HIF-1α/BNIP3信号通路对BCG诱导巨噬细胞自噬的影响

Role of HIF-1α/BNIP3 signaling pathway in regulating macrophage autophagy induced by BCG infection

摘要:

【背景】缺氧诱导因子1α(hypoxia-inducible factor 1-alpha，HIF-1α)是响应细胞低氧反应的关键因子，在红细胞生成、血管形成、能量代谢及调节宿主免疫代谢中发挥着重要作用。【目的】探讨HIF-1α/Bcl-2-腺病毒E1B相互作用蛋白3(Bcl-2-adenovirus E1B 19-kDa interacting protein 3，BNIP3)信号通路对牛分枝杆菌卡介苗(Bacillus Calmette-Guérin，BCG)诱导小鼠巨噬细胞RAW 264.7自噬的影响。【方法】构建HIF-1α的小干扰RNA (siHIF-1α)，转染RAW 264.7细胞后，结合BCG感染，采用流式细胞仪检测细胞自噬率，用Western blotting或免疫荧光技术检测HIF-1α、BNIP3、LC3、Beclin 1、Rheb和mTOR的表达水平。【结果】BCG感染显著上调巨噬细胞中LC3和HIF-1α的表达，用siHIF-1α结合BCG感染后显著下调巨噬细胞中HIF-1α、BNIP3、LC3、Beclin 1和细胞自噬率水平，并促进Rheb和p-mTOR的表达。【结论】在BCG感染RAW 264.7细胞过程中，干扰HIF-1α表达抑制了HIF-1α/BNIP3信号通路，进而激活了mTOR途径，抑制BCG感染诱导的细胞自噬。

Abstract:

[Background] Hypoxia-inducible factor 1-alpha (HIF-1α) is a key factor in response to cellular hypoxia and plays an important role in erythropoiesis, angiogenesis, energy metabolism and regulation of host immune metabolism.[Objective] To investigate the effects of HIF-1α/Bcl-2-adenovirus E1B 19-kDa interacting protein 3 (BNIP3) signaling pathway on the BCG infection-induced autophagy of macrophage RAW 264.7 cells. [Methods] Small interfering RNA of HIF-1α, siHIF-1α, was constructed, and after the transfection of siHIF-1α and/or Bacillus Calmette-Guerin (BCG) infection of RAW 264.7 cells, the autophagy rate of the cells was detected by flow cytometer. Western blotting or immunofluorescence technique was employed to determine the protein levels of HIF-1α, BNIP3, LC3, Beclin 1, Rheb and mTOR.[Results] BCG infection up-regulated the expression of LC3 and HIF-1α in RAW 264.7 cells. The transfection of siHIF-1α down-regulated the levels of HIF-1α, BNIP3, LC3, and Beclin 1 and decreased the autophagy rate of the macrophages after BCG infection. Moreover, siHIF-1α promoted the expression of Rheb and p-mTOR.[Conclusion] Our results indicated that the knockdown of HIF-1α inhibited the HIF-1α/BNIP3 signaling pathway, thereby activating the mTOR pathway and inhibiting autophagy in RAW 264.7 cells after BCG infection.