肠胶质细胞NLRP6介导的神经炎症通路在IBS内脏高敏感中的作用机制研究

Alterations in the intestinal microbiota have been proven to be associated with visceral hypersensitivity in irritable bowel syndrome (IBS), but the underlying mechanisms remain unknown. NLRP6 and IL-18 is activated by the intestinal microbiota and maintains the intestinal homeostasis. IL-18 enhanced the production and secretion of nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) which promote the nerve pain in the skin. Our preliminary study has found that the NLRP6 was expressed in enteric glial cells (EGC). Therefore, the intestinal microbiota in the luminal could activate the NLRP6-IL18-NGF/GDNF signal in EGC, and result in the visceral hypersensitivity. (1) The role of NLRP6 and its mediated neuroinflammatory signal in the underlying mechanism about visceral hypersensitivity induced by intestinal dysbiosis will be studied in the IBS animal model with wild type mice and conditionally NLPR6 knock out mice and conditionally IL18 knock out mice. (2) To further clarify the underlying mechanism about how NLRP6 regulated the production and secretion of NGF and GDNF in EGC, EGC were transfected with NLRP6 s SiRNA plasmid and NLRP6 overexpression of lentivirus vector respectively. This study will clarify the pathophysiologic mechanism about visceral hypersensitivity in IBS induced by intestinal dysbiosis.

肠道菌群紊乱导致肠易激综合征（IBS）内脏高敏感的机制不清。NLRP6及其下游分子IL18被肠道菌群活化后参与维持肠道稳态。IL18可刺激皮肤合成参与神经痛形成的神经因子NGF和GDNF。预实验发现，肠胶质细胞（EGC）表达NLRP6。因此，位于肠腔内的肠道菌群可能通过活化EGC上NLRP6-IL18-NGF/GDNF神经炎症通路导致内脏高敏感。①应用条件性肠胶质细胞NLRP6基因敲除小鼠及条件性肠胶质细胞 IL18基因敲除小鼠，将腹泻型IBS患者及正常人的粪菌分别移植至其肠道，以明确肠道菌群紊乱通过活化EGC上NLRP6-IL18-NGF/GDNF通路导致内脏高敏感。②NLRP6 SiRNA及慢病毒过表达载体转染EGC体外培养，从正反两面进一步阐明NLRP6调控EGC合成NGF和GDNF的机制。本研究将深入阐明肠道菌群紊乱导致IBS腹痛的分子机制，为益生菌治疗IBS提供更深远的理论依据。

肠道菌群紊乱导致肠易激综合征（IBS）内脏高敏感的机制不清。NLRP6及其下游分子IL18 被肠道菌群活化后参与维持肠道稳态。IL18可刺激合成参与神经痛形成的神经因子NGF和GDNF。为阐明肠胶质细胞（EGC）上是否存在NLRP6-IL18-NGF/GDNF通路及该通路在肠道菌群紊乱导致内脏高敏感中的作用，本研究①将IBS大鼠的粪菌移植至SPF级大鼠，构建内脏高敏感模型，同时设立益生菌组及假造模组，分离结肠EGC，检测EGC上NLRP6-IL18-NGF/GDNF分子的表达水平。结果发现，肠道菌群失调可导致内脏高敏感及肠通透性增加，并抑制EGC上NLRP6分子的表达、促进EGC上ASC、IL18Rα及结肠IL18、GDNF、NGF的表达。益生菌可减轻内脏高敏感，并促进结肠EGC上NLRP6分子的表达，抑制EGC上ASC、IL-18Rα及降低结肠IL18、GDNF、NGF的表达。②EGC细胞株培养，依次给予NLRP6 SiRNA、NLRP6特异性激活物、IL18 SiRNA、外源性IL18重组蛋白。结果发现，IL18可以调控EGC分泌NGF及GDNF，但NLRP6分子不具有该作用。③益生菌与EGC细胞株共培养，依次给予NLRP6 SiRNA、IL18 SiRNA。结果发现，益生菌通过下调EGC上IL18来降低NGF及GDNF的表达，这个过程与NLRP6无关；益生菌可上调EGC上NLRP6分子的表达。因此，我们的研究表明肠胶质细胞上存在IL18-NGF/GDNF通路，且表达NLRP6分子，但NLRP6不调控NGF/GDNF的表达；肠道菌群紊乱可能通过抑制EGC上NLRP6的表达、及激活EGC上IL18-NGF/GDNF通路导致内脏高敏感性；益生菌具有促进EGC上NLRP6表达，抑制EGC上IL18-NGF/GDNF通路的作用，这可能是益生菌改善内脏高敏感性的新作用机制。