Wang Ying, Guo Qulian, Yan Jianqin, Yang Minghua, Lin Yan, Lin Zhiping
Department of Anesthesiology, Teaching Hospital of Fujian Medical University/Fujian Cancer Hospital, Fuzhou 350014, China.
Department of Anesthesiology, Xiangya Hospital, Central South University, Changsha 410008, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2016 Jun 28;41(6):578-85. doi: 10.11817/j.issn.1672-7347.2016.06.005.
To explore the role of DNA methyltransferases (DNMTs) in the pathogenesis of neuropathic pain (NPP) in rats following sciatic nerve chronic constriction injury (CCI).
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.
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).
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.
探讨DNA甲基转移酶（DNMTs）在大鼠坐骨神经慢性压迫性损伤（CCI）后神经性疼痛（NPP）发病机制中的作用。
将成功植入腰段鞘内导管的27只成年雄性Sprague-Dawley大鼠随机分为3组：假手术+生理盐水组（假手术+NS组）、CCI+NS组和CCI+5-氮杂胞苷组（CCI+5-AZA组）（每组n = 9）。假手术+NS组和CCI+NS组大鼠接受生理盐水，而CCI+5-AZA组大鼠在CCI手术后第3天至第14天每天通过脊髓注射给予10 μmol/L的5-氮杂胞苷（一种DNMTs抑制剂）。在CCI手术前或手术后第3、5、7、10或14天测量3组大鼠同侧后肢的机械缩足阈值（MWT）和热缩足潜伏期（TWL）。实验结束时，所有大鼠在深度麻醉下处死，解剖其腰段脊髓，分别通过RT-PCR、Western印迹和免疫组织化学检测DNMT1、DNMT3a和DNMT3b的表达。
与假手术+NS组相比，CCI+NS组大鼠在手术后第3天至第14天MWT和TWL明显降低（均P<0.05）。与CCI+NS组相比，CCI+5-AZA组大鼠在手术后第5天至第14天MWT和TWL明显升高（均P<0.05），但与假手术+NS组相比仍降低（均P<0.05）。所有大鼠腰段脊髓背角中DNMT1、DNMT3a和DNMT3b均高表达，阳性信号主要位于细胞核。与假手术+NS组相比，CCI+NS组大鼠在手术后第14天DNMT1、DNMT3a和DNMT3b水平显著升高（均P<0.05）。与CCI+NS组相比，CCI+5-AZA组大鼠DNMT1、DNMT3a和DNMT3b表达显著降低（均P<0.05），但与假手术+NS组相比仍升高（均P<0.05）。
腰段脊髓中DNMTs的上调可能在CCI大鼠NPP的发病机制中起重要作用。DNMTs抑制剂（5-氮杂胞苷）可降低DNMTs表达并减轻CCI诱导的NPP，这可能是NPP的一种潜在治疗药物。
