基于AMPK/mTORC1自噬通路研究二氢杨梅树皮素对损伤A549细胞的保护作用

Protective effect of ampelopsin on injury of A549 cells based on AMPK/mTORC1 autophagy pathway

为研究二氢杨梅树皮素(APS)对脂多糖(LPS)诱导A549细胞损伤模型的保护作用及其对AMPK/mTORC1自噬通路的影响,随机分为正常组、模型组、APS高中低浓度组(80、40、20μg/mL)。除正常组外,其余各组用30μg/mL LPS诱导8 h构建A549细胞损伤模型,APS干预24 h后,MTT法检测A549细胞存活率、荧光法检测上清液LDH漏出量、Elisa法检测上清液TNF-α含量、MDC法细胞检测自噬体形成情况、自噬双标mRFP-GFP-LC3检测细胞自噬流情况,Western blot法检测p-AMPK/AMPK、p-mTOR/mTOR以及自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ、P62蛋白表达水平。AMPK抑制剂(CC)干预验证试验,随机分为正常组、模型组、LPS+APS组、LPS+CC组、LPS+APS+CC组,APS与CC共培养时间24 h后,采用Western blot法检测p-AMPK/AMPK、p-mTOR/mTOR以及自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ、P62蛋白表达水平。结果表明,APS使LPS损伤的A549细胞存活率明显升高,LDH漏出量、TNF-α含量明显减少,细胞内自噬体明显增加,自噬溶酶体明显增加,P62蛋白、p-mTOR/mTOR表达明显下调,LC3-Ⅱ/LC3-Ⅰ表达明显上调;用CC抑制AMPK/mTORC1自噬通路后,APS能够明显上调P62蛋白、p-AMPK/AMPK、LC3-Ⅱ/LC3-Ⅰ蛋白表达,显著下调p-mTOR/mTOR蛋白表达。APS通过激活AMPK/mTORC1自噬通路促进自噬,增强自噬流,发挥对LPS诱导A549细胞损伤的保护作用。

To investigate the protective effect of ampelopsin(APS)on lipopolysaccharide(LPS)-induced A549 cell injury model and its impact on the AMPK/mTORC1 autophagy pathway,the cells were randomly divided into the following groups:normal group,model group,APS high concentration group,APS medium concentration group,and APS low concentration group(80,40,20μg/mL).Except for the normal group,all other groups were treated with 30μg/mL LPS to induce A549 cell injury model for 8 hours.After 24 hours of APS intervention,the survival rate of A549 cells was measured using the MTT method.The leakage of LDH in the supernatant was detected using the fluorescence method,and the TNF-αcontent in the supernatant was detected using the Elisa method.Autophagosome formation in the cells was assessed using the MDC method,while cell autophagy flow was measured using autophagy doublelabeled mRFP-GFP-LC3.The p-AMPK/AMPK,p-mTOR/mTOR,autophagy-related protein LC3-II/LC3-I,and P62 protein expression levels were detected using the Western blot method.Additionally,an intervention validation trial using an AMPK inhibitor(CC)was conducted.The trial involved randomly dividing the subjects into different groups,including the normal group,model group,LPS+APS group,LPS+CC group,and LPS+APS+CC group.After co-culturing APS and CC for 24 hours,Western blot analysis was performed to measure the expression levels of p-AMPK/AMPK,p-mTOR/mTOR,and the autophagy-related protein LC3-Ⅱ/LC3-Ⅰ,as well as P62 protein.The results indicated that APS significantly increased the survival rate of A549 cells damaged by LPS,while reducing LDH leakage and TNF-αcontent.Moreover,APS led to a significant increase in the number of autophagosomes and lysosomes in the cells,along with a downregulation of P62 protein and p-mTOR/mTOR expression.Additionally,the expression of LC3-Ⅱ/LC3-Ⅰwas significantly upregulated.Furthermore,when the AMPK/mTORC1 autophagy pathway was inhibited using CC,APS was found to upregulate the expression of P62 protein,p-AMPK/AMPK and LC3-Ⅱ/LC3-Ⅰproteins,while downregulating pmTOR/mTOR protein expression.These findings suggested that APS promoted autophagy by activating the AMPK/mTORC1 autophagy pathway,thereby enhancing autophagy flow and exerting a protective effect against LPS-induced damage to A549 cells.