大麻素2型受体参与阻塞性黄疸病人角质形成细胞β-内啡肽表达升高的机制研究

It is well known that cholestatic liver disease is associated with increased levels of circulating endogenous opioids. Elevated plasma endogenous opioid peptides not only impair the cardiovascular, brain and renal functions, but also induce pruritus and antinociception in cholestatic liver disease. However, the specific mechanisms for this elevated opioid tone in jaundiced patients remains unclear. Our previous study had suggested that skin keratinocytes β- endorphin synthesis were elevated in patients with obstructive jaundice, and it may be contributed to the elevated endogenous opioid peptides during cholestasis. And we need specific mechanism to explain this experimental result. Recent studies had suggested that activation of cannabinoid receptor type 2(CB2) can increase the expression of β- endorphin in keratinocytes, and clinical evidence also suggested that the plasma level of endogenous CB2 receptor agonists were also elevated during cholestasis, these findings prompted us to test the hypothesis that CB2 receptor may contribute to up-regulation of β--endorphin in keratinocytes of patients with obstructive jaundice. To test this hypothesis, we will do clinical observational study, animal and cell study focus on CB2 receptor activation in keratinocytes may play an important role in the local synthesis of endogenous opioids during cholestasis. We believe the result of our study will provide new clinical and experimental evidence for approaches to prevent increased opioidergic tone associated complications in cholestatic patients.

阻塞性黄疸病程中升高的血浆内源性阿片肽常常引发顽固性瘙痒、心律失常、肾功能损害、脑功能改变等一系列严重的并发症。但是，对于黄疸病程中这一内源性阿片肽升高的具体机制仍不明确。我们的前期研究提示，阻黄病人皮肤角质形成细胞β-内啡肽合成增加，可能参与了这一内源性阿片肽升高的过程，但缺乏特异性的机制来解释这一实验现象。而现有证据表明大麻素2型受体的特异性激活可诱导角质形成细胞β-内啡肽的表达，同时临床试验也表明阻黄病程中大麻素2型受体的内源性激动剂表达升高，因此我们提出假设，阻黄病程中皮肤角质形成细胞大麻素2型受体被潜在性过度激活，是外周内源性β-内啡肽合成增加的可能机制。为验证这一假设，本课题将在人体、动物和细胞三个层面全面论证这条以大麻素2型受体为中心，皮肤角质形成细胞为靶细胞的内源性阿片肽合成通路，以期为胆汁淤积性疾病中内源性阿片肽升高相关并发症治疗的靶向药物设计提供新的临床和实验证据。

阻塞性黄疸病程中升高的血浆内源性阿片肽常常引发顽固性瘙痒、心律失常、肾功能损害、脑功能改变等一系列严重的并发症。但是，对于黄疸病程中这一内源性阿片肽升高的具体机制仍不明确。我们的前期研究提示，阻塞性黄疸病人皮肤角质形成细胞的β-内啡肽合成增加，可能参与了这一内源性阿片肽升高的过程，但缺乏特异性的机制来解释这一实验现象。本课题结合生物化学、分子生物学、免疫组织化学、原代细胞培养、动物行为学等实验技术手段从以下三个方面开展了研究：（1）比较了阻塞性黄疸病人和非黄疸病人血浆内源性大麻素受体激动剂（2-AG/AEA）的血浆浓度及皮肤β-内啡肽表达水平。（2）观察了CB2受体拮抗剂AM630对阻塞性黄疸大鼠模型外周β-内啡肽表达和基础痛阈值的影响。（3）角质形成细胞（HaCat）中CB2受体/MAPK/ STAT/β-内啡肽合成通路的验证。本课题的研究结果表明：（1） 阻塞性黄疸患者血浆内源性大麻素受体激动剂AEA及β-内啡肽浓度较对照组升高，存在CB2受体被内源性激活的可能。（2）阻塞性黄疸患者皮肤角质形成细胞中β-内啡肽的mRNA和蛋白表达水平均升高，表明角质形成细胞可能是体内β-内啡肽合成的部位。（3）在阻塞性黄疸大鼠模型中连续注射CB2受体拮抗剂AM630可降低模型大鼠血浆和皮肤角质形成细胞中的β-内啡肽表达水平，但无法改变其基础痛阈值，表明皮肤角质形成细胞上的CB2受体参与了β-内啡肽合成的过程，但并未参与基础痛阈值改变过程。（4）AEA通过CB2受体诱导HaCaT细胞β-内啡肽的表达和分泌，p42/p44 MAPK和p38 MAPK通路磷酸化参与了这一过程。本课题的研究结果提示阻塞性黄疸病程中存在着一条以CB2受体为中心的β内啡肽合成途径，解答了阻塞性黄疸患者内源性阿片肽升高的具体机制，并揭示了皮肤角质形成细胞在体内内源性阿片肽合成过程中的重要作用，丰富了内源性镇痛机制的相关理论，也为外周镇痛机制的研究提供了新的思路和方法。