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中华口腔医学研究杂志(电子版) ›› 2018, Vol. 12 ›› Issue (02) : 110 -117. doi: 10.3877/cma.j.issn.1674-1366.2018.02.007

所属专题： 文献；

基础研究

前扣带回皮质注射钙调磷酸酶对大鼠牙移动疼痛的镇痛作用及机制
黄沛娜1, 孟博文1, 程杨帆1, 刘楚峰2, 吴冬乐1, 曹阳1,†()   
  1. 1. 510055　广州，中山大学光华口腔医学院·附属口腔医院，广东省口腔医学重点实验室
    2. 510280　广州，南方医科大学口腔医院（广东省口腔医院）正畸科
  • 收稿日期:2017-12-17 出版日期:2018-04-01
  • 通信作者: 曹阳
  • 基金资助:
    国家自然科学基金(81170990); 广东省自然科学基金(2017A030313529)

The role and mechanism of calcineurin injecting into anterior cingulate cortex in alleviating pain in rats following experimental tooth movement

Peina Huang1, Bowen Meng1, Yangfan Cheng1, Chufeng Liu2, Dongle Wu1, Yang Cao1,†()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
    2. Department of Orthodontics, Guangdong Provincial Stomatological Hospital, Affiliated Stomatological Hospital of Southern Medical University, Guangzhou 510280, China
  • Received:2017-12-17 Published:2018-04-01
  • Corresponding author: Yang Cao
  • About author:
    Corresponding author:Cao Yang,Email:
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引用本文：

黄沛娜, 孟博文, 程杨帆, 刘楚峰, 吴冬乐, 曹阳. 前扣带回皮质注射钙调磷酸酶对大鼠牙移动疼痛的镇痛作用及机制[J]. 中华口腔医学研究杂志(电子版), 2018, 12(02): 110-117.

Peina Huang, Bowen Meng, Yangfan Cheng, Chufeng Liu, Dongle Wu, Yang Cao. The role and mechanism of calcineurin injecting into anterior cingulate cortex in alleviating pain in rats following experimental tooth movement[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2018, 12(02): 110-117.

目的

探索前扣带回皮质（ACC）注射钙调磷酸酶（CaN）溶液对实验性牙移动大鼠的镇痛作用及机制。

方法

将60只200~ 250 g SD大鼠采用随机数字表法分为4组（假处理组10只、单纯加力组10只、0.9%氯化钠溶液组20只、CaN溶液组20只），分别给予建立大鼠正畸牙移动模型不加力、建模并加力、加力并注射0.9%氯化钠溶液、加力并注射CaN溶液的处理，观察比较ACC埋管前、建模前、建模后2 d、给药后30 min、2 h、4 h、24 h、3 d、5 d、7 d大鼠挠嘴时间及面部表情评分（RGS）来评价疼痛状态，蛋白免疫印迹法（Western blot）检测ACC区脑组织α-氨基羟甲基异恶唑丙酸（AMPA）受体亚基GluA1的磷酸化情况及磷酸化蛋白激酶A（pPKA）、磷酸化蛋白激酶Cγ（pPKCγ）的表达变化。应用SPSS 23.0软件进行统计分析。

结果

建模后2 d，假处理组和单纯加力组的平均挠嘴时间分别为（43 ± 9）、（158 ± 21）s，RGS值分别为19 ± 3、53 ± 6。与假处理组相比，单纯加力组疼痛行为显著增加（t挠嘴= 15.876，P挠嘴<0.001；tRGS= 16.250，PRGS<0.001），ACC区pGluA1-831和pPKCγ表达增加，差异具有统计学意义（t831= 3.060，P831= 0.013；tPKCγ= 2.831，PPKCγ= 0.011）。ACC区给药后2、4、24 h，CaN溶液组的挠嘴时间分别为（64 ± 26）、（67 ± 18）、（93 ± 21）s，相比于0.9%氯化钠溶液组的（147 ± 31）、（136 ± 29）、（119 ± 30）s显著减少；同时给药后30 min、2 h、4 h、24 h、3 d，CaN溶液组的RGS值相比于0.9%氯化钠溶液组亦显著减少，差异均有统计学意义。给药后2 h，ACC区pGluA1-831和pPKCγ表达量减少，差异具有统计学意义（t831= 7.916，P831<0.001；tPKCγ= 3.230，PPKCγ= 0.005）。

