脊髓MCP-1-ERK-KIF17∕NR2 B信号通路在大鼠2型糖尿病神经痛维持中的作用

Role of spinal MCP-1-ERK-KIF17∕NR2B signaling pathway in maintenance of type 2 diabetic neuro-pathic pain in rats

目的：评价脊髓单核细胞趋化蛋白-1（ MCP-1）-细胞外信号调节激酶（ ERK）-驱动蛋白超家族17（ KIF17）∕含2B亚基的N-甲基-D-天冬氨酸受体（ NR2B）信号通路在大鼠2型糖尿病神经痛维持中的作用。方法高脂高糖喂养6周龄雄性SD大鼠（体重120～160 g）8周诱发胰岛素抵抗，单次腹腔注射1％链脲佐菌素（STZ）35 mg∕kg，3 d后空腹血糖≥16．7 mmol∕L为2型糖尿病模型制备成功。注射STZ 14 d时机械缩足阈（ MWT）和热缩足潜伏期（ TWL）下降至基础值的85％以下为2型糖尿病神经痛模型制备成功。采用随机数字表法，将2型糖尿病神经痛大鼠分为4组（ n＝36）：2型糖尿病神经痛组（ DNP组）、2型糖尿病神经痛＋MCP-1中和抗体组（ DM组）、2型糖尿病神经痛＋ERK抑制剂U0126组（ DE组）和2型糖尿病神经痛＋5％二甲基亚砜组（ DD组）。 DM组、DE组和DD组分别于注射STZ 14 d时鞘内注射0．1 ng∕μl MCP-1中和抗体、0．5μg∕μl U0126和5％二甲基亚砜各10μl，1次∕d，连续注射14 d。取36只大鼠喂以普通饲料作为正常对照组（ C组）。分别于注射STZ前及鞘内给药1、3、7、14 d（ T0～4）时测定MWT和TWL；并于T1～4时测定痛阈后，每组处死9只大鼠，取脊髓组织，采用Western blot法测定磷酸化ERK（ p-ERK）、KIF-17和磷酸化NR2B（ p-NR2B）的表达水平。结果与C组比较，DNP 组、DM组、DE组和DD组T1～4时MWT降低，TWL缩短，脊髓组织p-ERK、KIF17和p-NR2B的表达上调（ P＜0．05）；与DNP组比较，DM组T3～4时、DE组T2～4时MWT升高，TWL延长，DM组和DE组T2～4时脊髓组织p-ERK、KIF17和p-NR2B的表达下调（ P＜0．05），DD组上述各指标差异无统计学意义（ P＞0．05）。结论脊髓MCP-1-ERK-KIF17∕NR2B信号通路参与了大鼠2型糖尿病神经痛的维持。

Objective To explore the role of spinal monocyte chemoattractant protein-1 （ MCP-1） -extracellular signal-regulated protein kinase （ ERK）-kinesin superfamily motor protein 17 （ KIF17）∕N-methyl-D-aspartate receptor subunit 2B （ NR2B） signaling pathway in the maintenance of type 2 diabetic neuropathic pain （DNP） in rats. Methods Type 2 diabetes mellitus was induced by a high-fat and high-sucrose diet and intraperitoneal streptozotocin （ STZ） 35 mg∕kg, and confirmed by fasting blood glucose level≥16-7 mmol∕L 3 days later in male Sprague-Dawley rats aged 6 weeks. Type 2 DNP was confirmed when the mechanical paw withdrawal threshold （ MWT ） and thermal paw withdrawl latency （ TWL ） measured on day 14 after STZ administration decreased to〈 80% of the baseline value. The rats with type 2 DNP were randomly divided into 4 groups （ n=36 each） using a random number table： type 2 DNP group （group DNP）, type 2 DNP ＋MCP-1 neutralizing antibody group （group DM）, type 2 DNP ＋ERK inhibi-tor group （group DE） and type 2 DNP ＋ dimethyl sulfoxide group （ group DD）. In DM, DE and DD groups, 0-1 ng∕μl MCP-1 neutralizing antibody 10 μl, 0-5 μg∕μl U0126 10 μl and 5 % dimethyl sulfoxide 10 μl were injected intrathecally, respectively, once a day for 14 consecutive days starting from 14 days after administration of STZ. Another 36 normal rats fed a common forage diet were adopted as con-trol group （ group C） . MWT and TWL were measured before STZ injection and at 1, 3, 7 and 14 days after STZ injection （ T0-4 ） . Nine rats were sacrificed after measurement of pain thresholds at T1-4 , and the lumbar segments （ L4-6 ） of the spinal cord were removed for determination of the expression of phosphoryla-ted ERK （p-ERK）, KIF17 and phosphorylated NR2B （p-NR2B） by Western blot. Results Compared with group C, the MWT was significantly decreased, the TWL was shortened, and the expression of p-ERK, KIF17 and p-NR2B was up-regulated at T1-4 in DNP, DM, DE and DD groups. Compared with group DNP, the MWT at T3-4 in group DM and at T2-4 in group DE was significantly increased, the TWL at T3-4 in group DM and at T2-4 in group DE was prolonged, and the expression of p-ERK, KIF17 and p-NR2B was down-regulated at T2-4 in DM and DE groups, and no significant changes were found in the pa-rameters mentioned above in group DD. Conclusion Spinal MCP-1-ERK-KIF17∕NR2B signaling pathway is involved in the maintenance of type 2 DNP in rats.