二甲双胍调控NF-κB信号通路对急性肺损伤肺泡上皮细胞焦亡的影响

陈涛; 欧阳运萍; 李鹏; 赵博; 杨小军

二甲双胍调控NF-κB信号通路对急性肺损伤肺泡上皮细胞焦亡的影响

Effect of metformin regulating NF-κB signaling pathway on the pyroptosis of alveolar epithelial cells in acute lung injury

摘要

摘要:
目的： 探究二甲双胍（DMBG）通过核转录因子-κB（NF-κB）信号通路对过氧化氢（H₂O₂）诱导的人肺泡上皮细胞凋亡与焦亡的作用。
方法： 体外培养人肺泡上皮细胞A549，分为空白组（不做干预）、模型组（400 μmol/L H₂O₂处理24 h）、低/中/高浓度组（模型组基础上分别加入1.25、2.5、5 mmol/L DMBG）、DMBG组（模型组基础上加入5 mmol/L DMBG）、inhibitor组（模型组基础上加入5 μmol/L NF-κB通路抑制剂BAY 11-7082）、DMBG + inhibitor组（模型组基础上加入5 mmol/L DMBG + 5 μmol/L BAY 11-7082）和DMBG + activator组（模型组基础上加入5 mmol/L DMBG + 1 μmol/L NF-κB通路激活剂Prostratin）。药物干预24 h后，采用细胞计数试剂盒-8（CCK-8）、酶联免疫吸附试验（ELISA）、Hoechst 33258染色、实时荧光定量PCR（RT-qPCR）、蛋白免疫印迹（WB）法检测细胞活力、氧化和炎症因子丙二醇（MDA）、超氧化物歧化酶（SOD）、白细胞介素（IL）-1β、IL-18水平、凋亡率、半胱氨酸天冬氨酸蛋白酶-1（Caspase-1）、消皮素-D（GSDMD）、核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）mRNA和蛋白表达水平、NF-κB p65和p-NF-κB p65蛋白表达水平。
结果： 与空白组相比，模型组细胞活力、SOD水平降低（P ＜ 0.05），MDA水平升高（P ＜ 0.05）；高浓度组较模型组细胞活力、SOD水平升高（P ＜ 0.05），MDA水平降低（P ＜ 0.05），因此选择5 mmol/L DMBG用于后续实验。与空白组相比，模型组细胞凋亡率、IL-1β、IL-18、Caspase-1、GSDMD、NLRP3 mRNA和蛋白及p-NF-κB p65 蛋白表达水平升高（P ＜ 0.05）。与模型组相比，DMBG组和inhibitor组细胞凋亡率、IL-1β、IL-18、Caspase-1、GSDMD、NLRP3mRNA和蛋白表达水平、p-NF-κB p65蛋白表达水平降低（P ＜ 0.05）；与DMBG组相比，BAY 11-7082增强了、Prostratin则抑制了DMBG对H₂O₂诱导的A549细胞的上述作用（P ＜ 0.05）。
结论： DMBG通过阻遏NF-κB信号通路抑制细胞的凋亡与焦亡，从而发挥对A549细胞氧化损伤的保护作用。

Abstract:
Objective To investigate the effects of metformin (DMBG) on the apoptosis and pyrodeath of human alveolar epithelial cells induced by hydrogen peroxide (H₂O₂) through nuclear transcription factor-κB (NF-κB) signaling pathway.
Methods In vitro, the human alveolar epithelial cells A549 were divided into the blank group (no intervention), model group (treated with 400 μmol/L H₂O₂ for 24 h), low/medium/high concentration group (adding 1.25, 2.5 and 5 mmol/L DMBG on the basis of model group), and DMBG group (adding 5 mmol/L DMBG on the basis of model group), inhibitor group (adding 5 μmol/L BAY 11-7082 of NF-κB pathway inhibitor on the basis of model group), DMBG + inhibitor group (adding 5 mmol/L DMBG + 5 μmol/L BAY 11-7082 on the basis of model group) and DMBG + activator group (adding 5 mmol/L DMBG + 1 μmol/L Prostratin of NF-κB pathway activator on the basis of model group). After 24 hours of drug intervention, the cell counting Kit 8 (CCK-8), enzyme-linked immunosorbent assay (ELISA), Hoechst 33258 staining, real-time fluorescence quantitative PCR (RT-qPCR) and Western blot (WB) were used to detect the cell viability, levels of oxidation and inflammatory factors (MDA, SOD, IL-1β, IL-18), apoptosis rate, RNA and protein expression levels of Caspase-1, Apodermin-D(GSDMD) and nucleotide-binding oligomerized domain-like receptor protein 3(NLRP3), and protein expression levels of F-κB p65 and p-NF-κB p65.
Results Compared with the blank group, the cell vitality and SOD level in model group decreased (P ＜ 0.05), while the MDA level increased (P ＜ 0.05). Compared with the model group, the cell activity and SOD level in the high concentration group increased (P ＜ 0.05), while the MDA level decreased (P ＜ 0.05). Therefore, the 5 mmol/L DMBG was selected for subsequent experiments. Compared with the blank group, the apoptosis rate, mRNA levels of the IL-1β, IL-18, Caspase-1, GSDMD and NLRP3, and protein expression level of p-NF-κB p65 in the model group increased (P ＜ 0.05). Compared with the model group, the apoptosis rate, mRNA and protein levles of IL-1β, IL-18, Caspase-1, GSDMD and NLRP3, and protein expression level of p-NF-κB p65 in the DMBG group and inhibitor group decreased (P ＜ 0.05). Compared with the DMBG group, the BAY 11-7082 enhanced and Prostratin inhibited the above effects of DMBG on A549 cells induced by H₂O₂ (P ＜ 0.05).
Conclusions DMBG can inhibit apoptosis and pyrodeath of A549 cells by blocking NF-κB signaling pathway, thus which plays a protective role in oxidative damage of A549 cells.

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