脂多糖活化的大鼠肺泡巨噬细胞TNF-α产生的新机制

The mechanism of lipopolysaccharide-activated rat alveolar macrophages producing tumor necrosis factor alpha

目的:探讨脂多糖(LPS)活化的肺泡巨噬细胞中低氧诱导因子-1α(HIF-1α)的表达及其在肺泡巨噬细胞产生肿瘤坏死因子α(TNF-α)中的作用。方法:应用HIF-1α诱骗法(HIF-1αdecoy)抑制其作用,并用Westernblotting、半定量RT-PCR、酶联免疫吸附法(ELISA)分别检测HIF-1α蛋白、mRNA的表达和TNF-α的产生。结果:HIF-1α蛋白含量在LPS组(1.95±0.57)和HIF-1αdecoy组(1.89±0.59)均明显高于对照组(0.41±0.14,P<0.05);HIF-1αmRNA的表达在对照组(0.7838±0.3183)、LPS组(0.7622±0.3387)和HIF-1αdecoy组(0.8155±0.3594)之间无显著差异(P>0.05);LPS刺激24h后,大鼠肺泡巨噬细胞TNF-α的产生明显高于对照组(61ng/Lvs156ng/L,P<0.05),抑制HIF-1α后TNF-α的产生明显低于LPS刺激组(90ng/Lvs156ng/L,P<0.05),但HIF-1αdecoy组TNF-α的产生仍然高于对照组(61ng/Lvs94ng/L,P<0.05)。结论:大鼠肺泡巨噬细胞在LPS的刺激下HIF-1α蛋白的稳定性增强使其表达明显上调,并且促进TNF-α的产生,提示HIF-1α在肺部慢性炎症性疾病如COPD的发病机制中可能有重要作用。

AIM： To investigate the expression of hypoxia inducible factor- 1 alpha （HIF- 1 α） and the role of HIF - 1α in tumor necrosis factor alpha （TNF- α） production in rat alveolar macrophages activated by lipopolysaccharide （LPS）. METHODS： HIF- 1α function was inhibited by using the method of HIF-1α decoy. Western blotting and semiquantitative RT- PCR were applied to determine the expression of HIF- 1α protein and mRNA, respectively. The production of TNF- α was determined with ELISA. RESULTS： The content of HIF-1α protein in LPS group （1.95±0.57） and HIF- 1α decoy group （1.89±0.59） were 4.8 times and 4.6 times higher than that in control group （0.41 ± 0.14）, respectively. The expression of HIF- 1α mRNA showed no difference among three groups （F = 3.14, P 〉 0.05）. The production of TNF- α in LPS group was higher than that in control group （61 ng/L vs 156 ng/L, q = 5.12, P 〈 0.05） and HIF - 1α decoy group （90 ng/L vs 156 ng/L, q = 4.63, P 〈 0.05）, respectively. However, the content of TNF- α in HIF- 16 decoy group was still higher than that in control group （61 ng/ L vs 94 ng/L, q = 4.47, P 〈 0.05）. CONCLUSION： The enhanced Stability of HIF- 16 protein results in the marked upregulation of its protein and HIF- 1α is contributed to the production of TNF- α in LPS- stimulating rat alveolar macrophages. It is indicated that HIF - 16 plays important role in the pathogenesis of chronic inflammation involved in diseases such as COPD.