自噬在脂多糖诱导的A549细胞炎症反应中的作用及机制研究

The role and mechanism of autophagy in lipopolysaccharide-induced inflammatory response of A549 cells

摘要:

目的 探讨自噬在脂多糖（lipopolysaccharide，LPS）诱导的人肺泡上皮A549细胞炎症反应中的作用及机制。 方法 用LPS刺激A549细胞建立炎症反应模型，按浓度（0、1、5、10 μg/mL）和时间（0、4、8、12、24 h）分组（各组n=3）。用自噬抑制剂3-甲基腺嘌呤（3-methyladenine，3-MA）处理细胞，分为对照组、LPS组、3-MA组、3-MA+LPS组（各组n=3）；用自噬激动剂雷帕霉素（rapamycin，RAPA）处理细胞，分为对照组、LPS组、RAPA组、RAPA+LPS组（各组n=3）。通过Toll样受体4（Toll-like receptor 4，TLR4）过表达质粒和siRNA转染A549细胞，实验分为两部分，每部分各分4组（各组n=3）：TLR4过表达对照组、TLR4过表达组、TLR4过表达对照+LPS组、TLR4过表达+LPS组；TLR4沉默对照组、TLR4沉默组、TLR4沉默对照+LPS组、TLR4沉默+LPS组。采用CCK-8法检测细胞存活率，免疫印迹法检测炎症指标（NLRP3、Caspase-1、ASC）、自噬指标（LC3B、Beclin-1、P62）、TLR4蛋白表达水平。 结果 1 μg/mL浓度的LPS刺激12 h后，炎症指标（NLRP3、Caspase-1、ASC）、自噬指标（LC3B、Beclin-1、P62）和TLR4蛋白表达均明显升高并达峰值（P<0.05）。与LPS组比较，3-MA+LPS组自噬相关蛋白表达降低，炎症相关蛋白表达升高，TLR4蛋白表达上调；RAPA+LPS组自噬相关蛋白表达升高，炎症相关蛋白表达降低，TLR4蛋白表达下调（P<0.05）。TLR4过表达+LPS组较TLR4过表达对照+LPS组自噬相关蛋白表达降低，炎症相关蛋白表达升高；TLR4沉默+LPS组较TLR4沉默对照+LPS组自噬相关蛋白表达升高，炎症相关蛋白表达降低（P<0.05）。 结论 在LPS诱导的人肺泡上皮A549细胞炎症反应中，自噬流对A549细胞具有一定保护作用；TLR4介导自噬流对LPS诱导的人肺泡上皮A549细胞炎症反应进行负向调控。

Abstract:

Objective To study the role and mechanism of autophagy in lipopolysaccharide (LPS)-induced inflammatory response of human alveolar epithelial A549 cells. Methods A549 cells were stimulated with LPS to establish a cell model of inflammatory response, and were then grouped (n=3 each) by concentration (0, 1, 5, and 10 μg/mL) and time (0, 4, 8, 12, and 24 hours). The A549 cells were treated with autophagy inhibitor 3-methyladenine (3-MA) to be divided into four groups (n=3 each): control, LPS, 3-MA, and 3-MA+LPS. The A549 cells were treated with autophagy agonist rapamycin (RAPA) to be divided into four groups (n=3 each): control, LPS, RAPA, and RAPA+LPS. The A549 cells were transfected with the Toll-like receptor 4 (TLR4) overexpression plasmid to be divided into four groups (n=3 each): TLR4 overexpression control, TLR4 overexpression, TLR4 overexpression control+LPS, and TLR4 overexpression+LPS. The A549 cells were transfected with TLR4 siRNA to be divided into four groups (n=3 each): TLR4 silencing control,TLR4 silencing, TLR4 silencing control+LPS, and TLR4 silencing+LPS. CCK-8 assay was used to measure cell viability. Western blot was used to measure the protein expression levels of inflammatory indicators (NLRP3, Caspase-1, and ASC), autophagic indicators (LC3B, Beclin-1, and P62), and TLR4. Results After stimulation with 1 μg/mL LPS for 12 hours, the levels of inflammatory indicators (NLRP3, Caspase-1, and ASC), autophagic indicators (LC3B, Beclin-1, and P62), and TLR4 increased and reached the peak (P<0.05). Compared with the LPS group, the 3-MA+LPS group had reduced expression of autophagy-related proteins and increased expression of inflammation-related proteins and TLR4, while the RAPA+LPS group had increased expression of autophagy-related proteins and reduced inflammation-related proteins and TLR4 (P<0.05). The TLR4 overexpression+LPS group had reduced autophagy-related proteins and increased inflammation-related proteins compared with the TLR4 overexpression control+LPS group, and the TLR4 silencing+LPS group had increased autophagy-related proteins and reduced inflammation-related proteins compared with the TLR4 silencing control+LPS group (P<0.05). Conclusions In the LPS-induced inflammatory response of human alveolar epithelial A549 cells, autophagic flux has a certain protective effect on A549 cells. TLR4-mediated autophagic flux negatively regulates the LPS-induced inflammatory response of A549 cells.