脊髓自噬功能激活与大鼠2型糖尿病神经病理性疼痛的关系

doi:10.12047/j.cjap.5644.2018.073

摘要/Abstract

摘要： 目的：探讨脊髓自噬功能与大鼠2型糖尿病神经病理性疼痛（DNP）的关系。方法：雄性SD大鼠（42只）高糖高脂饲养8周，腹腔单次注射链脲佐菌素（STZ）制备大鼠2型糖尿病模型。两周后检测机械缩足阈值（MWT）和热缩足潜伏期（TWL），降至基础值80%以下者为2型糖尿病神经病理性疼痛大鼠，记为DNP组（24只）；未降至基础值80%以下者为2型糖尿病无神经病理性疼痛大鼠，记为DA组（18只）。另取18只大鼠为对照（control，C）组，普通饲料喂养。于确定DA与DNP分组后的第3、7和14天，测定机械缩足阈值（MWT）和热缩足潜伏期（TWL），并在行为学检测结束后各组随机取6只大鼠处死，取L4~L6脊髓膨大，采用Western blot法检测自噬特异性蛋白微管相关蛋白1（Beclin-1）、微管相关蛋白1轻链3（LC3）和P62的表达。另取6只7 d DNP组大鼠采用免疫荧光双染法检测脊髓背角P62与小胶质细胞、星形胶质细胞、神经元的共表达情况。结果：连续8周喂养高糖高脂饲料的SD大鼠的血浆胰岛素水平升高，胰岛素敏感指数下调，表明出现胰岛素抵抗；在腹腔注射STZ后，血糖升高达到2型糖尿病诊断标准（≥16.7 mmol/L）；与C组、DA组比较，DNP组大鼠在第3、7和14天时MWT降低，TWL缩短，并且脊髓背角LC3-Ⅱ、Beclin-1表达上调，P62表达下降（P<0.05）。免疫荧光双染色显示，P62在脊髓背角表达，主要与神经元共存，少量与小胶质细胞共存，几乎不与星形胶质细胞共表达。结论：2型糖尿病神经病理性疼痛大鼠脊髓LC3-Ⅱ、Beclin-1和P62表达的改变提示脊髓自噬功能激活；脊髓背角中神经元自噬激活在2型糖尿病大鼠DNP的发生和发展起着关键作用。

关键词: 2型糖尿病, 神经病理性疼痛, 脊髓自噬, 大鼠

Abstract: Objective: To investigate the relationship between autophagy function in spinal cord and type 2 diabetic neuropathic pain in rats. Methods: Forty-two male Sprague-Dawley rats were fed with a high-sugar, high-fat diet for 8 weeks to induce the insulin resistance, and then received a single intraperitoneal streptozocin (STZ) injection to establish type 2 diabetes rat model. Two weeks after STZ injection, mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of rats were detected, the rats with MWT and TWL decreasing to below 80% compared to baseline were chosen as type 2 diabetic neuropathic pain rats (group DNP, n=24), the rest of the rats were chosen as type 2 diabetic non-neuropathic pain rats (group DA, n=18). And another 18 normal rats randomly selected from the total were classified as control group (group C) and fed with common forage for 8 weeks. The MWT and TWL were measured again on the 3rd, 7th and 14th day after determining the grouping of DA and DNP, and then, the lumbar segments 4~6 of the spinal cord were removed from the executed rats for determination of the expressions of microtubule-associated protein light chain 3 (LC3)、Beclin-1and P62 by Western blot. The co-expressions of P62 with GFAP or OX-42 or NeuN in spinal dorsal horn were detected in another 6 lumbar segments of diabetic neuropathic pain (DNP) rats on the 7th day by immunofluorescence double dye method. Results: Compared with group C, the insulin level was increased and ISI decreased in SD rats fed with high-sugar, high-fat diet, that meant the rats in insulin-resistance. After STZ injection, blood glucose rose to the standard of type 2 diabetes mellitus, i.e. ≥ 16.7 mmol/L. Compared with group C and group DA, MWT was significantly decreased, TWL shortened and the expression of LC3-Ⅱ and Beclin-1 in the spinal dorsal horn up-regulated, P62 expression down-regulated on the 3rd, 7th and 14th day in group DNP (P<0.05). P62 was mainly localized in spinal dorsal horn and coexisted with neurons, and spots of P62 immunoreactivity could be detected in a few microglia but not observed in astrocyte. Conclusion: The changes in expression of LC3-Ⅱ、Beclin-1 and P62 in spinal cord of type 2 diabetes neuropathic pain rats means autophagy activation of spinal, up-regulated autophagy of neurons in spinal dorsal horn mainly involves in the formation and development of type 2 diabetic neuropathic pain in rats.

Key words: type 2 diabetes mellitus, neuropathic pain, autophagy, rats

朱雅冰, 贾改丽, 陆嘉辉, 张茂表, 李军, 曹红. 脊髓自噬功能激活与大鼠2型糖尿病神经病理性疼痛的关系[J]. 中国应用生理学杂志, 2018, 34(4): 318-323.

ZHU Ya-bing, JIA Gai-li, LU Jia-hui, ZHANG Mao-biao, LI Jun, CAO Hong. The relationship between autophagy activation in spinal cord and type 2 diabetic neuropathic pain in rats[J]. CJAP, 2018, 34(4): 318-323.

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