Role Of Mitochondrial Ca²⁺ In Hyperoxia-induced Apoptosis In Alveolar Type Ⅱ Epithelial Cells

Objective To explore the effect of short-term hyperoxia on mitochondrial Ca²⁺concentration in alveolar type Ⅱ epithelial cells（ATⅡ）and the role of mitochondrial Ca²⁺concentration in hyperoxia-induced apoptosis in alveolar type Ⅱ epithelial cells.Methods Rat Alveolar type Ⅱ epithelial cells lines（RLE-6TN）in logarithmic growth phase cells were divided into 3 groups using a random number table method:control group（group C）,hyperoxia group（group H）,hyperoxia+Ruthenium Red group（group HR）.Cells in Group C were cultured in a conventional incubator（5%CO₂-95%air）for 4 h.Then the H and HR groups were cultured in a sealed oxygen chamber（90%O₂-5%CO₂-5%air）with high oxygen concentration for 4 h,and the mitochondrial uniporter（MCU）inhibitor Ruthenium Red 2μmol/L were added to group HR before hyperoxia exposure.Cells morphology were observed under the inverted phase contrast microscope,and cells apoptosis was analyzed by flow cytometry.The intensity of Ca²⁺fluorescence in mitochondria was measured by Multiscan Spectrum and the concentration of Ca²⁺was calculated.The mRNA levels of Bcl-2、Bax、Caspase-3 and Caspase-9 were assessed by q PCR.Results Compared with group C,the mitochondrial Ca²⁺concentration and cells apoptosis rates were all increased（P<0.05）in group H,the Bcl-2 expression was down-regulated（P<0.05）,and the Bax/Bcl-2 was up-regulated（P<0.05）,but there was no statistical difference in the expression of Caspase-3 and Caspase-9;the mitochondrial Ca²⁺concentration decreased in the HR group（P<0.05）,and there was no significant difference in apoptosis rate,the expression of Bcl-2 and the Bax/Bcl-2,but the expression of Caspase-3 and Caspase-9 was down-regulated（P<0.05）;Compared with group H,the mitochondrial Ca²⁺concentration and cells apoptosis rates were all decreased（P<0.05）in group HR,the Bcl-2 expression was up-regulated（P<0.05）,the Bax/Bcl-2 and the expression of Caspase-3,Caspase-9was all down-regulated（P<0.05）.Compared with group C,cells were presenting morphological manifestations of early apoptosis in Group H（The cells shriveled into a spherical shape with reduced transmittance and detachment from surrounding cells）.The HR group improved significantly compared with H group.Conclusion Increased mitochondrial Ca²⁺concentration was involved in the hyperoxia-induced apoptosis process in alveolar type Ⅱ epithelial cells.