大鼠肺泡上皮细胞自噬对内质网应激的影响

The impact of autuphagy in rat alveolar epithelial cell to endoplasmic reticulum stress

目的观察大鼠肺泡上皮细胞CCL149自噬对内质网应激的影响。方法将绿色荧光蛋白-微管相关蛋白1轻链3（GFP-LC3）质粒导入CCL149细胞，筛选出稳定表达GFP-LC3的细胞株。在分别加入自噬激活剂雷帕霉素和自噬抑制剂3-甲基腺嘌呤（3-MA）后，观察大鼠肺泡上皮细胞经缺氧复氧2、4、6 h时细胞自噬变化及相应的内质网应激反应。结果在缺氧复氧2、4、6 h这3个时间点，加入3-MA组自噬基因GFP-LC3表达量显著低于对照组（t＝6.317，P＝0.024；t＝8.583，P＝0.013；t＝6.341，P＝0.026），Beclin-1表达量显著低于对照组（t＝4.658，P＝0.039；t＝7.120，P＝0.022；t＝10.320，P＝0.000），p62表达量显著低于对照组（t＝5.906，P＝0.015；t＝7.032，P＝0.019；t＝7.072，P＝0.032），而内质网应激基因免疫球蛋白结合蛋白（BIP）表达量显著高于对照组（t＝5.988，P＝0.012；t＝9.473，P＝0.004；t＝12.863，P＝0.000），X-盒结合蛋白（XBP-1）表达量显著高于对照组（t＝4.285，P＝0.043；t＝9.563，P＝0.008；t＝10.531，P＝0.000），C/EBP同源蛋白（CHOP）表达量显著高于对照组（t＝5.477，P＝0.031；t＝8.373，P＝0.016；t＝8.954，P＝0.025）；加入雷帕霉素组自噬基因GFP-LC3表达量显著高于对照组（t＝4.502，P＝0.046；t＝10.000，P＝0.010；t＝4.983，P＝0.041），Beclin-1表达量显著高于对照组（t＝5.161，P＝0.029；t＝5.767，P＝0.029；t＝9.342，P＝0.012），p62表达量显著高于对照组（t＝6.687，P＝0.026；t＝7.084，P＝0.016；t＝9.767，P＝0.000），而内质网应激基因BIP表达量显著低于对照组（t＝7.245，P＝0.016；t＝8.432，P＝0.005；t＝12.562，P＝0.000），XBP-1表达量显著低于对照组（t＝6.255，P＝0.022；t＝8.450，P＝0.029；t＝8.365，P＝0.035），CHOP表达量显著低于对照组（t＝7.048，P＝0.014；t＝11.566，P＝0.000；t＝10.144，P＝0.000）。结论在大鼠肺泡上皮细胞缺血缺氧早期，增强细胞自噬可减弱内质网应激。

Objective To investigate the impact of autophagy in rat alveolar epithelial cells CCL149 to endoplasmic reticulum stress.Methods Building green fluorescent protein （GFP）-microtubelesas sociated protein light chain 3 （LC3） eukaryotic expression vector, and this plasmid was imported into CCL149 cells by plasmid transfection technology. The stable expression cell lines were screened to further study. Observing the level of cell autophagy and degree of endoplasmic reticulum stress in the time of hypoxia-reoxygenation （H/R） 2 hours, 4 hours, 6 hours after giving the process of autophagy activation agent （rapamycin） and autophagy inhibitors 3-Methyladenine （3-M）.Results 3-MA pretreatment led to decreased LC3 （t=6.317, P=0.024; t=8.583, P=0.013; t=6.341, P=0.026）, Beclin-1 （t=4.658, P=0.039; t=7.120, P=0.022; t=10.320, P=0.000） and p62 （t=5.906, P=0.015; t=7.032, P=0.019; t=7.072, P=0.032）, and increased binding immunoglobulin protein （BIP） （t=5.988, P=0.012; t=9.473, P=0.004; t=12.863, P=0.000）, X-Box Binding Protein 1 （XBP-1） （t=4.285, P=0.043; t=9.563, P=0.008; t=10.531, P=0.000） and C/EBP homologus protein （CHOP） （t=5.477, P=0.031; t=8.373, P=0.016; t=8.954, P=0.025） following H/R treatment at 2, 4 and 6 h. On the contrary, LC3 （t=4.502, P=0.046; t=10.000, P=0.010; t=4.983, P=0.041）, Beclin-1 （t=5.161, P=0.029; t=5.767, P=0.029; t=9.342, P=0.012） and p62 （t=6.687, P=0.026; t=7.084, P=0.016; t=9.767, P=0.000） were enhanced, and BIP （t=7.245, P=0.016; t=8.432, P=0.005; t=12.562, P=0.000）, XBP-1 （t=6.255, P=0.022; t=8.450, P=0.029; t=8.365, P=0.035） and CHOP （t=7.048, P=0.014; t=11.566, P=0.000; t=10.144, P=0.000） were decreased by treated with rapamycin following H/R treatment at 2, 4 and 6 h.Conclusion In the early of hypoxia reoxygenation, the endoplasmic reticulum stress was decreased as the autophagy enhanced in alveolar epithelial cells of rats.