PKA-CREB通路参与电针对退变椎间盘AQP1、3基因表达的影响

Effects of PKA-CREB Pathway Participating in Expressions of AQP1 and AQP3 Genes in Degenerative Intervertebral Disc by Electroacupuncture

目的：研究电针夹脊穴对兔退变椎间盘组织中磷酸化环腺苷酸反应序列结合蛋白（p CREB）表达的影响,探讨PKA-CREB通路参与电针调控终板软骨细胞水通道蛋白1和3（AQP1、3）基因表达的作用可能机制。方法：50只健康的骨骼发育成熟的雄性新西兰大白兔,随机分为正常组、假模型组、模型组、模型＋电针组、模型＋电针＋H89组,每组各10只。采用克氏针横穿椎体后椎体间加压法复制椎间盘退变模型。电针组针刺L4、L5夹脊穴28 d。正常组和假模型组在到达造模周期后、余下3组均在造模成功后的第1天和第28天,分别各取5只行L4-5椎间盘MRI检查,然后取椎间盘组织,供酶联免疫吸附剂测定（ELISA）、Western Blot、Real-time PCR检测。结果：MRI（T2加权）平扫提示电针治疗后的退变椎间盘信号较模型组增强;造模成功后,c AMP依赖性蛋白激酶A（PKA）、p CREB蛋白及AQP1、3mRNA表达均降低（P〈0.05）;电针治疗干预后可上调PKA、p CREB蛋白及AQP1、3mRNA表达水平（P〈0.05）;H89能够起到抑制PKA的表达（P〈0.05）、翻转电针上调p CREB蛋白表达的作用（P〈0.05）,AQP1、3mRNA表达水平也随之下降（P〈0.05）。结论：电针夹脊穴通过上调PKA-CREB通路的表达水平,继而增加AQP1、3mRNA的转录,促进椎间盘AQP1、3蛋白的表达,延缓椎间盘退变。

Objective：To study the effect of electroacupuncture at Jiaji on the expression of phosphorylated camp response element binding protein（ p CREB） in degenerated intervertebral discs of rabbits and investigate the possible mechanism of PKA-CREB pathway involved in the regulation of Aquaporin 1,3（ AQP1,3） gene expressions in endplate chondrocytes by electroacupuncture. Methods：Fifty healthy male mature New Zealand rabbits were randomly divided into normal group,sham model group,model group,model ＋ electroacupuncture group,model ＋ electroacupuncture ＋ H89 group,10 rats in each group. The intervertebral disc degeneration model was replicated by the compression of Kirschner wire across the vertebral body. EA group was treated with acupuncture at L4 and L5 Jiaji points for 28 days. When the normal group and the sham model group finished the modeling course,the remaining three groups respectively took 5 rabbits for L4-5 disc MRI examination after the success of the modeling on the first day and twenty-eighth day and then we took out the disc tissue for enzyme linked immunosorbent assay（ ELISA）,Western Blot and Real-time PCR detection. Results：The MRI（ T2 weighted） scan showed that the degenerative intervertebral disc signal after electroacupuncture treatment was stronger than that in the model group. After the success of modeling,the expressions of c AMP dependent protein kinase A（ PKA）,p CREB and AQP1,3 mRNA decreased（ P〈0.05）. The expressions of PKA,p CREB and AQP1,3 mRNA after the intervention of electroacupuncture treatment were increased（ P〈0.05）. H89 could inhibit the expression of PKA（ P〈0.05）,reverse the effect of electroacupuncture on up regulation of p CREB expression（ P〈0.05） and decrease the expressions of AQP1,3 mRNA（ P〈0.05）. Conclusion：Electroacupuncture at Jiaji points can improve the expressions of PKA-CREB pathway,and then increase the transcription of AQP1,3 mRNA,promote the expression of AQP1,3 in intervertebral discs and delay the degeneration of intervertebral discs.