针刺治疗功能性便秘的肠道菌群和肠道动力交互作用机制研究

Functional constipation is a common clinical disease. Modern medicine has no effective interventions. Our research group has confirmed that acupuncture is effective and safe for the treatment of FC, and the previous project supported by National Natural Science Foundation of China found that acupuncture can promote the GDNF expression in EGC, regulate the ENS-ICC-SMC function network to promote intestinal motility. Recent studies show that intestinal flora disorder is closely related to the occurrence and development of FC, moreover, as the target cell of intestinal flora, EGC is the key to the interaction of flora and dynamics. A number of studies have shown that acupuncture has a good effect on intestinal flora and motility. According to the above description , our project team put forward the following hypothesis: Acupuncture may affect the interaction between intestinal flora and intestinal motility by regulating the TLR2-NF-κB-GDNF signaling pathway mediated by EGC, to restore the homeostasis and improve the intestinal function. In this study, functional constipation mice are used as the research object, and acupuncture is used as an intervention to improve the intestinal function. Using high-throughput sequencing, immunofluorescence labeling combined with laser confocal microscopy and other technologies, also utilizing germ-free mice and TLR2 knockout mice, to reveal the mechanism of EGC, intestinal flora, intestinal motility and TLR2 signaling pathway in improving intestinal function by acupuncture, and to provide the scientific basis for the popularization and application of acupuncture in treating dominant diseases.

功能性便秘（FC）是临床常见病，现代医学缺乏有效干预手段。课题组前期研究已证实针刺治疗该病疗效确切、安全性高。前一个面上项目发现针刺可促使EGC分泌GDNF、调控ENS-ICC-SMC功能网络以促进肠道动力。最新研究表明肠道菌群紊乱与FC的发生发展密切相关，而EGC作为肠道菌群作用的靶细胞，是菌群和动力交互作用的枢纽。针刺对肠道菌群和动力均有良好调节作用。据此，项目组提出如下假说：针刺可能通过调控EGC介导的TLR2-NF-κB-GDNF信号通路影响肠道菌群和肠道动力的交互作用，恢复二者动态平衡，改善肠道功能。因此，本项目以FC小鼠模型为研究载体，以针刺大肠合募穴为干预措施，采用高通量测序、免疫荧光结合激光共聚焦显微镜等技术，并引进无菌小鼠和TLR2-/-小鼠，从正、反两面揭示EGC、肠道菌群、肠道动力和TLR2信号通路在针刺改善肠道功能中的作用机制，为针灸优势病种的推广应用提供科学依据。

功能性便秘（FC）是临床常见病，严重影响患者生活质量。团队前期已证实针刺治疗该病疗效确切。前一个面上项目并发现针刺可促使EGC分泌GDNF、调控ENS-ICC-SMC功能网络，促进肠道动力。有研究表明肠道菌群紊乱与FC密切相关，而EGC作为肠道菌群的靶细胞，是菌群和动力交互作用的枢纽。针刺对肠道菌群和动力均有良好调节作用。故提出假说：针刺可能通过调控EGC介导的TLR2-NFκB-GDNF信号通路影响肠道菌群和肠道动力的交互作用，恢复二者动态平衡，改善肠道功能。. 本项目以FC模型小鼠为研究载体，以针刺大肠合募穴为干预措施，采用高通量测序、免疫荧光结合激光共聚焦显微镜等技术，引进无菌小鼠及TLR2基因敲除小鼠，从正、反两面揭示EGC、肠道菌群、肠道动力和TLR2信号通路在针刺改善肠道功能中的作用机制，为针灸优势病种的推广应用提供科学依据。. 本项目分别从针刺对FC野生型小鼠和无菌小鼠肠道动力、肠道菌群、短链脂肪酸影响及针刺对FC野生型小鼠和TLR2基因敲除小鼠肠道动力、肠道菌群、EGC及TLR2-NFκB-GDNF信号通路影响六个方面的实验进行研究。. 结果表明：1.针刺可缩短FC小鼠首粒黑便排出时间，增加8 h排便粒数、重量及粪便含水率、小肠推进率及胃排空率，促进肠道动力，但仅对无菌小鼠小肠推进率有促进作用，对无菌小鼠及TLR2基因敲除小鼠的其余指标调节作用不明显。2.针刺可上调FC小鼠葡萄球菌科微生物，也可促进FC小鼠丁酸产生，但对无菌小鼠改善作用不明显。且丁酸与针刺上调葡萄球菌科微生物具有相关性，提示针刺可能通过调节肠道菌群，影响短链脂肪酸，促进肠道动力。3.针刺可调节FC小鼠厚壁菌门、拟杆菌门、疣微菌门等微生物，但对TLR2基因敲除小鼠改善作用不明显。4.针刺可恢复EGC形态，抑制EGC异常活化，降低TLR2、MyD88、NFκB、TNFα表达，升高GDNF表达，但对TLR2基因敲除小鼠调节作用不明显。且TLR2通路与肠道菌群具有相关性，提示针刺可能通过调控TLR2信号通路，调节肠道菌群和肠道动力。