基于CXCR4-FAK信号通路观察鞘内注射右美托咪定改善大鼠脊髓缺血再灌注损伤后神经运动功能

Intrathecal injection of dexmedetomidine improves neuromotor function in rats with spinal cord ischemia-reperfusion injury based on the CXCR4-FAK signaling pathway

目的基于CXC趋化因子受体4-粘着斑激酶(CXCR4-FAK)信号通路探讨鞘内注射右美托咪定(DHI)改善大鼠脊髓缺血再灌注损伤(SCIRI)后神经运动功能。方法将60只10~12周龄的SPF级SD雄性大鼠随机分为4组(n=15):sham组、模型组、DHI组、DPA组。模型组、DHI组、DPA组开胸夹闭主动脉弓15 min构建SCIRI模型,sham组仅开胸游离主动脉弓,不夹闭处理。DHI组、DPA组大鼠在缺血前72 h鞘内分别注射DHI和Diprotin A,注射剂量30μL,浓度5μmol/L,sham组、模型组注射等量生理盐水,每日定点注射1次,连续3 d。造模后第4天采用改良的Tarlov评分方法、伊文思蓝(EB)染色、干湿法、电镜、TUNEL染色、DCFH-DA染色及试剂盒、Western blot检测各组大鼠神经运动功能评分、血-脊髓屏障(BSCB)结构完整性、脊髓组织含水量、脊髓组织超微结构改变、细胞凋亡、氧化应激反应以及CXCR4、p-FAK蛋白的表达水平。结果与sham组相比,模型组大鼠神经运动功能评分、超氧化物歧化酶(SOD)活性明显降低,脊髓组织的含水量、EB红色荧光强度、细胞凋亡率、活性氧(ROS)绿色荧光强度、丙二醛(MDA)的含量、CXCR4、p-FAK的表达水平明显升高;与模型组相比,DHI组、DPA组大鼠脊髓组织神经运动功能评分、SOD酶活性、CXCR4、p-FAK的表达水平明显升高,脊髓组织的含水量、EB红色荧光强度、细胞凋亡率、ROS绿色荧光强度、MDA的含量明显降低;差异均具有统计意义(P<0.05);DHI组和DPA组相比,差异不明显(P>0.05),不具有统计学意义。结论右美托咪定对SCIRI大鼠的保护作用,可能是通过激活CXCR4-FAK信号通路,降低细胞的氧化应激损伤,以发挥对SCIRI大鼠脊髓组织的保护作用。

Objective To investigate whether the protective mechanism by which intrathecal injection of dexmedetomidine improves neuromotor function in rats with spinal cord ischemia-reperfusion injury is based on the CXCR4-FAK signaling pathway.Methods Sixty SPF-grade SD male rats aged 10-12 weeks were randomly divided into four groups(n=15 per group):the sham group,model group,DHI group,and DPA group.The SCIRI model was constructed in the model,DHI,and DPA groups by opening the thoracic clipped aortic arch for 15 min.In the sham group,the thoracic free aortic arch was only opened,without clamping.After successful establishment of the model,each rat was fed in a single cage with conventional feed and a standard diet.The DHI and DPA groups were injected in the sheath 72 h before ischemia with DHI and Diprotin A,respectively,with a dose of 30μL at a concentration of 5μmol/L.The sham and model groups were injected with the same volume of normal saline,with a fixed point injection once daily for 3 consecutive days.On day4 after modeling,the neuromotor function score,blood-spinal cord barrier structural integrity,water content,ultrastructural changes,apoptosis,oxidative stress response,and the expression levels of CXCR4 and p-FAK were detected using the improved Tarlov scoring method,Evans blue(EB)staining,wet/dry weight method,electron microscopy,TUNEL staining,DCFH-DA staining kit,and western blot techniques,respectively.Results Compared with the sham group,the neuromotor function score and SOD activity of the model group were significantly decreased.In contrast,the water content of the spinal cord,EB red fluorescence intensity,apoptosis rate,ROS green fluorescence intensity,MDA content,and CXCR4 and p FAK expression levels were significantly increased.Compared with the model group,the neuromotor function score,SOD activity,and CXCR4 and p FAK expression levels in the spinal cord were significantly increased in the DHI and DPA groups.In contrast,the water content of the spinal cord,EB red fluorescence intensity,apoptosis rate,the green fluorescence intensity of ROS,and MDA content were significantly lower,and these differences were statistically significant(P<0.05).There were no significant differences between the DHI and DPA groups(P>0.05).Conclusions The protective effect of dexmedetomidine on SCIRI rats may be caused by the activation of the CXCR4-FAK signaling pathway,thus reducing oxidative stress injury in cells,and playing a protective role in the spinal cord of SCIRI rats.