DNA甲基转移酶类在大鼠神经病理性疼痛发病机制中的作用

目的：探索DNA甲基转移酶类(DNA methyltransferases，DNMTs)在坐骨神经慢性缩窄性损伤(chronic constriction injury，CCI)大鼠神经病理性疼痛(neuropathic pain，NPP)发病机制中的作用。方法：腰段鞘内置管成功的27只成年雄性Sprague-Dawley大鼠，随机分为假手术组+生理盐水组(sham+NS组)、CCI+NS组及CCI+5-氮杂胞苷(5-azacytidine，5-AZA)组(CCI+5-AZA组)，每组9只。CCI术后第3天至第14天，sham+NS组和CCI+NS组每天鞘内注射1次NS，CCI+5-AZA组每天鞘内注射1次10 μmol/L 5-AZA。分别于术前及术后第3，5，7，10，14天测定各组大鼠术侧后足机械痛阈和热痛阈。术后第14天各组大鼠于深麻醉下处死，取脊髓腰膨大，采用RT-PCR，Western印迹和免疫组织化学测定DNMT1，DNMT3a和DNMT3b的表达。结果：术后第3天至第14天，CCI+NS组的机械痛阈和热痛阈均较sham+NS组明显下降(均P<0.05)。术后第5天至第14天，CCI+5-AZA组的机械痛阈和热痛阈均较CCI+NS组明显上升(均P<0.05)，但仍低于sham+NS组(均P<0.05)。DNMT1，DNMT3a和DNMT3b在大鼠脊髓腰膨大背角处有致密表达，且以胞核分布为主。术后第14天CCI+NS组的DNMT1，DNMT3a和DNMT3b表达较sham+NS组均明显上升(均P<0.05)。CCI+5-AZA组的DNMT1，DNMT3a和DNMT3b表达均较CCI+NS组明显下降(均P<0.05)，但仍高于sham+NS组(均P<0.05)。结论：DNMTs在脊髓腰膨大处的表达上调很可能在CCI大鼠NPP发病机制中起重要作用。DNMTs抑制剂5-AZA可下调DNMTs的表达和缓解CCI诱导的NPP，可能成为NPP的潜在治疗药物。

Role of DNA methyltransferases in the pathogenesis of neuropathic pain in rats

Objective: To explore the role of DNA methyltransferases (DNMTs) in the pathogenesis of neuropathic pain (NPP) in rats following sciatic nerve chronic constriction injury (CCI). Methods: A total of 27 adult male Sprague-Dawley rats with successful implantation of lumbar intrathecal catheter were randomly divided into 3 groups: a sham + normal saline group (sham+NS group), a CCI+NS group, and a CCI+5-azacytidine group (CCI+5-AZA group) (n=9 in each group). The rats in the Sham+NS group and the CCI+NS group received NS, while the rats in the CCI+5-AZA group received 10 μmol/L of 5-AZA (a DNMTs inhibition) once a day through spinal injection from the 3th day to 14th day after CCI surgery. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of ipsilateral hinds in the 3 groups were measured before or at the 3th, 5th, 7th, 10th or 14th day after CCI surgery. At the end of experiments, all rats were killed under deep anesthesia and their lumbar spinal cords were dissected to examine the DNMT1, DNMT3a and DNMT3b expression by RT-PCR, Western blot and immunohistochemistry, respectively. Results: Compared with the sham+NS group, the MWT and TWL in the CCI+NS group were obviously reduced from the 3th day to the 14th day after surgery (both P<0.05). Compared with the CCI+NS group, the MWT and TWL in the CCI+5-AZA group were obviously increased from the 5th day to the 14th day after surgery (both P<0.05), but they were still reduced compared with the sham+NS group (both P<0.05). The DNMT1, DNMT3a and DNMT3b were highly expressed in the lumbar spinal dorsal horn in all rats, and the positive signals were mainly located in the nucleus. The DNMT1, DNMT3a and DNMT3b levels in the CCI+NS group were increased significantly compared with that in the sham+NS group on the 14th day after surgery (all P<0.05). The DNMT1, DNMT3a and DNMT3b expressions in the CCI+ 5-AZA group were decreased significantly compared with that in the CCI+NS group (all P<0.05), but they still increased compared with that in the sham+NS group (all P<0.05). Conclusion: Up-regulation of DNMTs in the lumbar spinal may play an important role in the pathogenesis of NPP in CCI rats. DNMTs inhibitors (5-AZA) could reduce expression of DNMTs and attenuate CCI-induced NPP, which might be a potential therapeutic drug for NPP.