多西环素通过上调自噬调控脂多糖诱导的THP-1细胞炎症因子水平

Doxycycline upregulates autophagy to modulate the levels of inflammatory cytokines in LPS-stimulated THP-1 cells

目的:探讨细胞自噬对多西环素调控脂多糖(LPS)刺激巨噬细胞引发炎症反应的影响及其分子机制。方法:利用LPS刺激人单核/巨噬细胞系THP-1建立炎症反应细胞模型,用多西环素进行干预。通过测定细胞培养上清液中肿瘤坏死因子α(TNF-α)和白细胞介素8(IL-8)等细胞因子评估炎症水平;通过Western blot法检测LC3B、核因子κB(NF-κB)和磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)等蛋白水平评估细胞自噬水平及对炎症细胞信号通路的影响。利用3-甲基腺嘌呤(3-MA)和雷帕霉素分别抑制和促进细胞自噬,研究自噬对多西环素调控LPS刺激THP-1细胞炎症因子水平的影响。结果:LPS刺激THP-1细胞诱导细胞因子TNF-α及IL-8快速释放,12 h左右达到峰值;多西环素有效地抑制了LPS诱导的细胞因子产生(P<0.05)。多西环素增加了LPS诱导的THP-1细胞自噬水平,且多西环素本身是一种自噬诱导剂。LPS上调p-mTOR蛋白水平,而多西环素抑制p-mTOR的蛋白水平,提示多西环素通过mTOR依赖的途径而LPS通过非mTOR依赖的途径诱导自噬。进一步研究显示,联用LPS、雷帕霉素及多西环素抑制了NF-κB的蛋白水平,雷帕霉素增加了多西环素对细胞因子生成的抑制;反之,自噬抑制剂3-MA减弱了多西环素对NF-κB的抑制效应,同时减弱了多西环素对炎症因子生成的抑制效应。结论:自噬参与多西环素调控LPS刺激单核/巨噬细胞引起的炎症反应。

AIM: To analyze the effect of autophagy on inflammatory response regulated by doxycycline in lipopolysaccharide(LPS)-stimulated THP-1 cells and to investigate its molecular mechanism. METHODS: A human monocyte/macrophage cell line THP-1 was stimulated with LPS to establish an cell model of inflammatory response, and the cells were treated with doxycycline. The cytokines, such as tumor necrosis factor-α(TNF-α) and interleukin-8(IL-8), in cell culture supernatant were measured by ELISA for evaluating the inflammatory levels. For determining the level of autophagy and its effect on inflammatory cell signaling pathways, the protein levels of LC3 B, nuclear factor κB(NF-κB) and phosphorylated mammalian target of rapamycin(p-mTOR) were determined by Western blot. 3-Methyladenine(3-MA), an autophagy inhibitor, and rapamycin, an autophagy inducer, were used to study the effect of autophagy on inflammatory response regulated by doxycycline in LPS-stimulated THP-1 cells. RESULTS: The levels of TNF-α and IL-8 were increased rapidly and peaked at 12 h in LPS-stimulated THP-1 cells(P<0.05). Doxycycline significantly inhibited LPS-induced cytokine production in the THP-1 cells. Doxycycline up-regulated LPS-induced autophagy in THP-1 cells and doxycycline itself was an autophagy inducer. The protein levels of p-mTOR was up-regulated by LPS and down-regulated by doxycycline, suggesting that doxycycline induced autophagy via mTOR-dependent pathway while LPS through mTOR-independent pathway. Further studies showed that the combination of LPS, rapamycin and doxycycline inhibited the protein levels of NF-κB, and rapamycin increased the inhibitory effect of doxycycline on cytokine releases. Conversely, 3-MA, the autophagy inhibitor, attenuated the inhibitory effect of doxycycline on NF-κB and cytokine production. CONCLUSION: Autophagy is involved in the process of doxycycline modulating LPS-induced inflammatory response in the THP-1 cells.