CREB激活促进神经痛大鼠脊髓背角HCN4转录的实验研究  

Experimental study of CREB activation promoting HCN4 transcription in spinal dorsal horn of neuropathic rats

作　　者：王蓉 万琪 谢晔 徐陶 曾俊伟 刘晓红 Wang Rong;Wan Qi;Xie Ye;Xu Tao;Zeng Junwei;Liu Xiaohong(Department of Physiology,Zunyi Medical University,Zunyi Guizhou 563099,China)

机构地区：[1]遵义医科大学生理学教研室,贵州遵义563099

出　　处：《遵义医科大学学报》2023年第1期7-13,共7页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:31860291,81960161);2018年贵州省教育厅创新群体重大研究项目(NO:黔教合KY字[2018]025)。

摘　　要：目的探讨CREB激活促进神经痛大鼠脊髓背角HCN4转录的信号机制。方法雄性成年SD大鼠随机分为6组:Sham组,Sham+Vehicle组,CCI+Vehicle组,CCI+H-89(PKA抑制剂),CCI+KN-93(CaMKⅡ抑制剂),CCI+Naphthol AS-E(CREB抑制剂)。各组大鼠在CCI术后分别注射0.005%DMSO、H-89、KN-93或Naphthol AS-E,在CCI术前,术后1、3、5、7 d检测各组大鼠给药1 h后的机械刺激缩足反射阈值(MWT);实时荧光定量PCR检测HCN4 mRNA表达;Western blot检测脊髓(L_(4)~L_(6))背角HCN4、PKA、p-PKA、CaMKⅡ、p-CaMKⅡ、CREB和p-CREB表达;EMSA检测CREB与HCN4启动子的DNA结合活性。结果与Sham组相比,CCI+Vehicle组在1、3、5、7 d的MWT显著降低(P<0.05),脊髓背角HCN4 mRNA表达明显上调(P<0.05),HCN4、PKA、p-PKA、CaMKⅡ、p-CaMKⅡ、CREB和p-CREB蛋白表达明显上调(P<0.05);与CCI+Vehicle组相比,H-89、KN-93或Naphthol AS-E抑制剂组MWT显著上调(P<0.05),HCN4、PKA、p-PKA、CaMKⅡ、p-CaMKⅡ、CREB和p-CREB蛋白表达显著下降(P<0.05),HCN4 mRNA表达显著减少(P<0.05);与Sham组相比,CCI+Vehicle组背角CREB与HCN4启动子的DNA结合活性升高,但H-89、KN-93或Naphthol AS-E处理后CREB与HCN4启动子的DNA结合活性减弱。结论神经痛大鼠脊髓背角PKA和CaMKII激活,导致转录因子CREB与HCN4基因启动子结合,促进HCN4转录和蛋白表达。Objective To explore the mechanism of CREB activation on HCN4 transcription in spinal dorsal horn of neuropathic pain rats.Methods Male adult SD rats were randomly divided into Sham,Sham+Vehicle,Chronic constriction injury(CCI)+Vehicle,CCI+H-89(PKA inhibitor),CCI+KN-93(CaMKⅡinhibitor),CCI+Naphthol AS-E(CREB inhibitor)groups.After operation,rats were intrathecal injected with 0.005%DMSO(10μl),H-89(8 nM),KN-93(50 nM)or Naphthol AS-E(5μg/10μl),respectively for 7 days.The mechanical withdrawal threshold(MWT)was measured before CCI and on 1,3,5 and 7 d after CCI.RT-PCR was used to detect mRNA expression of HCN4.Western blot was used to detect the expression of HCN4,PKA,p-PKA,CaMKⅡ,p-CaMKⅡ,CREB and p-CREB in spinal dorsal horn.The DNA binding activity of CREB and HCN4 promoter was detected by EMSA.Results The CCI+Vehicle group displayed significantly decreased MWT on days 1,3,5 and 7 compared with Sham rats(P<0.05),which were markedly elevated by H-89,KN-93 or Naphthol AS-E(all P<0.05).The expression of HCN4 mRNA and protein in CCI+Vehicle group was significantly up-regulated than that of the Sham group(P<0.05).The protein expressions of HCN4,PKA,p-PKA,CaMKⅡ,p-CaMKⅡ,CREB and p-CREB in CCI group were significantly increased(P<0.05).Compared with CCI+Vehicle group,H-89,KN-93 or Naphthol AS-E treatment significantly suppressed the increased expression of PKA,p-PKA,CaMKⅡ,p-CaMKⅡ,CREB and p-CREB after nerve injury(P<0.05).Compared with Sham group,the DNA binding activity of CREB and HCN4 promoter was increased in the CCI+Vehicle group,which was suppressed by H-89,KN-93 or Naphthol AS-E.Conclusion The activation of PKA and CaMKII in the spinal dorsal horn of CCI rats leads to the binding of the transcription factor CREB with the HCN4 gene promoter,which promotes the transcription and expression of HCN4.

关 键 词：脊髓背角 环磷腺苷效应元件结合蛋白 疼痛 超极化激活环核苷酸门控的阳离子通道4 电泳迁移率实验 

分 类 号：R338.2[医药卫生—人体生理学]