结论

大鼠正畸牙移动疼痛引起AMPA受体亚基GluA1在Ser831位点磷酸化增加，其可能由蛋白激酶Cγ介导。CaN溶液可通过引起GluA1去磷酸化而缓解正畸牙移动疼痛。

关键词: 钙神经素, 受体，α-氨基羟甲基异恶唑丙酸, 牙痛, 扣带回, 牙移动
Objective

To explore the analgesic effect and mechanism of injection of calcineurin in anterior cingulate cortex (ACC) on experimental tooth movement in rats.

Methods

According to random number table, 60 Sprague-Dawley rats (200-250 g) were randomly divided into four groups: sham group (10) , force group (10) , 0.9% sodium chloride solution group (20) and calcineurin (CaN) solution group (20) . The four groups were treated in different modes: tooth movement modeling but with no force, modeling and applying force, applying force and injecting saline, applying force and injecting CaN. Mouth wiping behaviors and rat grimace scale (RGS) were recorded at the time before brain cannulation surgery in ACC, before modeling, 2 d after modeling and 30 min, 2 h, 4 h, 24 h, 3 d, 5 d and 7 d after injection to assess pain perception in rats. The phosphorylation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluA1 and the expression of phosphorylated protein kinase A (pPKA) and phosphorylated protein kinase Cγ (pPKCγ) were detected with Western blot.

Results

At 2 d after tooth movement, the average mouth wiping time of the sham-operated group and the force group were (43 ± 9) s and (158 ± 21) s, respectively. Besides, the RGS values of these two groups were 19 ± 3 and 53 ± 6, respectively. Compared with the sham-operated group, pain behaviors (tMouthwiping= 15.876, PMouth wiping<0.001; tRGS= 16.250, PRGS<0.001) and the expressions of pGluA1-831 and pPKCγ in ACC were both increased in the force group (t831= 3.060, P831= 0.013; tPKCγ= 2.831, PPKCγ= 0.011) . At 2 h, 4 h and 24 h after injection into the ACC area, mean mouth wiping time of CaN solution group was (64 ± 26) , (67 ± 18) , and (93 ± 21) s, respectively, which was significantly less than that in the 0.9% sodium chloride solution group, with average mouth wiping time of (147 ± 31) , (136 ± 29) , (119 ± 30) s, respectively. Meanwhile, at 30 min, 2 h, 4 h, 24 h and 3 d after administration, the RGS values of the CaN solution group were also dramatically lower than those in the 0.9% sodium chloride solution group. In addition, the protein levels of pGluA1-831 and pPKCγ in ACC significantly decreased at 2 h after the injection of calcineurin (t831= 7.916, P831<0.001; tPKCγ= 3.230, PPKCγ= 0.005) .

Conclusions

Pain in rats following orthodontic tooth movement is associated with phosphorylation of AMPA receptor subunit GluA1 at Ser831, which is probably mediated by protein kinase C gamma. Calcineurin alleviates pain of orthodontic tooth movement by dephosphorylation of GluA1.

Key words: Calcineurin, Receptors, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, Toothache, Gyrus cinguli, Tooth movement
图1 SD大鼠正畸牙移动加力前后疼痛行为变化
图2 SD大鼠给药前后不同时间点疼痛行为变化
图3 SD大鼠正畸牙移动加力后GluA1的磷酸化情况
图4 SD大鼠ACC区注射CaN溶液后pGluA1-831表达变化及其与疼痛行为的相关性
图5 SD大鼠正畸牙移动加力及注射CaN溶液前后pPKCγ、pPKA磷酸化水平改变
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