氢溴酸山莨菪碱对敌草快诱导的小鼠巨噬细胞的保护作用及其机制探讨

Effects and mechanisms of Ani-HBr on DQ-induced injury in murine macrophages

摘要:

目的 探讨氢溴酸山莨菪碱（Ani-HBr）对敌草快（DQ）诱导的小鼠腹腔巨噬细胞（PMs）损伤的保护作用及其机制。方法 选取PMs进行培养，用DQ刺激PMs 12 h后，用不同浓度的Ani-HBr（10、20 μg/mL）干预24 h。采用CCK-8法检测细胞存活率；试剂盒检测ATP、ROS水平；流式细胞术分析巨噬细胞极化M1（F4/80CD86）和M2（F4/80CD206）比例；Western blotting检测线粒体生物合成核心因子核呼吸因子1（NRF1）、线粒体转录因子A（TFAM）、M1型［白细胞介素-6（IL-6）、IL-1β］和M2型［转化生长因子-β（TGF-β）、IL-10］巨噬细胞分泌细胞因子的表达，以及NF-κB通路关键蛋白P65及其磷酸化形式p-P65的表达。结果 DQ组细胞存活率低于Control组（P <0.05），DQ+Ani-HBr-10组、DQ+Ani-HBr-20组细胞存活率高于DQ组（P <0.05），且DQ+Ani-HBr-20组细胞存活率高于DQ+Ani-HBr-10组（P <0.05）。DQ组ATP水平低于Control组（P <0.05），ROS水平高于Control组（P <0.05），DQ+Ani-HBr-10组、DQ+Ani-HBr-20组ATP水平均高于DQ组（P <0.05），ROS水平低于DQ组（P <0.05）。DQ组NRF1、TFAM蛋白相对表达量均低于Control组（P <0.05），DQ+Ani-HBr-10组、DQ+Ani-HBr-20组NRF1、TFAM蛋白相对表达量均高于DQ组（P <0.05）。DQ组P65、p-P65蛋白相对表达量均高于Control组（P <0.05），DQ+Ani-HBr-10组、DQ+Ani-HBr-20组P65、p-P65蛋白相对表达量均低于DQ组（P <0.05）。DQ组M1型巨噬细胞比例高于Control组（P <0.05），M2型巨噬细胞比例低于Control组（P <0.05），DQ+Ani-HBr-10组、DQ+Ani-HBr-20组M1型巨噬细胞比例均低于DQ组（P <0.05），M2型巨噬细胞比例均高于DQ组（P <0.05）。DQ组IL-6、IL-1β蛋白相对表达量均高于Control组（P <0.05），TGF-β和IL-10蛋白相对表达量均低于Control组（P <0.05）。DQ+Ani-HBr-10组、DQ+Ani-HBr-20组IL-6、IL-1β蛋白相对表达量均低于DQ组（P <0.05），TGF-β和IL-10蛋白相对表达量均高于DQ组（P <0.05）。结论 Ani-HBr对DQ诱导的PMs损伤具有保护作用，其机制可能与上调NRF1/TFAM蛋白表达、改善线粒体功能、抑制NF-κB通路激活，以及调节巨噬细胞极化平衡有关。

Abstract:

Objective To investigate protective effects of anisodamine hydrobromide (Ani-HBr) against diquat (DQ)-induced injury in murine peritoneal macrophages (PMs) and to explore the underlying mechanisms.Methods PMs were cultured and stimulated with DQ for 12 h, followed by intervention with different concentrations of Ani-HBr (10 μg/mL and 20 μg/mL) for 24 h. Cell viability was measured by the CCK-8 assay. ATP and ROS levels were detected using commercial kits. Macrophage polarization was analyzed by flow cytometry using M1 (F4/80CD86) and M2 (F4/80CD206) markers. Western blotting was performed to determine the expressions of mitochondrial biogenesis factors nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM), M1-type cytokines [interleukin-6 (IL-6) and interleukin-1β (IL-1β) ], M2-type cytokines [transforming growth factor beta (TGF-β) and interleukin-10 (IL-10) ], and proteins associated with the NF-κB pathway including P65 and phosphorylated P65 (p-P65).Results Cell viability was significantly lower in the DQ group than in the control group (P < 0.05). Compared with the DQ group, cell viability was significantly increased in both the DQ+Ani-HBr-10 and DQ+Ani-HBr-20 groups (P < 0.05). In addition, cell viability was higher in the DQ+Ani-HBr-20 group than in the DQ+Ani-HBr-10 group (P < 0.05). ATP levels were significantly lower, and ROS levels were significantly higher in the DQ group than in the control group (both P < 0.05). Compared with the DQ group, ATP levels were significantly increased, and ROS levels were significantly decreased in the DQ+Ani-HBr-10 and DQ+Ani-HBr-20 groups (all P < 0.05). The expression levels of NRF1 and TFAM were significantly lower in the DQ group than in the control group (P < 0.05), whereas both were significantly increased in the DQ+Ani-HBr-10 and DQ+Ani-HBr-20 groups compared with the DQ group (P < 0.05). The expression levels of P65 and p-P65 were significantly higher in the DQ group than in the control group (P < 0.05), while both were significantly lower in the DQ+Ani-HBr-10 and DQ+Ani-HBr-20 groups than in the DQ group (P < 0.05). The proportion of M1 macrophages was significantly increased and the proportion of M2 macrophages was significantly decreased in the DQ group compared with the control group (P < 0.05). Compared with the DQ group, the proportions of M1 macrophages were significantly decreased and the proportions of M2 macrophages were significantly increased in the DQ+Ani-HBr-10 and DQ+Ani-HBr-20 groups (P < 0.05). The relative protein expression levels of IL-6 and IL-1β were significantly higher, whereas those of TGF-β and IL-10 were significantly lower, in the DQ group than in the control group (P < 0.05). Compared with the DQ group, the relative protein expression levels of IL-6 and IL-1β were significantly decreased, while those of TGF-β and IL-10 were significantly increased, in the DQ+Ani-HBr-10 and DQ+Ani-HBr-20 groups (P < 0.05).Conclusion Ani-HBr exerts significant protective effects against DQ-induced injury in PMs, which may be associated with upregulation of NRF1/TFAM expression, improvement of mitochondrial function, inhibition of NF-κB pathway activation, and modulation of macrophage polarization balance.