Mechanisms Of MTOR Pathway Regulating Fibroblasts Of Premature Mouse Lung In Cell Damage And Repair Induced By Oxidative Stress

Objective To investigate Lung fibroblasts proliferation and apoptosis after different concentrations and time oxygen exposure in premature mouse.To further elucidate the effect of rapamycin intervention on Lung fibroblasts proliferation and apoptosis after different concentrations and time oxygen exposure in premature mouse.To study Lung fibroblasts proliferation and apoptosis after m TORsi RNA transfection,meanwhile,gene and protein expression of updtream and downstream molecule in m TOR pathway were analysised.Further studies are required to understand protein levels of each collagen in extracellular matrix,apoptosis-related gene and pro-fibrogenic cytokine after high concentrations of oxygen,rapamycin and m TORsi RNA transfection.Methods Premature mouse lung fibroblasts were the experimental subjects.Cells cultured in fresh complete medium were exposured to 21%,40%, 60% and 90% oxygen for 3 days,7 days and 14 days.The morphological changes and the number of L929 cells were obsreved in inverted phase contrast microscope, apoptosis cells were detected with Annexin V-PI double staining method, MTT assay were used to evaluate cell proliferation. The cell through a small RNA interference will be divided into cell transfection group and non-transfected control group,MTT assay were used to evaluate cell proliferation, apoptosis cells were detected with Annexin V-PI double staining method, protein expression of updtream and downstream main molecule such as m TORC1, 4EBP1 and P70S6 K in m TOR pathway were analysised with western blot, gene expression of updtream and downstream main molecule such as m TORC1, 4EBP1 and P70S6 K in m TOR pathway were analysised with RT-PCR. The cell will be divided into 90% oxygen exposure group,transfection group, 90% oxygen and rapamycin group and control group,were measured by MTT, Type ?, ? collagen(Col ?,Col ?) and fibronectin(FN) in the medium were measured by ELISA. Protein expression of apoptosis-related gene such as bcl-2 and P53 were analysised with western blot, protein expression of pro-fibrogenic cytokine such as CTGF and TGF-? were analysised with western blot.Results 1. high concentration of oxygen Intervention: a. L929 cell proliferation activity decreased after hyperoxia, and there was a statistically significant difference with the air control group for L929 cell proliferation decreased more obvious as the oxygen concentration and oxygen exposure time prolonged(P<0.05); Hyperoxia inhibit cell proliferation, inhibition rate increases with oxygen concentration and exposure duration. b. Hyperoxia accelerate L929 apoptosis and necrosis,there was a statistically significant difference with the air control group the oxygen concentration and oxygen exposure time extended L929 apoptosis rate increased(P <0.05). c. Hyperoxia the refractive index of a cell reduced, intracytoplasmic particles increased, cell growth uneven,poor state, the number of cells with oxygen concentration and oxygen exposure time reduced. 2. The results of rapamycin and a high concentration of oxygen joint intervention:. A rapamycin and a high concentration of oxygen to intervene jointly L929 cell proliferation activity was significantly decreased, and there was significant difference with the air and rapamycin groups as the oxygen concentration, oxygen exposure time and duration of action of rapamycin extended L929 cell proliferation decreased more obvious(P <0.01); 60% hyperoxia exposure to high oxygen inhibition of proliferation is more obvious, the inhibition rate increased with the concentration of oxygen, and oxygen and rapamycin treatment time increased. b. Rapamycin and a high concentration of oxygen to jointly intervene accelerate L929 apoptosis and necrosis, there was a statistically difference with the air control group L929 apoptosis rate increased as oxygen concentration, duration of action of rapamycin and oxygen increased(P <0.05). c. Rapamycin and a high concentration of oxygen to intervene jointly to reduce the refractive index of the cell body, increasing particulate matter in the cytoplasm, cell growth uneven, poor state, the number of cells reduced with the oxygen concentration, oxygen and rapamycin action time extend. 3. Results of m TORsi RNA interference with L929 cell proliferation and apoptosis,protein and m RNA expression of m TOR signaling pathway upstream and downstream molecules : a.There was a significant differences between non-transfected group and m TORsi RNA interference group, m TORC1,P70S6 K and 4EBP1 protein expression decreased in transfection group(P<0.01). b. There was a significant differences between non-transfected group and m TORsi RNA interference group, m TORC1, P70S6 K and4EBP1 m RNA expression decreased in transfection group(P<0.01). c.There was a significant difference between m TORsi RNA interference group and non-transfected group, after transfected L929 cell proliferation decreased(P <0.01); m TORsi RNA interference can suppress L929 cell proliferation, the inhibition rate was 26.71%. d. There was a significant difference between m TORsi RNA interference group and non-transfected group, m TORsi RNA interference can accelerate L929 cell apoptosis(P<0.01). 4. Results of high concentrations of oxygen, rapamycin and m TORsi RNA intervention on matrix : 90% hyperoxia group, 90%hyperoxia and rapamycin lung fibroblasts extracellular matrix col-?, col-? and FN were increased compared with air control group, the difference was statistically significant(P <0.05); compared with the air control group, transfection group col-?, col-? and FN decreased, the difference was statistically significant(P <0.05). b. There was a significant difference compared with air control group, 90% hyperoxia group, 90% hyperoxia and rapamycin transfected lung fibroblasts bcl-2protein expression decreased, P53 protein expression increased(P <0.01).c. There was a significant difference compared with air control group,90% hyperoxia group, 90% hyperoxia and rapamycin group CTGF and TGF-?protein expression were increased, CTGF and TGF-?protein expression in transfected group decreased(P <0.01);Conclusions1. High concentrations of oxygen alone inhibit premature rat lung fibroblasts proliferation, accelerated its apoptosis in vivo.2. Rapamycin inhibits premature rat lung fibroblast proliferation,accelerate apoptosis and its possible mechanism is by m TOR signaling pathway.3. m TORsi RNA inhibit premature rat lung fibroblast proliferation,accelerate apoptosis.4. Rapamycin and m TORsi RNA suppress premature rat lung fibroblast proliferation, the possible mechanism is through the m TOR signaling pathway, specifically inhibit expression in premature rat lung fibroblasts m TORC1,4EBP1 and P70S6 K protein and m RNA.5. 90% high concentration of oxygen and rapamycin could promote hyperoxia exposure preterm mouse lung fibroblasts col-?, col-? and FN increased, blocking the mechanism may through m TOR signaling pathway.6. Bcl-2 is an anti-apoptotic factor, can inhibit lung fibroblast apoptosis;P53 is a pro-apoptotic factor, can promote lung fibroblast apoptosis. 90%of high concentrations of oxygen, rapamycin, m TORsi RNA transfection could promote lung fibroblast apoptosis.7. CTGF and TGF-? pro-fibrotic role in oxidative cell damage BPD pulmonary fibrosis possible mechanisms was by m TOR signaling pathway, m TOR signaling pathway stimulate the expression of CTGF and TGF-? thus promotes premature rat lung fibroblasts proliferation involved in the processes of BPD repair.8. TGF-? and CTGF are pro-fibrotic factor, while CTGF biological effects relatively specific, and its a pro-fibrotic factor downstream of TGF-?, may be an important target for pulmonary fibrosis in BPD.