DNA甲基化在坐骨神经慢性缩窄性损伤大鼠神经病理性疼痛发生发展中的作用机制

Neuropathic pain is a chronic pain, with the increased incidence year by year. Pathogenesis of neuropathic pain is complex and not discovered clearly, and the conventional painkillers such as opioids and non-steroidal anti-inflammatory drugs have poor effectiveness for it. It’s urgent to explore the pathogenesis of neuropathic pain clearly and find better therapic drugs for it. Recent evidence suggests that transcription and expression level of pro- or antinociceptive genes are highly involved in the generation and maintenance of neuropathic pain. DNA methylation is one of the earliest discovered and most characteristic epigenetic mechanisms in mammals, which can regulate gene transcription and expression. Our previous results showed up-regulated global DNA methylation, DNA methyltransferase 1 (DNMT1), DNMT3a, DNMT3b and methyl-CpG-binding protein 2 (MeCP2) expression were clearly detected in the lumbar spinal cord in rats following sciatic nerve chronic constriction injury (CCI) 14 days after surgery, accompanying with mechanical allodynia and thermal hyperalgesia. Mechanical allodynia and thermal hyperalgesia induced by CCI were attenuated by intrathecal 5-azacytidine (an demethylation reagent). That indicates DNA methylation may be highly involved in the neuropathic pain in CCI rats. For further study of the specific mechanisms of DNA methylation in neuropathic pain, this research project intends to create the methylation profiles of the genome in the lumbar spinal cord, to find the specific pain-related genes regulated by DNA methylation and the specific regulatory pathways, and to explore the specific mechanisms of intrathecal 5-azacytidine on neuropathic pain in CCI rats. There are few studies about the involvement of DNA methylation in neuropathic pain, and successful completion of the study may give some new ideas and new target points in the prevention and treatment of neuropathic pain.

神经病理性疼痛（NPP）发病机制不清，发病率逐年上升，疗效不佳，是亟待解决的研究难点。大量疼痛基因表达水平的改变是NPP发生发展中极其重要的机制，DNA甲基化是调控基因表达的重要表观修饰方式之一。申请人前期研究首次发现坐骨神经慢性缩窄性损伤（CCI）大鼠脊髓腰膨大处总体DNA甲基化水平、DNA甲基转移酶类和甲基化CpG连接蛋白2表达均显著增加，并伴有NPP的产生和维持，鞘内注射去甲基化试剂5-氮杂胞苷（5-AZA）可缓解CCI大鼠的疼痛，提示DNA甲基化机制参与NPP的发生发展。本课题进一步深入研究DNA甲基化参与CCI大鼠NPP的具体机制，采用DNA甲基化芯片、DNA焦磷酸测序及染色质免疫共沉淀等方法构建CCI大鼠脊髓腰膨大处基因组甲基化谱，找寻受DNA甲基化调控的具体疼痛基因和具体途径及明确鞘内注射5-AZA对CCI大鼠NPP调节作用的具体机制，以期为NPP的有效防治提供新思路和靶标。

我们前期研究结果显示坐骨神经慢性缩窄性损伤（chronic constriction injury，CCI）大鼠脊髓腰膨大处总体DNA甲基化水平和甲基化的CpG连接蛋白2（methyl-CpG binding protein 2，MeCP2）的表达水平显著增加，提示DNA甲基化机制参与了CCI大鼠神经病理性疼痛（neuropathic pain，NPP）的发生发展。本课题在前期研究基础上采用DNA甲基化芯片、甲基化DNA免疫共沉淀-实时定量PCR法（methylated DNA immunoprecipitation-qPCR，MeDIP-qPCR）、qRT-PCR及染色质免疫共沉淀PCR（Chromatin immunoprecipitation，CHIP-PCR）等方法进一步深入研究DNA甲基化在大鼠NPP发病机制中的重要作用。DNA甲基化芯片结果显示，CCI大鼠基因启动子中出现的差异富集峰（differential enrichment peaks，DEPs）共有1201个，DEPs所在的基因参与了长时程增强作用等多种功能通路，构建出了CCI大鼠脊髓腰膨大处基因组的DNA甲基化谱。采用MeDIP-qPCR对DNA甲基化芯片筛选出的8个重要疼痛相关基因Mir124-3、Grm4、Gad2、Pparg、Slc1a2、Adrb2、Kcnf1和Htr4进行验证。结果显示，Grm4和Htr4在CCI大鼠的甲基化水平均明显增高；Adrb2和Kcnf1甲基化水平在CCI大鼠升高，但差异尚无统计学意义，和甲基化芯片结果较一致。采用qRT-PCR检测CCI大鼠Grm4、Htr4、Adrb2和Kcnf1的表达改变。结果显示，CCI大鼠Grm4、Htr4、Adrb2和Kcnf1的表达水平均明显降低。最后，我们进一步观察腹腔注射5-氮杂胞苷（5-azacytidine，5-AZA）4mg/kg对CCI和脊神经结扎（spinal nerve ligation，SNL）大鼠NPP的影响和对Grm4、Htr4、Adrb2和Kcnf1表达的改变。结果显示，腹腔注射5-AZA可以明显缓解CCI和SNL大鼠的NPP，并能够显著提高Grm4、Htr4、Adrb2和Kcnf1的表达。本课题明确了DNA甲基化在NPP发病机制中的重要作用，为其有效防治和新药研发提供了新的方向。