Jia Lei, Zhang Yang, Qu Yu-Juan, Huai Juan, Wei Hui, Yue Shou-Wei
Department of Physical Medicine & Rehabilitation, Qilu Hospital, Shandong University, Jinan, China.
J Cell Biochem. 2019 May;120(5):8110-8119. doi: 10.1002/jcb.28090. Epub 2018 Nov 13.
Neuropathic pain is an abnormal pain, which is related to the activation of extracellular-regulated kinase (ERK) signaling. This study was to investigate the effects of ERK knockdown via lentivirus-mediated RNA interference on allodynia in rats with chronic compression of the dorsal root ganglia (DRG) and to uncover the potential mechanisms.
The model of chronic compression of the dorsal root ganglia (CCD) was established in rats by surgery. Gene silence was induced by injecting rats with lentivirus expressing ERK short hairpin RNA (shRNA). Behavioral test was performed by calculating paw withdrawal mechanical threshold (PWMT) and thermal paw withdrawal latency (TPWL).
We firstly generated lentivirus expressing ERK shRNA to downregulate ERK gene expression both in vitro and in vivo by using Western blot analysis and quantitative reverse transcription polymerase chain reaction. In CCD, ERK mRNA, and protein levels in DRG neurons were dramatically increased, accompanied with decreased PWMT and TPWL. Lentivirus-mediated RNA interference decreased ERK gene expression in DRG neurons and normalized the PWMT and TPWL in CCD rats, but not in rats infected with lentivirus expressing negative control shRNA. Further, calcium responses of DRG neurons to the hypotonic solution and 4α-phorbol 12,13-didecanoate were enhanced in CCD rats, which were suppressed by lentivirus-mediated ERK gene silence. Finally, the levels of transient receptor potential vanilloid 4 gene expressions in DRG neurons and L4 to L5 spinal cord isolated from CCD rats were dramatically upregulated, which were reversed by lentivirus-mediated ERK gene knockdown.
Lentivirus-mediated RNA interference (RNAi) silencing targeting ERK might reverse CCD-induced neuropathic pain in rats through transient receptor potential vanilloid 4.
神经性疼痛是一种异常疼痛，与细胞外调节激酶（ERK）信号通路的激活有关。本研究旨在探讨慢病毒介导的RNA干扰敲低ERK对背根神经节（DRG）慢性压迫大鼠痛觉过敏的影响，并揭示其潜在机制。
通过手术建立大鼠背根神经节慢性压迫（CCD）模型。向大鼠注射表达ERK短发夹RNA（shRNA）的慢病毒以诱导基因沉默。通过计算 paw withdrawal mechanical threshold（PWMT）和thermal paw withdrawal latency（TPWL）进行行为测试。
我们首先通过蛋白质免疫印迹分析和定量逆转录聚合酶链反应，制备了表达ERK shRNA的慢病毒，以在体外和体内下调ERK基因表达。在CCD模型中，DRG神经元中的ERK mRNA和蛋白水平显著升高，同时PWMT和TPWL降低。慢病毒介导的RNA干扰降低了DRG神经元中ERK基因的表达，并使CCD大鼠的PWMT和TPWL恢复正常，但在感染表达阴性对照shRNA慢病毒的大鼠中则未恢复。此外，CCD大鼠DRG神经元对低渗溶液和4α-佛波醇12,13-十二烷酸酯的钙反应增强，而慢病毒介导的ERK基因沉默抑制了这种反应。最后，从CCD大鼠分离的DRG神经元和L4至L5脊髓中瞬时受体电位香草酸亚型4基因表达水平显著上调，而慢病毒介导的ERK基因敲低则使其逆转。
慢病毒介导的靶向ERK的RNA干扰（RNAi）沉默可能通过瞬时受体电位香草酸亚型4逆转CCD诱导的大鼠神经性疼痛。
