肠道菌群免疫对话中CRF-NLRP6炎性小体相关通路的电针调节机制

Intestinal flora-immunity dialogue plays a key role in maintaining the balance of intestinal microenvironment. IBS is one of the most common intestinal flora-immunity dialogue disorders.Our group has confirmed that acupuncture can regulate the intestinal flora structure and maintain the balance of gut immunity in IBS, but the regulatory mechanism among acupuncture, flora and immunity needs to be explained.The latest study shows that CRF-NLRP6inflammasomes related pathways plays an important role in intestinal flora-immunity dialogue.Therefore, this study is designed into the fllowed steps:①The WAS method was used to establish the model of intestinal flora-immunity disorders in IBS mice. It is supposed to prove that WAS could induce abnormality of CRF-NLRP6inflammasomes related pathways, then affect the intestinal flora. ②The WAS model group will be carried out electroacupuncture intervention to confirm that electroacupuncture could down-regulate CRF, recruit NLRP6 inflammasomes, regulate immunity and recover homeostasis of flora. ③Using siRNA technique to make NLRP6 silence,and we observe the effect of electro-acupuncture on intestinal flora and immunity to confirm that CRF-NLRP6 inflammasomes related pathways is the key pathway of electroacupuncture effect. It could confirm the hypothesis that electroacupuncture stabilizes the intestinal flora-immunity dialogue by regulating the CRF-NLRP6 inflammasomes related pathways from the perspectives of the three steps above.It could also provide a new scientific basis for acupuncture treatment of IBS, and enrich the theoretical connotation of acupuncture.

肠道菌群免疫对话在维持肠道微环境平衡中起关键作用，IBS是最常见的肠道菌群免疫对话失调疾病之一。项目组已证实针刺可以调节IBS肠道菌群结构、维持肠道免疫平衡，但针刺、菌群、免疫三者之间调控机制凾待阐释。最新研究显示CRF-NLRP6炎性小体相关通路在肠道菌群免疫对话中发挥重要作用。因此，本项目拟①通过WAS法建立肠道菌群免疫失调IBS小鼠模型，证实WAS可以致CRF-NLRP6炎性小体相关通路异常而影响菌群；②WAS模型组实施电针干预，证实电针可以下调CRF，募集NLRP6炎性小体，调节免疫，恢复菌群稳态；③通过siRNA技术沉默NLRP6，观察电针对菌群、免疫的影响，证实CRF-NLRP6炎性小体相关通路是电针效应的关键途径。从上述三个层面验证“电针对肠道菌群免疫对话的稳定作用是通过调节CRF-NLRP6炎性小体相关通路实现的”假说。为针灸治疗IBS提供新的科学依据，丰富针灸学理论内涵。

肠易激综合征（IBS）是常见的功能性胃肠疾病，发病机制复杂，其中肠道菌群免疫对话失调是IBS重要的发病机制之一。胞内核苷酸结合寡聚化结构域蛋白样受体6（NLRP6）在可接收菌群信号并促进肠道免疫系统做出免疫应答反应，因此CRF-NLRP6炎性小体相关通路可能是阐明针刺对菌群-免疫调节的分子生物学机制新的切入点。本研究采用避水应激（WAS）法构建IBS小鼠模型，选取“天枢”、“足三里”作为电针干预穴位，并应用腺相关病毒体内干扰或过表达NLRP6，采用腹壁撤离反射实验、旷场实验、肠道动力测试评估IBS模型小鼠的内脏敏感性、焦虑抑郁情况和肠道动力，采用WB、PCR、免疫荧光等方法检测IBS小鼠结肠组织NLRP6炎性采用小体（NLRP6，ASC，Caspase 1）及其下游信号通路相关因子表达水平并采用16S rRNA检测肠道菌群。本研究结果发现电针可缓解WAS诱导的IBS模型小鼠的内脏高敏感、肠动力紊乱、焦虑抑郁状态，促进IBS模型小鼠结肠组织中NLRP6炎性小体的激活，降低IBS模型小鼠结肠组织中CRF的表达，增加IBS模型小鼠结肠组织中MUC2、E-cadherin、IL-18及抗菌肽（ITLN1， RELMβ，Ang4)的表达，并对肠道菌群具有调控作用。本研究得出结论电针缓解WAS导致的IBS相关症状可能与稳定肠道菌群免疫有关，而且其作用可能是通过调控NLRP6炎性小体相关通路实现的。本研究为针刺治疗IBS提供了科学依据，丰富了针灸学理论内涵，利于针灸疗法的临床推广应用。