苹果多酚通过激活AMPK/SIRT1信号通路对LPS诱导的肺上皮细胞自噬反应的影响

目的:探讨苹果多酚通过调节腺苷酸活化蛋白激酶/沉默信息调节因子1（AMP-activated protein kinase/Sirtuin1,AMPK/SIRT1)信号通路对脂多糖（lipopolysaccharide,LPS）诱导的人肺泡上皮细胞（A549）自噬反应的影响。方法:使用不同浓度的苹果多酚提取物（apple polyphenol extract，APE）预处理A549细胞2 h后，LPS诱导A549细胞培养24 h，MTT法检测增殖活性，筛选APE最佳预处理浓度；将A549细胞分为对照组、LPS组（3 mg/L LPS）、LPS+APE组（3 mg/L LPS+20 μg/mL APE）、APE+Compound C组（3 mg/L LPS+20 μg/mL APE+50 μmol/L Compound C），免疫荧光染色观察A549细胞自噬；流式细胞术检测A549细胞凋亡；Western blot法检测细胞中自噬相关蛋白及AMPK/SIRT1通路相关蛋白表达水平。结果:与对照组比较，经LPS诱导的A549细胞增殖活性、自噬水平、LC3Ⅱ/LC3Ⅰ、Beclin-1、SIRT1、p-ULK1/ULK1、p-AMPK/AMPK蛋白表达降低，p62蛋白表达及细胞凋亡率升高（P＜0.05）；与LPS组比较，LPS+APE组细胞增殖活性、自噬水平、LC3Ⅱ/LC3Ⅰ、Beclin-1、SIRT1、p-ULK1/ULK1、p-AMPK/AMPK蛋白表达水平显著升高，p62蛋白表达及细胞凋亡率显著降低（P＜0.05）；与LPS+APE组比较，APE+Compound C组A549细胞增殖活性、自噬水平、LC3Ⅱ/LC3Ⅰ、Beclin-1、SIRT1、p-ULK1/ULK1、p-AMPK/AMPK蛋白表达水平显著降低，p62蛋白表达及细胞凋亡率显著升高（P＜0.05）。结论:苹果多酚通过激活AMPK/SIRT1 信号通路提高LPS诱导的肺上皮细胞自噬，降低细胞凋亡。

The effect of apple polyphenol on the LPS-induced autophagy response of lung epithelial cells by activating AMPK/SIRT1 signaling pathway

Objective:To investigate the effect of apple polyphenol on lipopolysaccharide（LPS)-induced autophagy in human alveolar epithelial cells（A549) by regulating AMP-activated protein kinase/Sirtuin1（AMPK/SIRT1) signaling pathway.Methods:A549 cells were pretreated with different concentrations of apple polyphenol extract（APE) for 2 h,and A549 cells were cultured for 24 h by LPS.The proliferation activity was detected by MTT method,and the optimal pretreatment concentration of APE was screened.The A549 cells were divided into control group,LPS group（3 mg/L LPS),LPS+APE group（3 mg/L LPS+20 μg/mL APE) and APE+Compound C group（3 mg/L LPS+20 μg/mL APE+50 μmol/L Compound C).Immunofluorescence staining was used to observe autophagy of A549 cells.Flow cytometry was used to detect apoptosis of A549 cells.Western blot method was used to detect the expression levels of autophagy-related proteins and AMPK/SIRT1 pathway-related proteins in cells.Results:Compared with the control group,the proliferation activity,autophagy level,LC3Ⅱ/LC3Ⅰ,Beclin-1,SIRT1,p-ULK1/ULK1,p-AMPK/AMPK protein expression of LPS-induced A549 cells decreased,and the p62 protein expression and the apoptosis rate increased（P＜0.05).Compared with the LPS group,the proliferation activity,autophagy level,LC3Ⅱ/LC3Ⅰ,Beclin-1,SIRT1,p-ULK1/ULK1,p-AMPK/AMPK protein expression in the LPS+APE group increased,and the p62 protein expression and the apoptosis rate decreased（P＜0.05).Compared with the LPS+APE group,the proliferation activity,autophagy level,LC3Ⅱ/LC3Ⅰ,Beclin-1,SIRT1,p-ULK1/ULK1,p-AMPK/AMPK protein expression in the APE+Compound C group decreased,and the p62 protein expression and the apoptosis rate increased（P＜0.05).Conclusion:Apple polyphenol can increase LPS-induced autophagy of lung epithelial cells and reduce cell apoptosis by activating AMPK/SIRT1 signaling pathway.