慢性吗啡下调小胶质细胞焦亡介导中枢神经系统免疫抑制的作用及其调控机制研究

Morphine is the first line of medication for chronic pain and the clinical usage of morphine increases gradually. However, long-term exposure to morphine will result in neural cell injuries and abnormal immunity in central nervous system. We previously found that chronic morphine induced immunosuppression in central nervous system, which would further exacerbate neural disorders. However, mechanisms in initiation of neural immunosuppression induced by chronic morphine are unclear. We recently discovered that chronic morphine exposure down-regulated the pyroptosis signaling in the immune cells of the central nervous system – microglia, and pyroptosis of microglia was prone to disruption. Pyroptosis is one of the programmed cell death which interacts with inflammatory signaling. Whether down-regulation of pyroptosis of microglia can mediate immunosuppression in central nervous system is unclear. The underlying mechanisms still remain elusive. Activation of pyroptosis is dependent of mitochondrial functions, and our recent preliminary study showed that chronic morphine exposure lead to abnormal expression of mitophagy-inducer which regulates mitochondrial functions. Therefore, we hypothesize that chronic morphine exposure would regulate mitophagy and mitochondrial functions thereby downregulating pyroptosis of microglia, which consequently leads to immunosuppression. This study will verify that chronic morphine induce immunosuppression in central nervous system by downregulating pyroptosis of microglia. This study will help to discover the therapeutic target for immunosuppression in central nervous system after chronic morphine exposure.

吗啡作为慢性疼痛的一线治疗药物，其临床使用逐年增加；但其长期使用可导致中枢神经细胞损伤及免疫异常。我们前期研究发现，慢性吗啡摄入导致中枢神经系统免疫抑制，可协同加重神经功能障碍。然而慢性吗啡导致神经系统免疫抑制的机制仍很不明确。我们预实验发现：慢性吗啡可下调中枢神经系统免疫细胞——小胶质细胞的焦亡信号通路及焦亡（pyroptosis）发生。焦亡是由炎症信号参与激活的细胞程序性死亡，小胶质细胞焦亡信号的下调是否介导中枢神经系统免疫抑制？其作用、相互关系及内在机制并不明确。由于焦亡的激活依赖于线粒体功能，线粒体自噬可调控线粒体功能，而我们进一步预实验发现：慢性吗啡摄入后线粒体自噬诱导因子表达异常。因此，我们假设：慢性吗啡通过调控线粒体自噬，改变焦亡通路上游的线粒体功能而下调小胶质细胞的焦亡发生，导致免疫抑制。本课题将验证上述假设，为寻找慢性吗啡所致中枢神经系统免疫抑制的治疗靶点提供科学依据。

研究背景：吗啡是慢性疼痛的一线治疗药物，其使用人群及总体使用量日益增多，但长期的吗啡摄入可导致中枢神经系统免疫抑制。最近研究发现焦亡是炎症反应中的关键生物学过程，吗啡摄入是否导致中枢神经系统小胶质细胞焦亡过程的变化仍不清楚。本研究拟通过实验研究慢性吗啡摄入后对小胶质细胞焦亡发生的影响及机制。.研究方法和内容：使用皮下注射剂量递质的吗啡注射液建立小鼠慢性吗啡成瘾模型。使用原代小胶质细胞和BV2细胞系建立吗啡处理的细胞培养模型。使用LPS诱导炎症反应，使用ATP诱导焦亡发生。脑组织、细胞分别提取蛋白、mRNA进行western blot和PCR分析。测定细胞培养体系中LDH浓度检测细胞焦亡的发生。.结果：慢性吗啡摄入或处理后，小鼠脑组织、原代小胶质细胞、BV2细胞系均发现LPS诱导的焦亡信号通路分子NLRP3蛋白、caspase-1激活下调。LPS联合ATP处理原代培养小胶质细胞可导致细胞肿胀，随后崩解，释放IL-1β和LDH增多，提示小胶质细胞焦亡发生。吗啡处理后的原代小胶质细胞经过LPS-ATP联合处理后，肿胀减轻，释放IL-1β和LDH较少。BV2细胞系中，吗啡处理可逆转LPS诱导的NLRP3上调和caspase-1激活，吗啡处理也可减少LPS诱导的细胞释放IL-1β和LDH。研究发现，吗啡逆转LPS介导的焦亡分子伴随着吗啡介导的parkin上调，提示吗啡上调parkin是发生NLRP3下调的可能机制。.结论：慢性吗啡摄入可能通过上调小胶质细胞parkin从而减少NLRP3的表达及下调下游焦亡信号通路。