川芎嗪抑制LPS诱导的人Ⅱ型肺泡上皮细胞炎性反应

Tetramethylpyrazine inhibits LPS-induced inflammation of human type Ⅱ alveolar epithelial cells

目的体外探讨川芎嗪(TMP)对脂多糖(LPS)诱导的人Ⅱ型肺泡上皮细胞(HAECⅡ)炎性反应的保护作用及机制。方法体外培养HAECⅡ(A549细胞来源),LPS刺激建立炎性模型,分别加入TMP和前B细胞克隆增强因子(PBEF)抑制剂FK866进行干预。q-PCR和Western blot检测肿瘤坏死因子α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-8(IL-8)和PBEF的mRNA和蛋白表达;通过Western blot检测细胞核和细胞质内磷酸化P65蛋白来反映核因子κB(NF-κB)的激活。结果 LPS刺激A549细胞后,TNF-α、IL-1β、IL-8和PBEF mRNA及蛋白表达均较对照组明显增高(P<0.001),伴随着细胞核和细胞质磷酸化的P65蛋白增高(P<0.001);TMP干预后,上述炎性因子mRNA和蛋白表达下降,磷酸化P65蛋白表达降低(P<0.05);FK866干预后,TNF-α、IL-1β和IL-8表达以及磷酸化P65蛋白降低(P<0.01)。结论 TMP可能通过降低PBEF的表达,从而抑制NF-κB的激活,减轻肺泡上皮细胞炎性反应。

Objective To study the protective effect of TMP （Tetramethylpyrazine） on LPS （Lipopolysaccharides） - induced inflammatory response of human type 11 alveolar epithelial cells （ HAEC Ⅱ ） and its corresponding mechanism. Methods HAECⅡ （ A549 cells derived from human lung adenocarcinoma cells） were cultured in vitro. Inflammation model was established using A549 cells after LPS stimulation. TMP and FK866 （ a specific inhibitor for pre-B cell colony-enhancing factor） , were added to intervene respectively. Expression level of mRNA, inflammatory factors including tumor necrosis factor-α （TNF-α）, interleukin- 1β （ IL- 1β ）, interleukin- 8 （ IL- 8 ） and PBEF （pre-B cell colony-enhancing factor） were detected by q-PCR and Western blot, respectively. The activation of NF-κB（ Nuclear factor κB） was examined by Western blot to find the changes in phosphorylated P65 protein level in both nucleus and cytoplasm. Results Both the mRNA and protein level of TNF-α, IL-1β, IL-8 and PBEF in A549 cells were significantly higher after LPS stimulation than those in the control group（P 〈 0. 001 ）. Meanwhile, the phosphorylation of P65 protein in the nucleus and cytoplas was higher （ P 〈 0.001 ）. The expression of the aforementioned inflammatory factors and the phosphorylation of P65 protein were significantly lower after TMP intervention than those of LPS group （ P 〈 0. 05 ）. In comparison, after FK866 was added, the expression of TNF-α, IL-1β and IL-8 and the phosphorylation of P65 protein were also decreased（P 〈 0. 01 ）. Conclusions TMP may be involved in the reduction of PBEF expression, which therefore inhibits NF-κB activation, antagonizes alveolar epithelial cell inflammatory response.