阿托伐他汀预处理对脊髓缺血再灌注损伤大鼠损伤脊髓microRNA表达的影响

MicroRNA expression in spinal cord ischemia-reperfusion injury after atorvastatin preconditioning in rats

中文关键词:  脊髓  缺血再灌注损伤  阿托伐他汀  微小RNA

英文关键词:Spinal cord  Ischemia-reperfusion injury  Atorvastatin  MicroRNA

中文摘要:

  【摘要】 目的：检测他汀类药物预处理对脊髓缺血再灌注损伤大鼠损伤脊髓组织中microRNA（miRNA，miR）表达谱的影响，探讨他汀神经保护作用的机制。方法：18只雄性SD大鼠随机分为假手术组、手术组和药物组，每组6只，其中手术组和药物组以阻断腹主动脉的方法建立大鼠急性脊髓缺血再灌注损伤模型，假手术组在开腹后于腹主动脉上绕线而不阻断动脉。药物组大鼠给予胃内灌注10mg/kg/d阿托伐他汀14d后再行建模手术，手术组在建模术前仅给予等容量生理盐水。对各组分别在术后6、12、24和48h应用BBB评分评价脊髓神经功能。在术后48h处死动物，取腰骶段脊髓组织。对组织切片后采用HE染色和2，3，5，-triphenyltetrazolium chloride（TTC）染色明确神经组织病理变化及损伤情况，对缺血面积和脊髓横断面面积行定量检测并计算缺血百分比；应用miRNA芯片检测组织中miRNA表达谱；分别用假手术组和手术组RNA为模板，采用实时定量PCR对芯片结果进行验证。结果：手术组在术后各时间点的BBB评分较假手术组显著下降（P<0.05），药物组在术后各时间点的BBB评分均较手术组明显增高（P<0.05）但仍低于假手术组（P<0.05）。HE染色显示手术组和药物组均存在脊髓缺血性损伤，但药物组较手术组减轻。TTC染色显示假手术组、手术组和药物组的缺血百分比分别为（2.1±0.1）%、（77.3±5.1）%和（43±3.2）%，手术组缺血百分比较假手术组显著增大（P<0.05)，而药物组缺血百分比与手术组比较显著缩小（P＜0.05）但仍显著高于假手术组（P＜0.01）。芯片检测miRNA表达谱显示，手术组与假手术组相比，其中48种miRNA表达明显改变，38种上调1.6~4.9倍，另有10种miRNA表达下调；与手术组相比，药物组有13种miRNA表达发生改变，并可逆转缺血再灌注损伤后的8种miRNA，包括miR-365、miR-323、miR-672*、miR-760-5p、miR-376b-5p、miR-369-5p、miR-210和miR-199a。miRNA表达的实时定量PCR结果和芯片结果比较无显著性差异(P>0.05)。结论：阿托伐他汀预处理可对大鼠脊髓缺血再灌注损伤发挥神经保护作用，其机制可能与其对miRNA的调控有关。

英文摘要:

  【Abstract】 Objectives: To explore microRNA(miRNA, miR) expression in spinal cord ischemia-reperfusion(I/R) injury and the regulatory mechanism involving microRNA with statins precondition in rats. Methods: 18 male Sprague-Dawley(SD) rats were randomly divided into three groups. Experimental groups(n=6 per group) were as follows: sham, control(receiving only normal saline) and atorvastatin-pretreated(10mg/kg/day for 2 weeks before occlusion). Spinal cord I/R injury was induced in SD rats by occluding the descending thoracic aorta in control and atorvastatin-pretreated group, the sham group did not receive aorta occluding in operation. Neurological function was assessed by using BBB scores at 6, 12, 24 and 48h after operation. In 48h after operation, animals were sacrificed. In order to identify pathological changes and damage of the nervous tissue, lumbosacral spinal cord tissues were harvested for hematoxylin-eosin（HE） and 2, 3, 5, -triphenyltetrazolium chloride(TTC) staining, followed by quantitative measurement of ischemic area and cross-sectional area of spinal cord, and then calculation of ischemic percentages. MiRNA profile was also determined from isolated RNA by using miRNA microarrays, followed by validation with quantitative real-time PCR(qRT-PCR) in both sham and control group. Results: The BBB score in control group decreased significantly at each time point compared with ｔｈａｔ ｉｎ sham group(P<0.05). However, atorvastatin pretreatment induced a markedly improved BBB score at each time point compared with control group(P<0.05), but had obviously lower score than sham group(P<0.05 ). Spinal cord ischemic injury was identified by HE staining in both control and atorvastatin-pretreated group. However, the injury in atorvastatin-pretreated group was alleviated compared with ｔｈａｔ ｉｎ control ｇｒｏｕｐ. The ischemic percentage, characterized by pale TTC stains, wａｓ (2.1±0.1)%, (77.3±5.1)% and (43±3.2)% in sham, control and atorvastatin-pretreated group, respectively. The infarction in control ｇｒｏｕｐ increased significantly compared with ｔｈａｔ ｉｎ sham group(P＜0.05). Pretreatment with atorvastatin remarkably attenuated the infarction compared with control(P＜0.05). However, compared with sham group, the infarction in atorvastatin-pretreated group was significantly higher(P＜0.01). MiRNA microarrays showed a total of 48 typeｓ of miRNAs wａｓ significantly different from control group compared with sham group. Aｍｏｎｇ those, 38 miRNAs were up-regulated, with a 1.6-to 4.9 fold change, whereas 10 miRNAs were down-regulated. Compared with control group, a total of 13 types of miRNA levels was significantly different in atorvastatin-pretreated group. I/R injury samples pretreated with atorvastatin reversed the up or down regulation in control of 8 miRNAs, including miR-365, miR-323, miR-672*, miR-760-5p, miR-376b-5p, miR-369-5p, miR-210 and miR-199a. The miRNA expressions validated by qRT-PCR were similar to those in miRNA microarrays(P>0.05). Conclusions: Atorvastatin precondition can protect spinal cord against I/R injury. Altered expression of miRNAs may contribute to the mechanism of neuroprotection of statins in spinal cord I/R injury.