to explore the molecular mechanism of protection of hyperoxia-induced lung injury by ERS signal target.Methods:Wistar newborn rats were selected for creating of hyperoxia model,then divided into three groups① room air control group (RA); ② The hyperoxia group (85% O2); ③ hyperoxia+ IGF-1 (85% O2+rh-IGF-1) group. The treatment group received intraperitoneal injection of IGF-1, detected the lung wet and dry weight ratio and white blood cell count in BALF with Diff-quik stain, by the HE staining observed alveolar development and degree of injury, nuclear staining combined TUNEL method detected lung cell apoptosis, using westernblot methods detected lung tissue caspase-12, JNK, p-Jnk,CHOP,expression levels.Results:1. the lung W/D ratio of IGF-1 treatment group when hyperoxia for 3,7,14 days was less than the hyperoxia model group of the same days,there was statistically significant. While there was no change in the hyperoxia for 1 day group.the W/D of the same days(3,7,14 days) of hyperoxia group was increase significantly than that of normalcontrol group and the W/D of IGF-1 treatment group compared with the hyperoxia group was decreased significantly.2. when hyperoxia for 1,3,7 14 days,the white blood cell count in BALF of IGF-1 treatment group were reduced than that of the hyperoxic group,there was significant difference. Diff-quik staining results:inflammatory cell infiltration reduced in IGF-1 group than the hyperoxic lung epithelial cells was observed by electron microscope, observed lung cell apoptosis with nuclear staining combined TUNEL method; expression levels of Bcl2/BaX, Bip/GRP78, CHOP/GADD153 and caspase-12 were detected, using western blotting method.Results:1. The lung wet/dry (W/D) proportion on the 3rd,7th and 14th days after expoxure to hypreoxia was significantly increased in hyperoxia group in comper with that in control group.2. The white blood cells count on first; third and 7th days after exposure to hyperoxia was significantly higher in hyperoxic newborn rate of BALF than it was in normal group, and the cell count has increasingly rose up along with time in model group during first, third and 7th days. Diff-quik stain results:in contrast to normal group, the secretions and inflammatory cells increased in Hyperoxic BALF group, and infiltration of inflammatory cells has been increased along with time.3. HE staining results:inflammatory cell exudation was visible after three days exposure to hyperoxia. On the 7th day, the alveolar space was reduced, and inflammatory cells were increased. On 14t day, the pulmonary fibrosis was appeared.4. Transmission electron microscopy results revealed that the number of lamellar corpuscles was increased in hyperoxia group, and along with prolongation of hyperoxia, the emptying state increased, karyopyknosis to the edge and capitation was visible in the type II alveolar epithelial cells.5. Measured pulmonary apoptosis with TUNEL staining:pulmonary apoptotic index in hyperoxia groups was (5.6±0.8) in 3days, (6.3±0.7) in 7days and (6.4±0.1) in 14days, while it was (4.1±1.1) 3days, (4.2±0.9) 7days and (4.5±0.7) days in control group, it was significantly higher in model group and the difference was statistically significant.6. western blotting results revealed that,BaX expression in 3 days after exposure to hyperoxia was higher in model group than it was in control and it was gradually increased along with time. Bcl2 expression in 3 days was lower in model group than it was in control group and it was reduced along with time, the Bcl2/BaX of hyperoxia for 3 days group was lower than control group and it was gradually decreased along with time extension; Bip/GRP78, CHOP/GADD153, caspase-12 protein of hyperoxia group were upregulated, when the hyperoxia time extended,the protein content became higher, while there were no significant change in the hyperoxia for 1 day group.Conclusion: group,there was significant difference.3. Lung apoptotic index of model group on hyperoxia 3 days (5.3±0.7), hyperoxia 7 days (5.3±0.7), hyperoxia 14 days(6.2±0.3) were significantly higher than normal group on normal 3 days (4.2±0.9),7days (4.3±0.8), the normal 14 days (4.5±0.7)); while apoptosis index of IGF-1 treatment group on 3 days(4.6±0.7),7 days (5.0±0.7),14 days (5.1±0.3) were significantly reduced,had statistically difference. TUNEL staining showed that the number of apoptotic cells of IGF-1 treatment group on the 3rd,7th,14th day were lower than the same days of model group and had statistical difference.4. Westernblot was used to detect the protein,it showed that caspase-12、JNK、pJNK、CHOP protein content of model group on the 3rd,7th,14th day were significantly higher than IGF-1 treatment group on the same days, which shows that the IGF-1 can inhibit the protein expressions.Conclusion:1. IGF-1 can reduce hyperoxia-induced lung edema and inflammatory response, and inhibits hyperoxia-induced lung apoptosis.2. The protective effect of IGF-1 from hyperoxia-induced lung injury is correlated with the downregulations of ERS related caspase-12, JNK, CHOP protein expression.So IGF-1 can protect from hyperoxia-induced lung injury perhaps through the ERS and JNK signaling pathway. |
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