南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (8): 1256-1262.doi: 10.12122/j.issn.1673-4254.2022.08.20

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氙气后处理对大鼠脊髓缺血再灌注损伤起保护作用：基于下调mTOR通路和抑制内质网应激介导的神经元凋亡

罗 兰，佟家祺，李 璐，金 沐   

  1. 首都医科大学附属北京友谊医院麻醉科，北京 100050
  • 出版日期:2022-08-20 发布日期:2022-09-05

Xenon post-conditioning protects against spinal cord ischemia-reperfusion injury in rats by downregulating mTOR pathway and inhibiting endoplasmic reticulum stress-induced neuronal apoptosis

LUO Lan, TONG Jiaqi, LI Lu, JIN Mu   

  1. Department of Anesthesiology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China
  • Online:2022-08-20 Published:2022-09-05

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摘要： 目的 基于哺乳动物雷帕霉素靶蛋白（mTOR）通路和内质网应激（ERS）-凋亡通路探讨氙气后处理治疗大鼠脊髓缺血再灌注损伤（SCIRI）的作用机制。方法 将50只雄性SD大鼠随机分为5组：假手术组（Sham组），脊髓缺血再灌注组（I/R组），氙气后处理组（Xe组），脊髓缺血再灌注+雷帕霉素组（I/R+Rapa组），氙气后处理+雷帕霉素组（Xe+Rapa组）（10 只/组）。通过夹闭腹主动脉85 min，再灌注4 h建立大鼠SCIRI模型。手术前3 d，I/R+Rapa组和Xe+Rapa组的大鼠每天予以腹腔注射雷帕霉素（Rapa， 4 mg/kg），其余3组大鼠注射等体积的溶剂。缺血再灌注后1 h时，Xe组和Xe+Rapa组的大鼠经呼吸机吸入50%氙气+50%氧气混合气1 h。其余3组的大鼠予以50% 氮气+50%氧气混合气1 h。于再灌注4 h观察记录大鼠后肢运动功能后收集标本，进行HE染色、尼氏染色计数正常神经元数量，Western blot和RT-PCR方法分别检测脊髓组织中内质网应激通路［葡萄糖调节蛋白78（GRP78）、激活转录因子6（ATF6）、肌醇需要酶1α（IRE1α）、RNA依赖的蛋白激酶样ER激酶（PERK）］、mTOR通路（mTOR和磷酸化mTOR（p-mTOR））以及凋亡信号（Bax、Bcl-2和Caspase-3）蛋白和mRNA表达。结果 与 Sham组相比，其余各组大鼠后肢运动功能评分显著降低（P<0.01），正常神经元数量减少；I/R组的GRP78、ATF6、IRE1α、PERK mRNA含量和蛋白水平明显升高（P<0.05或0.01），p-mTOR/mTOR比值、Bax/Bcl-2的比值及Caspase-3 mRNA含量和蛋白水平显著升高（P<0.01）。与I/R组相比，Xe组的GRP78、ATF6、IRE1α、PERK mRNA含量和蛋白水平明显降低（P<0.01），p-mTOR/ mTOR比值、Bax/Bcl-2的比值及Caspase-3 mRNA含量和蛋白水平显著下降（P<0.05或0.01）；I/R+Rapa组的GRP78、ATF6 mRNA含量和蛋白水平明显降低（P<0.01），p-mTOR/mTOR蛋白的比值、Bax/Bcl-2 mRNA的比值及Caspase-3含量和蛋白水平显著下降（P<0.01）。与Xe组相比，I/R+Rapa组和Xe+Rapa组的BBB评分和Tarlov评分明显降低（P<0.01）；Xe+Rapa组Caspase-3蛋白表达和Bax/Bcl-2 mRNA的比值显著见下降（P<0.05或0.01）。结论 氙气后处理通过抑制内质网应激和神经元凋亡对大鼠脊髓缺血再灌注损伤起保护作用，而mTOR通路部分介导了氙气后处理的脊髓保护作用。

关键词: 脊髓缺血再灌注损伤；氙气后处理；mTOR；内质网应激；凋亡

Abstract: Objective The purpose of this study was to determine whether xenon post-conditioning affects mTOR signaling as well as endoplasmic reticulum stress (ERS)-apoptosis pathway in rats with spinal cord ischemia/reperfusion injury. Methods Fifty male rats were randomized equally into sham-operated group (Sham group), I/R model group (I/R group), I/R model + xenon post-conditioning group (Xe group), I/R model+rapamycin (a mTOR signaling pathway inhibitor) treatment group (I/R+ Rapa group), and I/R model + xenon post- conditioning with rapamycin treatment group (Xe + Rapa group). In the latter 4 groups, SCIRI was induced by clamping the abdominal aorta for 85 min followed by reperfusion for 4 h. Rapamycin (or vehicle) was administered by daily intraperitoneal injection (4 mg/kg) for 3 days before SCIRI, and xenon post-conditioning by inhalation of 1∶1 mixture of xenon and oxygen for 1 h at 1 h after initiation of reperfusion; the rats without xenon post-conditioning were given inhalation of nitrogen and oxygen (1∶1). After the reperfusion, motor function and histopathologic changes in the rats were examined. Western blotting and real-time PCR were used to detect the protein and mRNA expressions of GRP78, ATF6, IRE1α, PERK, mTOR, p-mTOR, Bax, Bcl-2 and caspase-3 in the spinal cord. Results The rats showed significantly lowered hind limb motor function following SCIRI (P<0.01) with a decreased count of normal neurons, increased mRNA and protein expressions of GRP78, ATF6, IRE1α, PERK, and caspase-3, and elevated p-mTOR/mTOR ratio and Bax/Bcl-2 ratio (P<0.01). Xenon post-conditioning significantly decreased the mRNA and protein levels of GRP78, ATF6, IRE1α, PERK and caspase-3 (P<0.05 or 0.01) and reduced p-mTOR/mTOR and Bax/Bcl-2 ratios (P<0.01) in rats with SCIRI; the mRNA contents and protein levels of GRP78 and ATF6 were significantly decreased in I/R +Rapa group (P< 0.01). Compared with those in Xe group, the rats in I/R+Rapa group and Xe+Rapa had significantly lowered BBB and Tarlov scores of the hind legs (P<0.01), and caspase-3 protein level and Bax/Bcl-2 ratio were significantly lowered in Xe+Rapa group (P<0.05 or 0.01). Conclusion By inhibiting ERS and neuronal apoptosis, xenon post-conditioning may have protective effects against SCIRI in rats. The mTOR signaling pathway is partially involved in this process.

Key words: spinal cord ischemia/reperfusion injury; xenon post-conditioning; mTOR; endoplasmic reticulum stress; apoptosis