Hydrogen Peroxide Induced Repairment, Survival And Apoptosis Of Alveolar Type â…¡ Epithelial Cell And The Regulation Mechanism Mediated By P38 MAP Kinase

Objective Oxygen therapy is widely used for patients to improve blood oxygen saturation and reduce tissue hypoxia, and has saved lots of patients.However,supplemental O2 of prolonged high concentration administered to patients may result in bronchopulmonary dysplasia(BPD). The development of BPD has something to do with alveolar typeâ…¡epithelial cell (AT-â…¡) AT-â…¡can function as a progenitor for regeneration after lung injury. p38 MAP kinase (p38 MAPK) plays a role in cell proliferation, survival and apoptosis, which may have effect on survival or death of AT-â…¡under oxidative stress. In the present study, we investigated the survival and apoptotic responses of primary cultured rat AT-â…¡induced by hydrogen peroxide (H₂O₂) and the regulation mechanism mediated by p38 MAPK.Methods AT-â…¡cells from rat were isolated by trypsin digestion and differential adherence to plates coated with IgG. Primary cultured rat AT-â…¡cells were then stimulated by H₂O₂ to simulate ROS attack in vivo. Cells were divided into control group(cells in serum-free culture medium) , H₂O₂-treated group(cells in serum-free culture medium with 10,100,500 and 1000μM H₂O₂) and p38 MAPK inhibition group. Morphological changes were viewed with inverted microscope and electron microscope. Cell viability, apoptotic ratios and the expression of phosphorylated p38 MAPK were measured by MTT assay,flow cytometry and western blot analysis, respectively. In some groups, cell viability and -apoptotic ratios of AT-â…¡was detected after pretreatment with SB203580 and succedent H₂O₂-incubation.Results We developed a method of isolating AT-â…¡cells based on trypsin digestion to dissociate lung cells prior to adherence onto IgG-coated plates, which recovered 1-2×10⁷ cells per rat with purity and viability above 90%. The osmiophilia lamellar bodies, which are thinked to be the special constructions of ATâ…¡cells, were observed by transmission electron microscopy. ATâ…¡cells were stimulated by H₂O₂ for 180min, and when compared with control group, 10μM H₂O₂-treated group showed no morphological changes. Intercellular space slightly widened when cells were treated with 100μM H₂O₂. 500μM H₂O₂-treated group showed wider intercellular space and cells turned shrinked,some cells broke off from culture plate . More cells turned round, shrinked, ruptured and broke off from culture plate when treated with 1000μM H₂O₂. And apoptotic bodies were observed under transmission electron microscope too. MTT and flow cytometry assay demonstrated that H₂O₂-incubation at the concentration of 10μM and 100μM for 180min had no impact on cell viability and apoptosis(P>0.05),but led to decreased cell viability from 97.50±2.588 to 80.67±6.121% and 44.50±2.429% (P<0.05) and increased apoptotic ratios from 7.8960±4.4065% to 29.2420±4.2045% and 48.8800±8.6261% (P<0.05) at the concentration of 500μM and 1000μM in AT-â…¡cells, respectively. When compared with 500μM H₂O₂-treated group, SB203580 and succedent 500μM H₂O₂-treated group showed wider intercellular space, more cells turned shrinked, ruptured and broke off from culture plate . 500μM H₂O₂ induced rapid activation of p38MAPK. Phosphorylated p38MAPK peaked at 15 min after stimulation by 500μM H₂O₂ and returned to almost control values within 3 hours; specific inhibition of p38MAPK using SB203580 decreased the activation of p38MAPK. And specific inhibition of p38MAPK using SB203580 decreased cell viability and increased apoptotic ratios after 500μM H₂O₂ exposure for 180min(P<0.05).Conclusions1. A method for isolating rat AT-â…¡cells by trypsin digestion and differential adherence to plates coated with IgG exhibits high cell yield, purity and viability, can be used for our investigation.2. Reduced cell viability and increased apoptosis of AT-â…¡appear relating to the level of oxidative stress.3. p38MAPK signaling pathway plays a role in the regulation of apoptosis and cell viability and may be protective for AT-â…¡cells under oxidative stress.