天津医药

天津医药 ›› 2019, Vol. 47 ›› Issue (7): 705-708.doi: 10.11958/20190390

• 实验研究 • 上一篇    下一篇

mTOR-自噬通路对脓毒症小鼠海马小胶质细胞表型的影响

庄欣琪，蒋毅，杨曼 ，王瑶琪 ，卢悦淳，吕国义 ，谢克亮，于泳浩   

  1. 1天津医科大学总医院麻醉科，天津市麻醉学研究所（邮编 300052）；2天津医科大学第二医院麻醉科
  • 收稿日期:2019-02-13 修回日期:2019-04-09 出版日期:2019-07-15 发布日期:2019-08-01
  • 通讯作者: 庄欣琪 E-mail:zhuang_xinqi@163.com

The role of mTOR-autophagy pathway on microglia phenotype in hippocampus of septic mice

ZHUANG Xin-qi,JIANG Yi,YANG Man,WANG Yao-qi,LU Yue-chun,LYU Guo-yi,XIE Ke-liang,YU Yong-hao   

  1. 1 Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin Institute of Anesthesiology,Tianjin 300052, China; 2 Department of Anesthesiology, the Second Hospital of Tianjin Medical University
  • Received:2019-02-13 Revised:2019-04-09 Published:2019-07-15 Online:2019-08-01
  • Contact: Xinqi Zhuang E-mail:zhuang_xinqi@163.com

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摘要： 目的 探讨哺乳动物雷帕霉素靶蛋白（mTOR）-自噬通路对脓毒症小鼠海马小胶质细胞表型的影响。方法 54 只雄性 ICR 小鼠，按照随机数字表法分为 3 组（n=18）：假手术组（Sham 组）、脓毒症组（CLP 组）和脓毒症+mTOR抑制剂雷帕霉素组（CLP+Ra组）。Sham组只进行开腹手术操作；CLP组采用盲肠结扎穿孔术制备脓毒症小鼠模型；CLP+Ra 组于造模前 6 h腹腔注射雷帕霉素（1.5 mg/kg）。于造模后 24 h各组取 6只，采用酶联免疫吸附试验（ELISA）法检测海马组织肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、IL-10和转化生长因子（TGF）-β水平；采用免疫荧光染色法检测海马组织切片离子钙接头蛋白分子（Iba）-1/CD86和 Iba-1/CD206阳性细胞数量；采用蛋白免疫印迹法测定海马组织 mTOR磷酸化（p-mTOR）水平、自噬相关蛋白微管相关蛋白 1轻链 3Ⅱ（LC3Ⅱ）和 Beclin-1表达情况。结果 与 Sham组比较，CLP组 TNF-α、IL-1β、IL-10和 TGF-β水平升高，Iba-1/CD86和 Iba-1/CD206阳性细胞数量增加，p-mTOR水平上调，LC3Ⅱ和 Beclin-1表达下调（均 P＜0.05）。与 CLP组比较，CLP+Ra组 TNF-α和 IL-1β水平降低，IL-10和 TGF-β水平升高，Iba-1/CD86阳性细胞数量减少，Iba-1/CD206阳性细胞数量增加，p-mTOR水平下调，LC3Ⅱ和 Beclin-1表达上调（均 P＜0.05）。结论 mTOR-自噬通路通过调节小胶质细胞表型转化，影响脓毒症小鼠海马炎症反应。

关键词: 脓毒症, 脑, 海马, 小神经胶质细胞, 自噬, 哺乳动物雷帕霉素靶蛋白

Abstract: Objective To investigate the role of mammalian target of rapamycin (mTOR) - autophagy pathway on microglia phenotype in hippocampus of septic mouse model. Methods Fifty-four male ICR mice were randomly allocated into three groups (n=18 for each group): Sham operation group (group Sham), cecal ligation and puncture group (group CLP)and CLP plus mTOR inhibitor rapamycin group (group CLP+Ra). Only laparotomy was performed in group Sham. Sepsis mouse model was performed by cecal ligation and puncture in group CLP. Rapamycin 1.5 mg / kg was injected intraperitoneally 6 hours before cecal ligation and puncture in group CLP+Ra. The enzyme-linked immunosorbent assay (ELISA) was conducted to assess the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-10 and transforming growth factor (TGF)-β in hippocampus. Immunofluorescence was used to calculate the number of ionized calcium binding adapter molecule (Iba) - 1 / CD86 and Iba-1 / CD206 positive microglia in hippocampal sections. The expression levels of phosphorylated mTOR (p-mTOR), microtubule associated protein 1 light chain 3Ⅱ (LC3Ⅱ) and Beclin-1 in hippocampus were detected by Western blot assay. Results Compared with group Sham, the levels of TNF-α, IL-1β, IL-10, TGF-β, the number of Iba-1 / CD86 and Iba-1 / CD206 positive microglia and the expression of p-mTOR were increased while the expression of LC3Ⅱ and Beclin-1 were decreased in group CLP (P＜0.05). Compared with group CLP, the levels of IL-10,TGF-β, the number of Iba-1/CD206 positive microglia and the expression of LC3Ⅱ and Beclin-1 were increased while the levels of TNF-α, IL-1β, the number of Iba-1/CD86 positive microglia and the expression of p-mTOR were decreased in group CLP+Ra (P＜0.05). Conclusion mTOR-autophagy pathway affects hippocampal inflammation in septic model mice by regulating microglial phenotype transformation.

Key words: sepsis, brain, hippocampus, microglia, autophagy, mTOR protein