Zhu Ya-Bing, Jia Gai-Li, Lu Jia-Hui, Zhang Mao-Biao, Li Jun, Cao Hong
Department of Anesthesiology, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325027, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2018 Apr 8;34(4):318-323. doi: 10.12047/j.cjap.5644.2018.073.
To investigate the relationship between autophagy function in spinal cord and type 2 diabetic neuropathic pain in rats.
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, =24), the rest of the rats were chosen as type 2 diabetic non-neuropathic pain rats (group DA, =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.
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 (<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.
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.
探讨大鼠脊髓自噬功能与2型糖尿病性神经病理性疼痛之间的关系。
42只雄性Sprague-Dawley大鼠采用高糖高脂饮食喂养8周以诱导胰岛素抵抗，随后单次腹腔注射链脲佐菌素（STZ）建立2型糖尿病大鼠模型。STZ注射后2周，检测大鼠的机械缩足阈值（MWT）和热缩足潜伏期（TWL），将MWT和TWL较基线下降至80%以下的大鼠选为2型糖尿病性神经病理性疼痛大鼠（DNP组，n = 24），其余大鼠选为2型糖尿病非神经病理性疼痛大鼠（DA组，n = 18）。另外从总数中随机选取18只正常大鼠作为对照组（C组），用普通饲料喂养8周。在确定DA组和DNP组分组后的第3、7和14天再次测量MWT和TWL，然后，从处死的大鼠中取出脊髓腰段4~6，通过蛋白质免疫印迹法测定微管相关蛋白轻链3（LC3）、Beclin-1和P62的表达。在糖尿病性神经病理性疼痛（DNP）大鼠的另外6个腰段脊髓上，于第7天采用免疫荧光双染法检测脊髓背角中P62与胶质纤维酸性蛋白（GFAP）或离子钙接头蛋白1（OX-42）或神经元核抗原（NeuN）的共表达情况。
与C组相比，高糖高脂饮食喂养的SD大鼠胰岛素水平升高而胰岛素敏感指数降低，这意味着大鼠出现胰岛素抵抗。STZ注射后，血糖升至2型糖尿病标准，即≥16.7 mmol/L。与C组和DA组相比，DNP组在第3、7和14天MWT显著降低，TWL缩短，脊髓背角中LC3-Ⅱ和Beclin-1的表达上调，P62表达下调（P<0.05）。P62主要定位于脊髓背角并与神经元共存，在少数小胶质细胞中可检测到P62免疫反应性斑点，但在星形胶质细胞中未观察到。
2型糖尿病性神经病理性疼痛大鼠脊髓中LC3-Ⅱ、Beclin-1和P62表达的变化意味着脊髓自噬激活，脊髓背角神经元自噬上调主要参与大鼠2型糖尿病性神经病理性疼痛的形成和发展。
