摘要
目的探讨α1抗胰蛋白酶(AAT)对脂多糖(LPS)诱导肺泡上皮细胞炎症因子分泌的影响及其机制。方法选择具有肺泡上皮细胞特征的非小细胞肺癌细胞系A549为研究对象,将其分为对照组、AAT(0.5mg/mL)组、LPS(10μg/mL)组、AAT+LPS组4组。分别加入AAT和LPS进行干预和诱导,细胞培养10h后收集并提取蛋白,采用蛋白免疫印迹法(Western Blot)检测核转录因子-κB(NF-κB)抑制蛋白IκBα和NF-κB p65的表达水平;培养18h后分别收集细胞培养上清和细胞,采用酶联免疫吸附法(ELISA)检测细胞上清中肿瘤坏死因子-α(TNF-α)、白细胞介素-8(IL-8)和单核细胞趋化因子-1(MCP-1)的含量,采用实时荧光定量PCR检测细胞中白细胞介素-1β(IL-1β)的mRNA表达水平。结果与LPS组比较,AAT+LPS组细胞培养上清中TNF-α、IL-8、MCP-1的含量明显降低,细胞中IL-1βmRNA的水平降低,组间比较差异有统计学意义(P<0.05);Western blot结果显示,与LPS组比较,AAT+LPS组IκBα的磷酸化和细胞核内NF-κB p65的蛋白水平明显下降。结论 AAT能够有效地抑制LPS诱导肺泡上皮细胞炎症的因子的分泌,此过程可能是通过抑制NF-κB的激活和进入细胞核而发挥作用。
Objective To investigate the effects and underlying mechanisms of al-antitrypsin （AAT） on in-flammatory cytokines production induced by lipopolysaccharide （LPS） in lung alveolar epithelial cells. Methods Human alveolar epithelial cells A549 were divided into control group, AAT （0.5 mg/mL） group, LPS （10 μg/mL） group and AAT ＋ LPS group, after 10 h incubation, the expression levels of phosphorylated IκBα and NF-κB p65 subunit were determined by Western Blot. And after 18 h incubation, TNF-α, IL-8 and monocyte chemotactic factor-1 （MCP-1） contents in cell supernatant were determined by ELISA, and the mRNA level of IL-1β was quantified by quantitative real time PCR. Results alantitrypsin eliminated the phosphorylation of IκBα and the nuclear distribution of NF-κB induced by LPS. In addition, compared with the LPS-treated cells, the levels of TNF-α, IL-8, MCP-1 in cell culture medi-um and the IL-1β mRNA level were significantly lower in LPS plus AAT treated cells （P 〈0.05）. Western Blot showed that compared with the LPS group, the phosphorylation of IκBα and the nuclear translocation of NF-κB p65 were significantly reduced in LPS ＋ AAT group. Conclusion α1-antitrypsin inhibits pro-in-flammatory cytokines production induced by LPS via reducing the NF-κB nuclear translocation.
出处
《新疆医科大学学报》
CAS
2017年第8期1061-1064,共4页
Journal of Xinjiang Medical University
基金
新疆维吾尔自治区高校科研计划青年教师科研启动基金(XJEDU2009S53)
