青蒿琥酯抑制巨噬细胞释放前炎症细胞因子的分子作用机制研究

Molecular mechanism of artesunate attenuates the release of proinflammatory cytokines from macrophages

目的:革兰阴性菌细胞膜上的脂多糖(lipopolysaccharide,LPS)与重症急性胰腺炎(severe acute pancreatitis,SAP)的发生发展密切相关。局部和全身单核细胞/巨噬细胞在SAP炎症过程中起着重要作用。青蒿琥酯(artesunate,AS)通过减少前炎症细胞因子的释放来保护重症急性胰腺炎大鼠。本研究进一步探讨AS抗炎作用的分子机制。方法:采用酶联免疫吸附试验研究上清液中前炎症细胞因子的释放水平;实时定量荧光PCR方法检测PI3K-Ⅲ及其信号通路关键分子m RNA的表达。最后,通过免疫印迹法检测PI3K-Ⅲ的磷酸化水平,观察AS发挥抗炎作用的分子机制与PI3K-Ⅲ磷酸化水平之间的联系。结果:与LPS(100 ng/mL)组相比,LPS+AS组能显著抑制LPS诱导的小鼠巨噬细胞释放前炎症细胞因子;LPS+3-甲基腺嘌呤(3-methyladenine,3-MA)组能显著抑制LPS诱导小鼠巨噬细胞释放TNF-α。与Medium组相比,LPS组能显著增加小鼠腹腔巨噬细胞中PI3K-Ⅲ及其关键分子的mRNA表达。最后,与LPS(100 ng/mL)组相比,LPS+AS组能显著抑制LPS诱导的小鼠腹腔巨噬细胞中PI3K-Ⅲ磷酸化水平的增加。结论:AS的抗炎机制与抑制PI3K-Ⅲ磷酸化水平密切相关。

AIM:Lipopolysaccharide(LPS)on the cell membrane of gram negative bacteria is closely related to the occurrence and development of severe acute pancreatitis(SAP).Local and systemic monocyte/macrophages play an important role in the inflammatory process of SAP.Artesunate(AS)was reported to protect rats with severe acute pancreatitis by reducing the release of proinflammatory cytokines.This study further explored the molecular mechanism of anti-inflammatory effect of AS.METHODS:The release of proinflammatory cytokines in the supernatant were studied by enzymelinked immunosorbent assay.Then,the mRNA expressions of PI3K-III and its key molecules in signaling pathway were detected by real-time quantitative PCR.Finally,the phosphorylation levels of PI3K-Ⅲwere detected by Western blot.RESULTS:AS could significantly inhibit the release of proinflammatory cytokines from mouse macrophage induced by LPS.Autophagy inhibitor 3-methyladenine(3-MA)could significantly inhibit the release of TNF-αfrom mouse macrophages induced by LPS;LPS significantly increased the mRNA expression of PI3K-Ⅲand its key molecules in mouse peritoneal macrophages(PMs).Finally,AS could significantly inhibit the increase of PI3K-Ⅲ phosphorylation induced by LPS in PMs.CONCLUSION:The anti-inflammatory mechanism of AS is closely related to the inhibition of PI3K-Ⅲ phosphorylation.