| 陈彬彬,张一肖,王振,万海方.脊髓背角环磷酸腺苷直接激活的交换蛋白在大鼠炎性痛中的作用[J].浙江中西医结合杂志,2022,32(9): |
| 脊髓背角环磷酸腺苷直接激活的交换蛋白在大鼠炎性痛中的作用 |
| The role of exchange protein directly activated by cyclic adenosine monophosphate in inflammatory pain of rats in spinal dorsal horn |
| 投稿时间：2021-10-25 修订日期：2022-06-22 |
| DOI： |
| 中文关键词: 脊髓背角 环磷酸腺苷直接激活的交换蛋白 炎性痛 弗氏完全佐剂 |
| 英文关键词:Spinal dorsal horn Exchange protein directly activated by cyclic adenosine monophosphate Inflammation pain Freund's complete adjuvant |
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| 中文摘要: |
| 【摘要】 目的 探讨大鼠脊髓背角环磷酸腺苷（cAMP）直接激活的交换蛋白（Epac）在炎性痛调节过程中的作用。方法 清洁级健康成年雄性SD大鼠84只，3月龄，体重230~250 g，其中非模型鼠20只，模型鼠64只；采用双侧后爪趾底皮下注射弗氏完全佐剂（CFA）100 μL建立炎性痛模型（以下称为建模）。采用Western blot法于建模前、建模后1、3、6、9、14天大鼠脊髓腰膨大检测Epacl、Epac2蛋白相对含量，每组4只；采用免疫荧光法检测脊髓背角Epacl阳性细胞数，每组4只。实验随机分为三组：对照组（C组）、生理盐水组（NS组）和Epac激动剂组（8p-CPT组），每组12只，C组为非模型鼠，未做任何处理，NS组和8p-CPT组为模型鼠，分别于建模后6天，鞘内给予NS、Epac激动剂8p-CPT（8p-CPT-2′-O-Me-cAMP）10 μL（以下称为给药）。于建模前1 h、给药前1 h、给药后30 min、1 h、2 h和3 h测定热刺激缩足潜伏期（TWL），观察大鼠痛行为学变化；于给药后1 h采用免疫荧光法检测三组大鼠脊髓背角即刻早期基因蛋白c-Fos阳性细胞数。结果 建模后3、6天脊髓腰膨大Epacl蛋白相对含量明显低于建模前和建模后1天（0.74±0.09和0.77±0.08分别比1.00±0.00和 0.99±0.07，P < 0.05），Epac2蛋白相对含量各时点差异均无统计学意义。建模后3、6、9、14天脊髓背角Epac1阳性细胞数明显低于建模前[（34.25±4.99）个、（24.00±8.04）个、（31.50±3.51）个和（43.00±8.98）个分别比（61.75±4.99）个，P < 0.05]，建模后3、6、9天脊髓背角Epac1阳性细胞数明显低于建模后1天[（34.25±4.99）个、（24.00±8.04）个和（31.50±3.51）个分别比（58.00±5.35）个，P < 0.05]。给药前1 h、给药后30 min、1 h、2 h和3 h NS组和8p-CPT组TWL均明显低于C组[（6.8±1.0）s和（7.1±0.8）s分别比（13.3±1.6）s，（7.3±0.9）s和（9.2±1.0）s分别比（13.2±1.5）s，（6.9±1.2）s和（9.9±1.3）s分别比（13.9±1.8）s，（7.0±0.7）s和（8.7±1.2）s分别比（13.2±1.2）s，（7.1±0.9）s和（7.0±0.9）s分别比（13.2±1.4）s，P < 0.05]，给药后30 min、1 h和2 h 8p-CPT组TWL均明显高于NS组[（9.2±1.0）s比（7.3±0.9）s，（9.9±1.3）s比（6.9±1.2）s，（8.7±1.2）s比（7.0±0.7）s，P < 0.05]。给药后1 h NS组和8p-CPT组脊髓背角c-Fos阳性细胞数明显高于C组，8p-CPT组脊髓背角c-Fos阳性细胞数明显低于NS组[（75.25±8.42）个和（40.50±6.81）个分别比（25.25±5.25）个，（40.50±6.81）个比（75.25±8.42）个，P < 0.05]。结论 CFA诱导的慢性炎性痛可以降低脊髓背角Epac1蛋白含量、引起局部神经元异常激活、增强外周伤害性刺激应激反应。 |
| 英文摘要: |
| 【Abstract】 Objective To investigate the role of exchange protein directly activated by cyclic adenosine monophosphate (Epac) in the regulation of inflammatory pain of rats in spinal dorsal horn. Methods A total of 84 cleaning grade healthy adult male Sprague-Dawley rats, aged 3 months, weighing 230-250 g, including 20 non-model rats and 64 model rats; Freund's complete adjuvant (CFA) 100 μL were injected on double hind claw preparaing for the model of inflammatory pain (the following is called modeling). Western blot technique was used to detect the expression of Epac1 and Epac2 protein in spinal intumescentia lumbalis of groups before modeling and 1, 3, 6, 9, and 14 days after modeling (n=4 rats per group); Immunofluorescene technique was used to detect level of Epac1 positive cells in