[Role of ZHX2 in regulating dorsal root ganglion μ-opioid receptor expression in mice with peripheral nerve injuryinduced pain hypersensitivity]

Abstract

Objective: To investigate the role of zinc-fingers and homeoboxes 2 (ZHX2) in regulating μ-opioid receptor expression in the dorsal root ganglion (DRG) in mice with peripheral nerve injury-induced pain hypersensitivity.

Methods: Forty-eight male adult C57BL6J mice were randomized into 4 groups and subjected to chronic constriction injury (CCI) of the sciatic nerve or sham operation followed by microinjection of a specific small interfering RNA (siRNA) of ZHX2 or a negative control siRNA sequence (siNC) into the DRG. Seven days later, the mice were examined for changes in the hind paw withdrawal frequency (PWF), after which the DRG tissue was collected for detecting the expressions of μ-opioid receptor at the mRNA and protein levels using RT-qPCR and Western blotting. In another experiment, the DRG tissues were collected from 6 mice (21-day-old) for primary culture of the DRG neurons, which were transfected with ZHX2 siRNA or the siNC to observe the changes in the expressions of ZHX2 and μ-pioid receptor.

Results: Microinjection of ZHX2 siRNA into the ipsilateral L3 and L4 DRGs significantly reversed CCI-induced μ-pioid receptor downregulation in the injured DRG and alleviated CCI-induced mechanical allodynia in the mice. In the cell experiment, ZHX2 knockdown obviously upregulated the mRNA and protein expressions of opioid receptor in the primary cultured DRG neurons.

Conclusions: ZHX2 knockdown in the DRG reverses CCI-induced down-regulation of μ opioid receptor to alleviate periphery nerve injury-induced pain hypersensitivity in mice.

Keywords: dorsal root ganglia; nerve injury; neuropathic pain; zinc-fingers and homeoboxes 2; μ-opioid receptor.

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反转录实时定量PCR检测敲减CCI引起的DRG Zhx2增加对Oprm1 mRNA表达的变化 Effect of microinjection of ZHX2 siRNA (siRNA) or the negative control siRNA (siNC) into the ipsilateral L3/4 DRGs on Oprm1 expression in the DRGs on day 7 after CCI or sham operation (n=6). Unilateral L3/4 DRGs from two mice were pooled together. ^aP < 0.01 vs siNC+sham group; ^bP < 0.05 vs siNC+ CCI group

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Western blotting检测敲减CCI引起的DRG ZHX2增加对MOR表达的变化 Effect of microinjection of ZHX2 siRNA or siNC into the ipsilateral L3/4 DRGs on m opioid receptor (MOR) expression on day 7 after CCI or sham operation (n=6). Unilateral L3/4 DRGs from two mice were pooled together. ^aP < 0.01 vs siNC+sham group. ^bP < 0.05 vs siNC+CCI group

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4组小鼠患侧后爪PWF的比较 Comparison of hind paw withdrawal frequency on the ipsilateral side in the 4 groups 7 days after CCI injury or sham operation with ZHX2 siRNA or siNC into the ipsilateral L3/4 DRGs (n=6). ^aP < 0.01 vs siNC+sham group. ^bP < 0.05 vs siNC+CCI group

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4组小鼠对侧后爪PWF的比较 Comparison of hind paw withdrawal frequency on the contralateral side of the mice on day 7

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敲减DRG神经元细胞ZHX2表达对Oprm1基因表达的影响 Effect of ZHX2 knockdown in the DRG on Oprm1 mRNA expression in DRG primary neurons (Mean±SD, n=3). ^aP < 0.01 vs siNC group

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敲减DRG神经元细胞ZHX2表达对MOR蛋白质表达的影响 Effect of ZHX2 knockdown on m opioid receptor (MOR) protein expression in DRG primary neurons (n=3). ^aP < 0.01 vs siNC group