右美托咪定减轻兔脊髓缺血再灌注损伤的机制研究*

Therapeutic effects and mechanisms of dexmedetomidine on spinal cord ischemia-reperfusion injury in rabbits*

摘要:

目的 探讨右美托咪定（Dex）对兔脊髓缺血再灌注损伤（SCIRI）的保护作用及潜在分子机制。方法 采用成年新西兰大耳白兔复制SCIRI模型。实验一：42只兔随机分为7组，分别于灌注前（0?h）及再灌注后3?h、6?h、12?h、24?h、36?h及48?h 7个不同时间点，用Western blotting法检测脊髓组织磷酸化的Janus激酶2（p-JAK2）、磷酸化的信号转导与转录激活因子3（p-STAT3）的表达。实验二：36只兔随机分为对照组（Sham组）、缺血再灌注组（I/R组）及Dex干预组（Dex+I/R组）。Sham组分离腹主动脉后，不做缺血处理。I/R组和Dex+I/R组，分离腹主动脉并夹闭30?min，以诱导脊髓组织缺血。脊髓缺血前30?min，Dex+I/R组静脉给予Dex [10?μg/（kg·h），直至再灌注后1?h。再灌注后48?h对兔后肢运动功能进行Tarlov评分，苏木精-伊红（HE）染色观察脊髓组织病理学改变，原位末端转移酶标记（TUNEL）检测神经细胞凋亡情况，伊文思蓝检测血-脑屏障的渗透性，Western blotting法检测脊髓组织p-JAK2、p-STAT3、活化型半胱天冬酶-3（Cleaved caspase-3）、肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）的表达。结果 实验一：与灌注前（0?h）比较， 再灌注后6?h、12?h、24?h及36?h脊髓组织p-JAK2、p-STAT3的表达增加（均P?<0.05），并于24?h到达峰值。实验二：再灌注后48?h，Dex+I/R组的Tarlov评分增加（P?<0.05），脊髓组织损伤减轻；Dex处理能够降低脊髓神经细胞凋亡率（P?<0.05），并减少脊髓组织中伊文思蓝的渗透量（P?<0.05）；Dex上调p-JAK2、p-STAT3的表达（P?<0.05），并下调Cleaved caspase-3、TNF-α和IL-6的表达（P?<0.05）。结论 再灌注阶段，存在JAK2/STAT3保护性通路的激活，但这种激活作用并未维持；Dex可通过激活JAK2/STAT3信号通路、抑制凋亡蛋白（Cleaved Caspase-3）和炎症因子（TNF-α，IL-6）表达，从而减少神经细胞凋亡并维持血-脑屏障的完整性，减轻SCIRI。

Abstract:

Objective To investigate the role of a combination of per- and post-conditioning of dexmedetomidine (Dex) in rabbits with spinal cord ischemia-reperfusion injury (SCIRI). Methods Adult New Zealand white rabbits were used to replicate the SCIRI model. Experiment 1: Animals (42) were randomly divided into seven groups to evaluate the time-course expression of p-JAK2 and p-STAT3 after reperfusion. Experiment 2: Animals (36) were randomly divided into three groups: Sham, I/R and Dex+I/R. For the I/R group and I/R+DEX group, the abdominal aorta of the rabbits was clamped for 30 min to establish the model of SCIRI, while the rabbits in the Sham group only undergoing arterial dissociation. Dex (10?μg/(kg·h) was administered intravenously 30 min before ischemia until 1?h after reperfusion. Hind limb motor function was assessed using the modified Tarlov scale score at 48?h after reperfusion. Histopathological changes and neuronal apoptosis in the ventral grey matter were assessed by hematoxylin-eosin (HE) staining and TdT-mediated dUTP nick end labeling (TUNEL). The permeability of the blood-spinal cord barrier (BSCB) was examined via Evans blue (EB) extravasation. The protein expressions of p-JAK2, p-STAT3, Cleaved Caspase-3, TNF-α, and IL-6 were detected by Western blotting. Results Experiment 1: Compared with the 0?h group, the expression of p-JAK2 and p-STAT3 in the spinal cord tissue were increased at 6?h, 12?h, 24?h, and 36?h after reperfusion (all P?