丙酸和丁酸通过HCN2介导的5-HT合成参与腹泻型肠易激综合征脑-肠-微生态轴异常的研究

肠道菌群的发酵产物SCFAs导致5-HT紊乱，参与腹泻型肠易激综合征（IBS-D）脑-肠-微生态轴异常，但5-HT的调节信号通路不明。在预实验发现IBS-D患者血清丙酸和丁酸升高、HCN2在EC细胞中表达并与5-HT共定位基础上，我们推测丙酸和丁酸在肠道EC细胞和中枢神经元中分别通过其受体GPR43/41介导的cAMP调控HCN2，实现对5-HT合成关键酶Tph1和2的调节从而控制其合成，参与IBS-D脑-肠-微生态轴紊乱。在已有基础上，本项目拟在动物模型及IBS-D患者中干预肠道菌群，观察其对SCFAs、5-HT及中枢的影响来分析三者间的相互作用；并进一步运用体内外模型及HCN2 KO小鼠，阐明丙酸和丁酸通过HCN2参与IBS-D脑-肠-微生态轴交互异常的信号传导机制。研究可望揭示SCFAs通过HCN2参与IBS-D脑-肠-微生态轴紊乱的新机制，并为治疗IBS-D提供潜在的新靶点。

Irritable bowel syndrome (IBS) is a common functional bowel disease and seriously impacts the health-related quality of life, but the pathophysiological mechanisms of IBS are various and unclear. The most common subtype of IBS is diarrhea-predominant IBS (IBS-D). Recently, IBS is considered as a disorder of gut-brain axis by Rome Ⅳ committee. The abnormal interaction of microbiome-gut-brain axis might be involved in the initiation and progression of IBS-D. Short-chain fatty acids (SCFAs), the product of gut microbiota fermentation, is found to be involved in regulating the synthesis of 5-HT, but the specific signal pathway has not yet been elucidated. Actually, we found that the serum levels of propionic acid and butyric acid were increased in IBS-D patients as compared to healthy controls in our previous preliminary study. Moreover, we found that there were alterations of gut microbiota in IBS-D patients and rifaximin could modify gut microbiota. We also found that HCN2 is expressed in enterochromaffin cells (EC) in the intestinal epithelium and colocalized with 5-HT. Hence, we hypothesize that propionic acid and butyric acid act by its receptor GPR41/43 and mediated by its second messenger cAMP regulation for HCN2 to regulate the Tph1 and Tph2 which is the key enzymes for biosynthesis of 5-HT in intestinal EC cells and the central cells in the hippocampus. This might play an important role in the abnormal interaction of microbiome-gut-brain axis of IBS-D. On the bases of preliminary study, we will firstly investigate the relationships between the gut microbiota, SCFAs such as propionic acid and butyric acid and 5-HT in the abnormal interaction of microbiome-gut-brain axis in IBS-D. At this stage, we will observe the top-bottom effects of stress on intestinal symptoms and gut microbiota in mouse model of chronic unpredictable mild stress (CUMS). We then will explore the bottom-up effects of the modification of the gut microbiota on gut-brain axis in germ-free mice, visceral hypersensitive mice and IBS-D patients. Lastly, we will illustrate the mechanism about the involvement of propionic acid and butyric acid in the microbiome-gut-brain axis interaction via modulating 5-HT synthesis mediated by HCN2 in IBS-D by double immunohistochemical staining (5-HT antibody and HCN2 antibody) in larger sample, cell experiments (BON cell line, Human neuroblastoma cell SH-SY5Y, primary EC cells) and animal experiments (visceral hypersensitive mice and HCN2 knockout mice). The results of this study can provide an experimental basis for revealing the involvement of propionic acid and butyric acid through HCN2 in the disorder of IBS-D microbiome-gut-brain axis and find the potential new targets for the treatment of IBS-D.