背根神经节ZHX2在外周神经损伤痛觉过敏中的作用研究

Effect of zinc-fingers and homeoboxes 2 in the dorsal root ganglion on peripheral nerve injuryinduced pain hypersensitivity

目的探讨背根神经节（DRG）锌指和同源框蛋白家族（ZHX）在外周神经损伤痛觉过敏中的作用，为神经病理性疼痛（NP）的分子生物学机制提供新的思路。方法（1）24只8周龄雄性C57BL6小鼠，按随机数字表法分为坐骨神经慢性缩窄性损伤模型（CCI）组、假手术组，每组12只，造模前1d、造模后7d检测小鼠机械缩爪反应频率（PwF）和热缩爪反应潜伏期（PWL）的变化。造模后7d采用反转录实时定量PCR（RT-qPCR）检测小鼠DRGZHX1、ZHX2、ZHX3mRNA的表达；采用Westernblotting检测小鼠DRGZHX2蛋白的表达。（2）36只8周龄雄性C57BL6小鼠，按随机数字表法分为DRG内注射等剂量溶剂、无义阴性对照序列（siNC）、ZHX2小f扰RNA（siRNA）的CCI组和DRG内注射等剂量溶剂、siNC、ZHX2siRNA的假手术组．每组6只。注射药物前1d、注射药物后7d观察6组小鼠机械缩爪反应频率（PWF）和热缩爪反应潜伏期（PWL）的变化：RT-qPCR检测小鼠DRGZHX2mRNA的表达。结果（1）造模后7d，与假手术组患侧比较，CCI组小鼠患侧后爪PWF明显增加、PWL明显缩短，差异均有统计学意义（P〈0．05）：与假手术组比较，CCI组小鼠DRGZHX2mRNA的表达增加1．71倍，差异有统计学意义（P〈0．05）；与假手术组患侧比较，CCI组小鼠患侧DRGZHX2蛋白的表达增加2．15倍，差异有统计学意义（P〈0．05）；（2）与注射溶剂、siNC、siRNA的假手术组比较，注射溶剂、sinC的CCI组小鼠患侧PWF增加、PWL降低、DRGZHX2mRNA的表达增加，差异均有统计学意义（P〈0．05）。与注射溶剂、siNC的CCI组比较，注射siRNA的CCI组小鼠患侧PWF降低、PWL增加、DRGZHX2mRNA的表达降低，差异均有统计学意义（P〈0．05）。结论外周神经损伤上调受损DRG内ZHX2的表达，抑制ZHX2高表达可缓解神经损伤引起的痛觉过敏行为。

Objective To explore the roles of zinc-fingers and homeoboxes （ZHX） in the dorsal root ganglia （DRG） in peripheral nerve injury-induced pain hypersensitivity, and provide a new idea for molecular mechanism ofneuropathic pain （NP）. Methods （1） Twenty-four 8-week-old male C57BL6 mice were randomly divided into sham-operated group and chronic constriction injury （CCI） of the sciatic nerve model group （n=12）. One d before modeling and 7d after modeling, the changes of paw withdrawal frequency （PWF） to mechanical stimuli and paw withdrawal latency （PWL） to thermal stimulation were detected. Seven d after modeling, reverse transcription real-time quantitative PCR （RT-qPCR） was used to detect the ZHX1, ZHX2, and ZHX3 mRNA expressions, and Western blotting was employed to detect the ZHX2 protein expression. （2） Thirty-six 8-week-old male C57BL6 mice wererandomly divided into equivalent dose solvent CCI group, nonsense negative control sequence [siNC] CCI group and ZHX2 siRNA CCI group, and equivalent dose solvent sham-operated group, siNC sham-operated group and ZHX2 siRNA sham-operated group （n=6）; each treatment was given to the DRG. One d before drug injection and 7 d after drug injection, the changes of PWF to mechanical stimuli and PWL to thermal stimulation were detected; RT-qPCR was used to detect the ZHX2 mRNA expression. Results （1） Seven d after modeling, PWF was significantly increased and PWL was statistically shorten in the hind-paw of the ipsilateral side in the CCI group as compared with those in the sham-operated group （P〈0.05）; DRG ZHX2 mRNA expression in the CCI group increased for 1.71 times as compared with that in the sham-operated group, with significant difference （P〈0.05）; DRG ZHX2 protein expression in the CCI group increased for 2.15 times as compared with that in the sham-operated group, with significant difference （P〈0.05）. （2） As compared with the equivalent dose solvent sham-operated group, siNC sham-operated group and ZHX2 siRNA sham-operated group, equivalent dose solvent CCI group and siNC CCI group had significantly increased PWF and DRG ZHX2 mRNA expression, and statistically shorten PWL （P〈0.05）; and ZHX2 siRNA CCI group had significantly decreased PWF of the ipsilateral side and DRG ZHX2 mRNA expression, and statistically longer PWL of the ipsilateral side as compared with equivalent dose solvent CCI group and siNC CCI group （P〈0.05）. Conclusion Knockdown periphery nerve injury-induced DRG ZHX2 up-regulation attenuates pain hypersensitivity following periphery nerve injury, and ZHX2 may be a new potential target to treat NP.