CREB调控FTO介导的Bdnf mRNA m6A修饰在吗啡诱发痛觉过敏中的机制

Repeated use of morphine can lead to opioid-induced hyperalgesia (OIH), which is one of common issues in the treatment of clinical pain, but the mechanism is unclear. Previous working of applicant showed that in the environment of OIH, the expression level of brain-derived neurotrophic factor (BDNF) protein was several times that of the gene level, suggesting that BDNF post-transcriptional regulation plays an important role in it. The pilot data showed that on the OIH mouse model, N6-methyladenosine (m6A) of Bdnf mRNA in the dorsal root ganglion (DRG) neurons decreased, the RNA demethylation enzyme, fat mass and obesity-associated (FTO) increased and the transcription factor, cAMP response element-binding protein (CREB) was also increased. In view of this, we hypothesized that repeated application of morphine can make CREB phosphorylated and promote FTO high expression, resulting in decreased m6A modification on Bdnf mRNA, increasing BDNF protein production, and ultimately causing OIH. In the present study, we plan to repeat subcutaneous injection of morphine to prepare mouse models of OIH, using viral overexpression of CREB, conditional knockout FTO and other interventions to detect the behavior test, expression level of BDNF and FTO, phosphorylation level of CREB and m6A expression level of Bdnf mRNA in the DRG neurons to prove the hypothesis. This study is the depth and expansion of the previous research working of the applicant. It will help to reveal the mechanism of OIH, also provide a new target for clinical pain treatment and prevention of OIH.

反复应用吗啡可导致痛觉过敏(OIH)，是临床疼痛治疗常见的严重问题之一，但其机制不明。申请者前期研究显示，OIH环境下，BDNF蛋白水平表达是基因水平表达的数倍，提示BDNF转录后调控起重要作用。预实验发现，在OIH小鼠模型中，背根神经节（DRG）神经元Bdnf mRNA m6A减少，RNA去甲基化酶FTO增高，且转录因子CREB亦增高。鉴于此，提出假说：反复应用吗啡，使CREB磷酸化，促进FTO高表达，导致Bdnf mRNA m6A修饰减少，增加BDNF蛋白生成，最终引起OIH。本研究拟通过反复皮下注射吗啡，制备小鼠OIH模型，采用病毒过表达CREB，条件性敲除FTO等技术干预后，检测行为学、DRG神经元BDNF、FTO、CREB磷酸化水平及Bdnf mRNA m6A水平证明以上假说。本研究是申请者前期研究的深入和扩展，有助于揭示OIH的机制，为临床疼痛治疗并预防OIH发生提供新靶点。

反复应用吗啡可导致痛觉过敏(OIH)，是临床疼痛治疗常见的严重问题之一，但其机制不明。申请者前期研究显示，OIH环境下，BDNF蛋白水平表达是基因水平表达的数倍，提示BDNF转录后调控起重要作用。预实验发现，在OIH小鼠模型中，背根神经节（DRG）神经元Bdnf mRNA m6A减少，RNA去甲基化酶FTO增高，且转录因子CREB亦增高。鉴于此，提出假说：反复应用吗啡，使CREB磷酸化，促进FTO高表达，导致Bdnf mRNA m6A修饰减少，增加BDNF蛋白生成，最终引起OIH。本研究通过反复皮下注射吗啡，制备小鼠OIH模型，采用病毒过表达CREB，条件性敲除FTO等技术干预后，检测行为学、DRG神经元BDNF、FTO、CREB磷酸化水平及Bdnf mRNA m6A水平证明以上假说。研究结果证实，OIH模型中，小鼠DRG神经元BDNF蛋白表达水平，Bdnf基因mRNA转录水平增加，对DRG组织中发生m6A修饰的所有基因行聚类分析，发现BDNF mRNA发生负相m6A修饰。检测了m6A擦写酶FTO蛋白表达水平显著增加，Fto mRNA表达水平显著上升，Creb mRNA表达水平显著上升，CREB磷酸化水平显著上升，Bdnf mRNA和蛋白表达水平显著上升。应用FTO的特异性抑制剂FB23-2后，小鼠行为学疼痛阈值提高，FTO蛋白表达水平显著下降，Bdnf mRNA m6A水平显著增高, BDNF蛋白表达水平显著下降，CREB磷酸化水平显著下降。OIH是影响患者阿片类药物治疗效果和生活质量的重要因素，然而由于OIH的发病机制仍不清楚，使得相关的治疗难有突破性进展。本课题结果显示吗啡引起痛觉过敏的模型中，使CREB磷酸化，促进FTO高表达，导致Bdnf mRNA m6A修饰减少，增加BDNF蛋白生成，最终引起痛觉过敏。若本课题假说被证明是正确的，则可将FTO作为新的干预靶点，通过表观遗传学调控对OIH进行有效治疗，缓解病人的痛苦，提高生活质量。