Toll样受体/白细胞介素-1受体8减轻肿瘤坏死因子-α诱导的A549细胞的氧化损伤

Toll-like receptor/interleukin-1 receptor 8 alleviated oxidative damage induced by tumor necrosis factor-α in A549 cells

目的研究Toll样受体/白细胞介素-1受体8(TIR8)对肿瘤坏死因子-α(TNF-α)诱导的人Ⅱ型肺泡上皮细胞来源的A549细胞氧化损伤的影响。方法 A549细胞感染TIR8过表达慢病毒(LV-TIR8),经TNF-α刺激后,采用实时荧光定量聚合酶链反应(qRT-PCR)和蛋白质印迹法(Western blot)检测TIR8的表达。采用噻唑蓝(MTT)法检测细胞存活率,硫代巴比妥酸法检测细胞中丙二醛(MDA)含量,黄瞟吟氧化法检测细胞中超氧化物歧化酶(SOD)活性,流式细胞术检测细胞凋亡,Western blot检测cleaved Caspase-3蛋白水平,结果 LV-TIR8病毒感染可以增加细胞中TIR8的表达过表达TIR8的A549细胞经TNF-α诱导后,细胞存活率从(71.25 ±6.23)%升至(84.65 ±5.48)%,细胞中 MDA 含量从(164.62 ± 14. 25)μmol/mg 降至(124. 15 ± 10. 86)μmol/mg,SOD 活性从(59.60 ±5. 17)Unit/mg升至(80. 12 ± 8. 01) Unit/mg,细胞凋亡率从(21.36 ±2. 86)%降至(14.83 ±1.24)%,细胞中cleaved Caspase-3蛋白水平下降,差异均有统计学意义(P <0.05)。结论过表达TIR8可以减轻TNF-α诱导对A549细胞的氧化损伤,减少细胞合成MDA,提高细胞中SOD活性,减少细胞凋亡。

Objective To study the effect of toll-like receptor/interleukin-1 receptor8 ( TIR8) on oxidative damage induced by tumor necrosis factor-α(TNF-α) in human type Ⅱ alveolar epithelial A549 cells. Methods A549 cells were infected by TIR8 overexpression lentivirus ( LV-TIR8 ) virus and stimulated by TNF-α. The expression level of TIR8 was measured by quantitative real time polymerase chain reaction( qRT-PCR) and Western blotting. Methyl thiazolyl tetrazolium ( MTT) assay method was used to determine the proliferation activity of cells. The content of malondialdehyd ( MDA) in cells was detected by thiobarbituric acid method. The activity of superoxide dismutas ( SOD ) in the cells was detected by xanthine oxidation. Cell apoptosis was detected by flow cytometry. The level of cleaved Caspase-3 protein was detected by Western blotting. Results LV-TIR8 virus infection increased the expression level of TIR8 in cells. Tlie A549 cells which were overexpressed by TIR8,were induced by TNF-α, cell proliferative activity increased from (71.25 ±6. 23)% to ( 84. 65 ± 5.48)%. The content of MDA in cells decreased from ( 164. 62 ± 14. 25 )μmol/mg to ( 124. 15 ± 10. 86)μmol/mg. The activity of SOD increased from (59. 60 ±5. 17 ) Unit/mg to ( 80. 12 ±8.01) Unit/mg. The rate of apoptosis decreased from (21.36 ±2. 86 )% to ( 14. 83 ± 1. 24 )%. The level of cleaved Caspase-3 protein in cells decreased. Compjared with the cells did not overexpress TIR8 ,the differences were statistictilly significant( P < 0. 05). Conclusion Overexpression of TIR8 can reduce oxidative damage induced by TNF-α in A549 cells. It can reduce cell synthesis of MDA , increase the SOD activity and reduce cell apoptosis.