摘要
目的探讨细胞自噬对神经病理性疼痛(NP)大鼠N -甲基- D -天冬氨酸(NMDA)受体及其亚基NR2B和行为学的调控作用。方法将雄性SD大鼠按随机数字表法分为假手术(Sham)组、NP组、自噬抑制剂3 -甲基腺嘌呤(3-MA)预处理组(3-MA+NP组)和自噬激动剂雷帕霉素(Rap)预处理组(Rap+NP组),每组22只。采用结扎坐骨神经的方法制备NP动物模型;Sham组仅暴露坐骨神经但不结扎。两个预处理组分别于术前1 h腹腔注射3-MA 15 mg/kg或Rap 10 mg/kg。各组大鼠于术前及术后1、3、7、14 d检测行为学指标机械缩足反应阈(MWT)和热缩足潜伏期(TWL)。各组于术后1 d、7 d取脊髓组织,透射电镜下观察自噬体形成情况;采用蛋白质免疫印迹试验(Western Blot)检测自噬相关蛋白微管相关蛋白1轻链3 -Ⅱ(LC3 -Ⅱ)、Beclin1以及NMDA和NR2B亚基的表达;采用免疫荧光法检测LC3的阳性表达。结果与Sham组比较,NP组大鼠MWT随术后时间延长逐渐下降,TWL逐渐缩短;且1 d时自噬体及脊髓背角LC3 -Ⅱ、Beclin1、NMDA、NR2B蛋白表达和LC3阳性表达即明显增加,并持续至7 d,说明NP可导致痛敏发生并激活自噬活性。给予3-MA预处理可使NP大鼠MWT进一步降低、TWL进一步缩短,自噬体减少,脊髓背角LC3 -Ⅱ、Beclin1表达下降,NMDA、NR2B表达进一步增加,术后1 d与NP组比较差异即有统计学意义〔MWT(g):29.4±2.4比42.5±6.6,TWL(s):7.2±1.0比8.8±1.1,LC3 -Ⅱ/β-actin:0.38±0.03比0.52±0.07,Beclin1/β-actin:0.29±0.06比0.59±0.05,NMDA/β-actin:0.62±0.06比0.50±0.06,NR2B/β-actin:0.57±0.03比0.46±0.03,均P<0.05〕,且免疫荧光染色证实LC3阳性表达明显减少;而给予Rap预处理可使NP大鼠MWT增加、TWL延长,自噬体增加,脊髓背角LC3 -Ⅱ、Beclin1表达增加,NMDA、NR2B表达下降,也于术后1 d与NP组比较即表现出统计学差异〔MWT(g):49.4±4.4比42.5±6.6,TWL(s):10.5±1.2比8.8±1.1,LC3 -Ⅱ/β-actin:0.67±0.09比0.52±0.07,Beclin1/β-actin:0.71±0.08比0.59±0.05,NMDA/β-actin:0.40±0.05比0.50±0.06,NR2B/β-actin:0.34±0.04比0.46±0.03,均P<0.05〕,且免疫荧光显示LC3阳性表达增加,并持续至7 d。说明Rap可以增加自噬的活性,减少痛敏的发生,抑制NMDA受体及其NR2B亚基的表达。结论 NP可以诱发细胞自噬的增加,且通过自噬可以调控NMDA受体及其亚基的表达和痛敏的变化。
Objective To investigate the effects of autophagy on N-methy-D-aspartate (NMDA) receptor and its subunit NR2B and behavioral test in a rat model of neuropathic pain (NP). Methods Male Sprague-Dawley (SD) rats were divided into sham group, NP group, autophagy inhibitor 3-methyladenine (3-MA) pretreatment group (3-MA+NP group) and autophagy inducer rapamyein (Rap) group (Rap+NP group) by random number table with 22 rats in each group. NP animal model was reproduced by ligating sciatic nerve, while sciatic nerve of the rats in the sham group were only exposed but not ligated. The rats in two pretreatment groups were intraperitoneally challenged with 3-MA 15 mg/kg or Rap 10 mg/kg injection 1 hour before operation. Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before and 1, 3, 7, 14 days after operation in each group. Spinal cord tissues were harvested at 1 day and 7 days after operation for autophagosome observation by electron microscope. The expressions of autophagy protein microtubule-associated protein 1 light chain 3 -Ⅱ(LC3 -Ⅱ), Beclin1, and NMDA, NR2B were determined by Western Blot. The positive expression of LC3 was detected by immunofluorescence. Results Compared with sham group, the MWT and TWL of rats in NP group were decreased gradually with the prolongation of operation time, the number of autophagosome, the expressions of LC3 -Ⅱ, Beclin1, NMDA, NR2B, and the positive expression of LC3 in spinal cord were significantly increased at 1 day after operation and till 7 days, which indicated that NP led to hyperpathia and autophagy activation. Compared with NP group, MWT was significantly further decreased, TWL was further shortened, the number of autophagosome was decreased, the expressions of LC3 -Ⅱ and Beclin1 in spinal cord were decreased, and NMDA and NR2B expressions were further increased after 3-MA pretreatment, with significant differences at 1 day after operation [MWT (g): 29.4±2.4 vs. 42.5±6.6, TWL (s): 7.2±1.0 vs. 8.8±1.1, LC3 -Ⅱ/β-actin: 0.38±0.03 vs. 0.52±0.07, Beclin1/β-actin: 0.29±0.06 vs. 0.59±0.05, NMDA/β-actin: 0.62±0.06 vs. 0.50±0.06, NR2B/β-actin: 0.57±0.03 vs. 0.46±0.03, all P < 0.05]. Immunofluorescence staining confirmed that the positive expression of LC3 was significantly decreased. Rap pretreatment could increase MWT, TWL and the number of autophagosome, increase LC3 -Ⅱ and Beclin1 expressions in spinal cord, and decrease NMDA and NR2B expressions in NP rats, and significant differences at 1 day after operation were found as compared with those in NP group [MWT (g): 49.4±4.4 vs. 42.5±6.6, TWL (s): 10.5±1.2 vs. 8.8±1.1, LC3 -Ⅱ/β-actin: 0.67±0.09 vs. 0.52±0.07, Beclin1/β-actin: 0.71±0.08 vs. 0.59±0.05, NMDA/β-actin: 0.40±0.05 vs. 0.50±0.06, NR2B/β-actin: 0.34±0.04 vs. 0.46±0.03, all P < 0.05], and immunofluorescence showed that the positive expression of LC3 was increased and lasted for 7 days. It indicated that Rap could increase the activity of autophagy, alleviate the occurrence of hyperalgesia, and reduce the expressions of NMDA receptor and its NR2B subunit. Conclusion NP could regulate the variety of NMDA/NR2B and hyperalgesia via increasing autophagy.
作者
田礼华
张杨
谢克亮
陈红光
Tian Lihua;Zhang Yang;Xie Keliang;Chen Hongguang(Department of Pain, Tianjin Wuqing Hospital of Traditional Chinese Medicine, Tianjin 301700, China;Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin 300054, China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2019年第3期341-345,共5页
Chinese Critical Care Medicine
基金
国家自然科学基金(81601667).