spinal dorsal horn (n=4 rats per group). The experiment were randomized into 3 groups: C group、NS group and 8p-CPT group (n=12 rats per group). C group were non-model rats with no treatments, NS group and 8p-CPT group were model rats of day 6 after double hind claw injection of CFA by intrathecal administration of NS 10 μL and 8p-CPT (8p-CPT-2′-O-Me-cAMP) 10 μL (the following is called administration), the agonist of Epac respectively. Thermal withdrawal latency (TWL) was measured at 1 hour before modeling、1 hour before administration、30 minutes, 1 hour, 2 hours and 3 hours after administration to observe the changes of rats pain behavior. Immunofluorescene technique was used to detect level of c-Fos positive cells in spinal dorsal horn of groups at 1 hour after administration. Results The expression of Epac1 in spinal intumescentia lumbalis was significantly reduced on 3 and 6 days after modeling than that before modeling and 1 day after modeling (0.74±0.09 and 0.77±0.08 vs. 1.00±0.00 and 0.99±0.07, P < 0.05); the expression of Epac2 had no significantly differences. Level of Epac1 positive cells in spinal dorsal horn was decreased significantly on 3, 6, 9, and 14 days after modeling than that before modeling (34.25±4.99、24.00±8.04、31.50±3.51 and 43.00±8.98 vs. 61.75±4.99, P < 0.05); Level of Epac1 positive cells in spinal dorsal horn was decreased significantly on 3, 6 and 9 days after modeling than that 1 day after modeling (34.25±4.99、24.00±8.04 and 31.50±3.51 vs. 58.00±5.35, P < 0.05). TWL of NS group and 8p-CPT group were significantly lower than that of C group at 1 hour before administration and 30 minutes, 1 hour, 2 hours, 3 hours after administration (6.8±1.0 and 7.1±0.8 vs. 13.3±1.6 s, 7.3±0.9 and 9.2±1.0 vs. 13.2±1.5 s, 6.9±1.2 and 9.9±1.3 vs. 13.9±1.8 s, 7.0±0.7 and 8.7±1.2 vs. 13.2±1.2 s, 7.1±0.9 and 7.0±0.9 vs. 13.2±1.4 s, P < 0.05). TWL of 8p-CPT group were significantly higher than that of NS group at 30 minutes, 1 hour and 2 hours after administration (9.2±1.0 vs. 7.3±0.9 s, 9.9±1.3 vs. 6.9±1.2 s, 8.7±1.2 vs. 7.0±0.7 s, P < 0.05). Compared with C group, level of c-Fos positive cells in spinal dorsal horn of NS group and 8p-CPT group were significantly increased at 1 hour after administration; Compared with NS group, level of c-Fos positive cells in spinal dorsal horn of 8p-CPT group was decreased significantly at 1 hour after administration (75.25±8.42 and 40.50±6.81 vs. 25.25±5.25, 40.50±6.81 vs. 75.25±8.42, P < 0.05). Conclusion Chronic inflammation pain by CFA reduced the expression of Epac1 protein in spinal dorsal horn, caused abnormal activation of local neurons and enhanced the peripheral nociceptive stimulus stress response. |
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