Effect of silent information regulator 1 on type Ⅱ alveolar epithelial cell injury in lipopolysaccharide-induced rats
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摘要:目的
探讨沉默调节蛋白1(SIRT1)在脂多糖(LPS)诱导的大鼠Ⅱ型肺泡上皮细胞损伤中的作用及机制。
方法分离大鼠原代Ⅱ型肺泡上皮细胞, 并分为对照组、模型组和实验组。模型组和实验组分别用空白和SIRT1 shRNA慢病毒感染后, 再加入LPS诱导细胞损伤。采用实时荧光定量聚合酶链式反应(PCR)和Western blot检测SIRT1的表达; 采用CCK-8实验检测细胞活力; 采用实时荧光定量PCR和酶联免疫吸附测定(ELISA)检测肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)的表达; 采用JC-1染色法检测线粒体膜电位; 采用化学显色法检测细胞丙二醛(MDA)水平和超氧化物歧化酶(SOD)活性。
结果与对照组相比, 模型组Ⅱ型肺泡上皮细胞SIRT1 mRNA和蛋白表达水平升高, 细胞活力下降, 促炎因子TNF-α和IL-1β的mRNA表达水平和分泌水平升高, 线粒体膜电位下降, MDA水平升高, SOD活性下降, 差异均有统计学意义(P < 0.05)。与模型组相比, 实验组Ⅱ型肺泡上皮细胞SIRT1 mRNA和蛋白表达水平下降, 细胞活力升高, 促炎因子TNF-α和IL-1β的mRNA表达水平和分泌水平下降, 线粒体膜电位升高, MDA水平下降, SOD活性升高, 差异均有统计学意义(P < 0.05)。
结论SIRT1通过影响线粒体活性和细胞氧化还原稳态促进LPS诱导的Ⅱ型肺泡上皮细胞损伤。
Abstract:ObjectiveTo investigate the role and mechanism of silent information regulator 1 (SIRT1) on type Ⅱ alveolar epithelial cell injury in lipopolysaccharide (LPS) induced rats.
MethodsPrimary type Ⅱ alveolar epithelial cells were isolated from rats and divided into control group, model group and experimental group. Model group and experimental group were infected with blank and SIRT1 shRNA lentivirus respectively, and then LPS was added to induce cell injury. The expression of SIRT1 was detected by real-time quantitative polymerase chain reaction (PCR) and Western blot; the cell viability was detected by CCK-8 assay; the expressions of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA); the mitochondrial membrane potential was detected by JC-1 staining assay; the level of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were detected by chemical chromogenic assay.
ResultsCompared with the control group, SIRT1 mRNA and protein expression levels of type Ⅱ alveolar epithelial cells in the model group increased significantly, cell viability decreased significantly, mRNA expression levels and secretion levels of pro-inflammatory factors such as TNF-α and IL-1β increased significantly, mitochondrial membrane potential decreased significantly, MDA level increased significantly, and SOD activity decreased significantly (P < 0.05). Compared with the model group, SIRT1 mRNA and protein expression levels of type Ⅱ alveolar epithelial cells in the experimental group decreased significantly, cell viability increased significantly, mRNA expression levels and secretion levels of pro-inflammatory factors such as TNF-α and IL-1β decreased significantly, mitochondrial membrane potential increased significantly, MDA level decreased significantly, and SOD activity increased significantly (P < 0.05).
ConclusionSIRT1 can promote LPS-induced type Ⅱ alveolar epithelial cell injury by affecting mitochondrial activity and cell redox homeostasis.
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表 1 3组细胞培养基中TNF-α及IL-1β水平比较(x±s)
pg/mL 组别 TNF-α IL-1β 对照组 1 377.69±242.75 89.76±10.31 模型组 5 893.24±496.12* 372.63±38.55* 实验组 3 974.26±400.11# 284.67±21.39# TNF-α: 肿瘤坏死因子-α; IL-1β: 白细胞介素-1β。
与对照组比较, *P < 0.05; 与模型组比较, #P < 0.05。
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表 3 3组细胞MDA、SOD水平比较(x±s)
组别 MDA/(nmol/mL) SOD/(U/mL) 对照组 27.34±5.62 19.62±3.98 模型组 56.87±8.44* 8.07±2.16* 实验组 39.85±6.03# 14.55±2.52# MDA: 丙二醛; SOD: 超氧化物歧化酶。
与对照组比较, *P < 0.05; 与模型组比较, #P < 0.05。
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