不同时段电针对急性脊髓损伤大鼠作用机制的蛋白质组学分析

Proteome Analysis on the Mechanism of Electroacupuncture in Relieving Acute Spinal Cord Injury at Different Time Courses in Rats

目的:检测电针急性脊髓损伤(ASCI)大鼠6、24、48h差异蛋白变化情况,初步分析其作用机制。方法:105只SD大鼠随机分为正常组、6h模型组、6h电针组、24h模型组、24h电针组、48h模型组和48h电针组,每组15只。采用自制的Allen’s打击装置造ASCI模型。电针"大椎""命门"。取脊髓损伤区脊髓组织对样本蛋白进行提取、定量、蛋白双向电泳图像分析,找出差异点进行质谱分析和数据库检索。结果:本研究共鉴定出了10个丰度变化大于1.5倍的差异蛋白。在6h段,有5种差异蛋白,与正常组比,模型组4种差异蛋白表达上调,1种下调,电针后4种差异蛋白的表达被逆转;在24h段,有7种差异蛋白,造模后6种差异蛋白上调,1种下调,电针后6种蛋白的表达被逆转;在48h段,有8种差异蛋白,造模后6种差异蛋白上调,2种下调,电针后其中5种差异蛋白的表达变化被逆转。与电针效应有关的蛋白功能涉及细胞能量代谢、信号转导、DNA修复、细胞凋亡、构建细胞骨架等。随着损伤时间的增加,被鉴定出的脊髓内差异表达蛋白也增多。24h组与6h组比较,多了2种差异蛋白:核苷二磷酸激酶及磷酸丙糖异构酶1,造模后其表达上调,电针后下调。48h组与24h组比较,多了3种差异蛋白:二氢硫辛酰胺脱氢酶、苹果酸脱氢酶1及三磷酸甘油醛脱氢酶,这3种蛋白在造模后表达分别上调、下调、上调,电针后均表现为上调,减少了2种蛋白:核苷二磷酸激酶及E2泛素结合酶。结论:电针可逆转急性损伤脊髓内多数差异蛋白表达的变化,其作用可能是通过抑制神经细胞凋亡、改善细胞能量代谢、调节异常蛋白等途径促进脊髓损伤的修复。随着时间和针刺次数的增加,电针可能逐渐侧重于发挥改善细胞能量代谢和抑制其凋亡的作用,而对受损蛋白的调节作用在减弱。

Objective To observe the effect of electroacupuncture （EA） on the differentially expressed proteins in the spinal cord at different time courses after acute spinal cord injury （ASOl） in the rat, so as to study its underlying mechanism in improving spinal traumatic injury. Methods A total of 105 male SD rats were randomized into normal control, model-6 h, EA-6 h, model-24 h, EA-24 h, model-48 h, EA-48 h groups, with 15 cases in each. ASOl model was established by using modified Allen＇s method. EA （2 Hz, 2-5 mA） was applied to ＂Mingmen＂ （GV 4） and ＂Dazhui＂ （GV 14） for 30 min. The injured spinal cord tissue （T10 -T11 ） was collected 6 h, 24 h and 48 h after ASOl and EA treatment, weighted and stored under -80 ℃ till detection. Twodimensional gel electrophoresis （2-DE） was used to separate total proteins of the spinal tissue, followed by protein extraction and quantitation, 2-D gel image analysis, matrix assisted laser desorption ionisation time-of-flight mass spec-trometry （MALDI-TOF-MS）, and databases-searching for identification of the differentially-expressed proteins. Results A total of 10 differentially expressed proteins were identified in the present study. At 6 h, compared with control group, of the 5 types of spinal differential proteins, 4 were upregulated in the expression after ASOl, and the rest one was downregulated; while after EA, ASOl-induced expression changes in 4 of the 5 differential proteins were reversed. At 24 h after ASOI, 7 types of differential proteins were identified. Compared with control group, 6 differential proteins were upregulated, and the rest one was downregulated in model group. Compared with model group, ASOl-induced expression changes in 6 of the 7 differential proteins were reversed by EA. At 48 h after ASCl, a total of 8 types of differential proteins were identified. Compared with control group, 6 differential proteins were upregulated in the expression, and the rest two downregulated in model group. Compared with model group, ASCl-induced expression changes in 5 of the 8 differential proteins were reversed by EA. Along with the increased time and treatment, 24 h vs 6 h, two more differential proteins were identified, i.e., nucleoside diphosphate kinase and triosephosphate isomerase 1 （TPI1 ）. 48 h vs 24 h, 3 more differential proteins were identified, i.e., dihydrolipoamide dehydrogenase, malate dehydrogenase 1, and glyceraldehyde-3-phos- phate dehydrogenase （GAPDH） ; but two proteins disappeared, i.e., nucleoside diphosphate kinase, and ubiquitin-conjugating enzyme E2N. The identified differential proteins involving the effects of EA in regulating cellular energy metabolism, DNA repair, cellular generation, differentiation, apoptosis, etc. Conclusion Proteome analysis indicates that in ASCl rats, some differentially expressed proteins involving energy metabolism, neuronal apoptosis reduction, protein-degradation inhibition may contribute to the effect of EA in repairing the traumatic spinal tissue.