组蛋白去乙酰化酶调控阿片受体参与慢性胰腺炎疼痛的机制研究

Chronic pancreatitis (CP) is a common disease of digestive system threatening the health of people. Pain is the most cardinal symptom of CP which still lacking effective treatment, and the mechanisms are poorly understood. Pain related gene expression alters without gene sequence changes in CP was most probably regulated by epigenetic mechanisms, epigenetics is a set of reversible and heritable changes in gene functions or other cell phenotypes that occur without changes in DNA sequence (genotype). These changes may be spontaneously induced or in response to environmental factors. And the most important epigenetic mechanisms is the histone acetylation/deacetylation, which regulated by HAT(histone acetyltransferase) and HDAC(histone deacetylase). In a rat CP model induced by trinitrobenzene sulfonic acid (TNBS), we observed that the HDAC expression level was significantly increased in the spinal dorsal horn, with the μ-opioid receptor(MOR) down-regulated simultaneously, and there’s a significant correlation between them. In addition, the astrocyte and microglia activation were also observed in the CP status, these changes might be the key mechanism of CP pain, but the mechanism has not been elucidated. Because of the significance of the enhanced HDAC in the CP and its role in the pain induction and maintenance, it will be worth to investigating the mechanisms of these expression changes. Thus, we proposed a hypothesis: under the CP status, epigenetic modification such as the histone acetylation/deacetylation activated by the glia activation, the enhanced HDAC expression down-regulated the μ-opioid receptor, then the balance of endogeneous analgesic system interrupted, which will promote the central sensitization of pain. And HDAC inhibitors may significantly alleviate the CP pain. For solving these problems and providing evidences for the hypothesis, our project intends to use the TNBS induced rat CP pain model, through a series of neuroscientific researches, aimed on: (1)Reveal and verify the impact of HDAC on the MOR expression and the effect on the pain behavior changes; (2)Verify the specific analgesic effect of the HDAC inhibitors; (3)Reveal the synaptic plasticity changes in the spinal cord dorsal horn; (4)Verify the impact of glial cells on HDAC expression, and explore the molecular regulate mechanism of HDAC on the endogenous opioid analgesic system. The project will clarify the HDAC regulating mechanisms involved in the formation of CP pain, help preventing CP induced pain and supply novel concept on special analgesic drugs research.

慢性胰腺炎(CP)是临床常见疾病，疼痛是其最突出的临床症状，且发生机制不明。在CP状态下痛相关基因的表达主要受组蛋白乙酰化/去乙酰化为主的表观遗传学调控。我们已经在大鼠CP模型中观察到脊髓背角组蛋白去乙酰化酶（Histone deacetylase,HDAC）的表达明显增强，且μ受体（MOR）表达显著下调，两者有明显相关性。另外，CP时胶质细胞也有明显活化，这些变化可能是CP时导致疼痛的关键机制，但其机理仍未阐明。本项目拟以三硝基苯磺酸（TNBS）诱导的大鼠CP疼痛模型为基础，使用现代神经科学手段开展相关研究，目的在于：①揭示和验证HDAC对MOR表达的影响及其与痛行为的关系；②验证HDAC抑制剂的特异性镇痛效应；③明确脊髓背角突触可塑性变化；④明确胶质细胞对HDAC的影响，及HDAC调控内源性阿片受体镇痛系统的分子机理。对进一步阐明CP疼痛的形成机制、预防CP致痛和新药研发有重要意义。

慢性胰腺炎(CP)是临床常见疾病，疼痛是其最突出的临床症状，且发生机制不明，现有药物疗效不佳，副作用多。在CP状态下痛相关基因的表达主要受组蛋白乙酰化/去乙酰化为主的表观遗传学调控。在本研究中，我们在三硝基苯磺酸（TNBS）诱导的大鼠CP模型中观察组蛋白去乙酰化酶2（HDAC2）介导CP疼痛的机制。通过研究脊髓背角HDAC2的表达和变化趋势，通过HDAC2抑制剂测试其与疼痛行为学改变和阿片受体表达变化的相关性分析，膜片钳记录脊髓背角自发/诱发EPSC幅值和频率变化，以及脊髓背角相关炎症因子（IL-1β、IL-6、TNF-α）的改变，PI3K、JNK信号通路的变化，揭示HDAC2在CP痛敏中的作用机制。结果显示脊髓背角HDAC2的表达明显增强，且μ受体（MOR）表达显著下调，两者有明显相关性。鞘内注射HDAC2抑制剂可显著减轻CP引起的机械性痛觉过敏。结果显示，CP诱导胶质HDAC2表达于神经元，可能通过促进神经胶质细胞活化释放炎性细胞因子IL-1β、IL-6和TNF-α。此外，对于神经胶质细胞的抑制作用也表现出抗炎和缓解痛敏的作用。HDAC2抑制MOR活动的机制可能与导致JNK和PI3K信号通路的激活有关。结论：在TNBS诱导的CP状态下，胶质细胞的活化会引起TNF-α、IL-1β、IL-6等细胞因子的释放，进而组蛋白乙酰化/去乙酰化的表观遗传学调控机制受到激活，HDAC2的表达升高，通过调节JNK、PI3K等相关信号通路，进一步引起MOR的表达抑制，内源性镇痛系统失衡，造成中枢敏化，形成慢性胰腺炎的痛觉敏化状态。