神经调节素对缺血再灌注损伤大鼠脊髓MMP-9和TIMP-1表达的影响

Effect of Neuregulin on the expression of MMP-9 and TIMP-1 in spinal cord ischemia reperfusion injury of rats

中文关键词:  脊髓损伤  神经调节素  缺血再灌注  基质金属蛋白酶-9  金属蛋白酶组织抑制剂-1  大鼠

英文关键词:Spinal cord injury  Neuregulin  Ischemia reperfusion  Matrix metalloproteinases-9  Tissue inhibitor of metalloproteinase-1  Rat

中文摘要:

  【摘要】 目的：观察神经调节素-1β（neuregulin-1β，NRG-1β）对大鼠脊髓缺血再灌注损伤后脊髓组织基质金属蛋白酶-9（MMP-9）、金属蛋白酶组织抑制剂-1（TIMP-1）表达的影响，并初步探讨其作用与机制。方法：48只SD大鼠随机分为对照组（n=16）、缺血再灌注组（模型组，n=16）、NRG-1β治疗组（n=16），对照组仅分离暴露腹主动脉，其余两组采用腹主动脉阻断法制备动物模型。治疗组在打开动脉夹后立即经尾静脉注射NRG-1β（10μg/kg），缺血再灌注模型组在打开动脉夹后立即经尾静脉注射等量的0.1mol/L的PBS缓冲液。于取材前3min根据改良Tarlov评分标准进行神经功能评分。分别于术后3h、6h、12h、24h取材（每个时间点4只），HE染色观察脊髓病理变化，免疫组化和Real-time PCR分别检测MMP-9、TIMP-1蛋白和mRNA水平的表达。结果：模型组在术后各时间点的Tarlov评分较对照组显著下降（P＜0.05），治疗组在术后6h、12h、24h的Tarlov评分较模型组显著增高（P＜0.05）。HE染色显示模型组和治疗组均存在脊髓组织损伤，但治疗组较模型组减轻。免疫组化结果显示，对照组未见MMP-9、TIMP-1阳性表达细胞；模型组MMP-9免疫阳性细胞数（个/高倍视野）在术后3h、6h、12h、24h分别为9.00±1.63、23.80±1.71、28.30±1.50、34.80±2.63，治疗组分别为8.50±0.58、17.80±0.96、20.80±3.50、30.00±2.16，其中6h、12h、24h治疗组与模型组相比阳性细胞数明显减少（P＜0.05）。模型组TIMP-1免疫阳性细胞数（个/高倍视野）在术后3h、6h、12h、24h分别为11.80±0.96、12.30±1.50、7.80±0.96、7.80±1.50，治疗组分别为12.30±0.96、13.80±0.96、10.50±1.73、10.30±0.96，其中12h、24h治疗组与模型组相比阳性细胞数明显增多（P＜0.05）。对照组MMP-9在术后3h、6h、12h、24h的mRNA表达量分别为4.93±0.21、4.95±0.24、4.96±0.25、4.98±0.23，模型组分别为5.38±0.25、6.53±0.31、6.87±0.29、7.53±0.33，治疗组分别为5.35±0.26、5.56±0.22、5.74±0.27、5.90±0.31，其中6h、12h、24h模型组与对照组比较MMP-9的mRNA表达量明显增加（P＜0.05），6h、12h、24h治疗组MMP-9的mRNA表达量较模型组明显减少（P＜0.05）。对照组TIMP-1在术后3h、6h、12h、24h的mRNA表达量分别为4.74±0.23、4.76±0.21、4.73±0.25、4.76±0.24，模型组分别为4.53±0.32、4.62±0.21、3.83±0.20、3.65±0.32，治疗组分别为4.55±0.26、4.65±0.27、4.28±0.22、4.25±0.24，其中12h、24h模型组与对照组比较TIMP-1的mRNA表达量明显减少（P＜0.05），12h、24h治疗组TIMP-1的mRNA表达量较模型组明显增加（P＜0.05）。结论：神经调节素可在大鼠脊髓缺血再灌注损伤过程中发挥神经保护作用，该作用的实现可能与MMP-9的表达下调和TIMP-1的表达上调有关。

