脊髓ROS激活自噬参与调节2型糖尿病神经病理性疼痛

ROS modulates type 2 diabetic neuropathic pain via activating autophagy in rat spinal cord

摘要:

【】 目的：探讨脊髓中活性氧(ROS)是否通过激活自噬参与大鼠2型糖尿病神经病理性疼痛(DNP)的形成和发展。方法：清洁级雄性SD大鼠高脂高糖喂养8周,后STZ单次小剂量 (35mg/kg)腹腔注射,注射后3d血糖≥16.7mmol /L为成功诱导2型糖尿病发生；注射后14d行为学测定痛阈下降至基础值的85%以下,即为成功制备出大鼠2型DNP模型,否则为2型糖尿病不痛组(PL组)。将造模成功的2型DNP大鼠随机分为3组,每组10只：DNP组、DNP+ROS清除剂组(DNP+PBN组)和溶剂对照组(SC组)。另取10只正常SD大鼠为对照组(C组),普通饲料喂养,给药后3d、7d、14d时测定所有组别大鼠的机械缩足反应阈值(MWT)和热缩足潜伏期(TWL),随后处死大鼠,取脊髓腰膨大L4-L6,用免疫印迹法测定脊髓中Beclin1、LC3Ⅱ/LC3Ⅰ和p62的表达水平；用流式细胞仪测定脊髓组织中活性氧(ROS)的含量。结果：与C组和PL组比较,DNP组和SC组于模型制备成功后3d、7d和14d时MWT下降,TWL缩短,脊髓ROS、Beclin1和LC3Ⅱ/LC3Ⅰ表达上调(p<0.05),而p62表达下降(p<0.05)；与DNP组比较,DNP+PBN组在3d、7d和14d时MWT升高、TWL延长,脊髓Beclin1和LC3Ⅱ/LC3Ⅰ表达下降(p<0.05),而p62表达上调(p<0.05)；DNP组与SC组各指标比较差异均无统计学意义。结论：脊髓ROS通过激活自噬参与调节2型糖尿病神经病理性疼痛的产生和维持。

Abstract:

Objective: To explore whether ROS modulates the initiation, development and maintenance of type 2 diabetic neuropathic pain (DNP) via activating autophagy. Methods: Pathogen-free male Sprague-Dawley rats were fed with a high-fat and high-sugar diet for 8 weeks, and then received a single intraperitoneal streptozocin (STZ) injection with a dose of 35mg/kg. Three days after the STZ injection, rats with blood glucose level of 16.7 mmol/L or higher were considered type 2 diabetes mellitus. Diabetic rats with neuropathic pain were defined as both MWT and TWL ≤ 85% of base value on 14d after STZ injection, while both TWL and MWT>85% base value was defined as PL group. The type 2 DNP rats were randomly divided into 3 groups (n=10) : DNP group, DNP+PBN group (DNP rats treated with ROS scavenger PBN) and SC group (solvent control group). Another 10 normal SD rats were selected as control group (C group) and were fed with common diet. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured at 3d, 7d and 14d after dosing. The lumbar segments 4-6 of the spinal cord were removed at the same time for determination of the expression of Beclin1, LC3Ⅱ/ LC3Ⅰ,and p62 by Western blot; the production of ROS was measured by flow cytometry. Results: Compared with C and PL groups, MWT was significantly decreased, TWL was shortened, ROS production and the expression of Beclin1 and LC3Ⅱ/LC3Ⅰin the spinal cord was up-regulated at 3, 7 and 14 d in DNP and SC groups (p<0.05); while the expression of p62 was decreased (p<0.05). Compared with DNP group, MWT was significantly increased, TWL was prolonged, the expression of Beclin1 and LC3Ⅱ/LC3Ⅰin spinal cord was decreased and p62 was increased at 3, 7 and 14 d in DNP+PBN group (p<0.05). No significant difference in the MWT, TWL, and the expression of Beclin1, LC3Ⅱ/LC3Ⅰand p62 between DNP group and SC group were observed (p>0.05). Conclusion: ROS modulates the initiation, development and maintenance of type 2 diabetic neuropathic pain (DNP) via activating autophagy.