英文摘要:

  【Abstract】 Objectives: Objectives: To observe the effect of Neuregulin-1β(NRG-1β) on the expression of matrix metalloproteinase-9(MMP-9) and tissue inhibitor of metalloproteinase-1(TIMP-1) in rats with spinal cord ischemia reperfusion injury, and to investigate its function and mechanism. Methods: 48 SD rats were randomly divided into control group(n=16), ischemia reperfusion model group(n=16), NRG-1β treatment group(n=16). Abdominal aorta was only exposed in control group. Spinal cord ischemia reperfusion injury was in?鄄duced by occluding the abdominal aorta in the other two groups. The NRG-1β(10μg/kg) was injected through tail vein immediately after opening the artery clamp in the treatment group, and the ischemia reperfusion model group was injected with an equal amount of 0.1mol/L PBS buffer solution after opening the artery clamp. Neurological function was assessed by using the modified Tarlov standard. Sampling test was performed at 3h, 6h, 12h, 24h respectively after injury(4 rats at each time point). The pathological changes were observed by HE staining. Protein and mRNA expressions of MMP-9 and TIMP-1 were assessed by immunohistochemistry and real-time PCR. Results: The Tarlov scorein model group decreased significantly at each time point compared with that in control group(P<0.05). Neuregulin treatment group induced a markedly improved Tarlov score at 6h, 12h, 24h compared with model group(P<0.05). Spinal cord injury was identified by HE staining in both model and treatment group. However, the injury in treatment group was alleviated compared with that in model group. Immunohistochemistry result showed that there was no positive expression of MMP-9 or TIMP-1 in control group. The number of MMP-9 positive cells in 3h, 6h, 12h, 24h model group was 9.00±1.63, 23.80±1.71, 28.30±1.50, 34.80±2.63 respectively, which was 8.50±0.58, 17.80±0.96, 20.80±3.50, 30.00±2.16 respectively in treatment group. The number of positive cells significantly decreased in 6h, 12h, 24h treatment group compared with that in model group(P<0.05). The number of TIMP-1 positive cells in 3h, 6h, 12h, 24h model groups was 11.80±0.96, 12.30±1.50, 7.80±0.96, 7.80±1.50　respectively,　which was 12.30±0.96, 13.80±0.96, 10.50±1.73, 10.30±0.96 respectively in treatment group. The number of positive cells significantly increased in 12h, 24h treatment group compared with that in model group(P<0.05). Real-time PCR result showed that the mRNA expression of MMP-9 in 3h, 6h, 12h, 24h control group was 4.93±0.21, 4.95±0.24, 4.96±0.25, 4.98±0.23 respectively, 5.38±0.25, 6.53±0.31, 6.87±0.29, 7.53±0.33 respectively in model group, and 5.35±0.26, 5.56±0.22, 5.74±0.27, 5.90±0.31 respectively in treatment group, the mRNA expression of MMP-9 in 6h, 12h, 24h model group significantly increased compared with that in control group(P<0.05). and the mRNA expression of MMP-9 in 6h, 12h, 24h treatment group significantly decreased compared with that in model group(P<0.05). The mRNA expression of TIMP-1 in 3h, 6h, 12h, 24h control group was 4.74±0.23, 4.76±0.21, 4.73±0.25, 4.76±0.24 respectively, was 4.53±0.32, 4.62±0.21, 3.83±0.20, 3.65±0.32 respectively in model group, and 4.55±0.26, 4.65±0.27, 4.28±0.22, 4.25±0.24 respectively in treatment group, the mRNA expressions of TIMP-1 in 12h, 24h model groups decreased significantly compared with that in control groups, and the mRNA expression of TIMP-1 in 12h, 24h treatment group significantly increased compared with that in model group(P<0.05). Conclusions: Neuregulin plays a role in neural protection in spinal cord ischemia reperfusion injury, and the activation mechanism may be caused by the down-regulated expression of MMP-9 and the up-regulated expression of TIMP-1.