应激通过Paneth 细胞引起肠道微生态改变在肠易激综合征中的作用及机制研究

Although there have been many studies attempting to divulge the pathoohysiology of Irritable bowel syndrome (IBS), the disease is still not clearly understood. Stress plays an important role in the pathogenesis of IBS, which not only enhances the activity of immune cells and alter the threshold of visceral sensitivity, but also induces the alteration of microbiome in digestive tract. However, the exact mechanism is unclear. The study carried by us previously indicated that stress decreased the production of enteric α-defensin, which was secreted by Paneth cells. Paneth cells, which reside in the crypts of the small intestine, are the main producers of antimicrobial peptides, including lysozyme and α-defensin. The function and homeostasis of Paneth cells play key role in maintaining the gastrointestinal function, especially the intestinal microbiome. In this study, we will establish two well-established rat model of IBS, including the neonatal maternal seperation model and water avoidance stress rat model. We will detect the functions of Paneth cells in small intestine, such as the number,secretion, autophagy, differentiation and proliferation. In these experiments, we will carry the following molecular biologic methods, such as real-time PCR, immunofluorescence and ELISA, combined with morphologic observation.The objective of this study is to investigate the influence of stress on the function of Paneth cells, resulted in gut dysbiosis, and the role in the pathogenesis of IBS. We hope to investigate the pathogenesis of GERD, and provide novel target to treat GERD.

肠易激综合征(IBS)的发病机制尚未被完全阐明，应激在IBS的发病机制中发挥重要作用，应激不仅可增强免疫细胞活性，改变消化道运动和内脏感受阈值，亦可引起胃肠菌群显著改变，但具体机制不明。预实验中我们发现应激可导致大鼠小肠黏膜抗菌肽α防御素表达下降，而Paneth细胞是小肠内α防御素的重要分泌细胞。目前研究提示，Paneth细胞的功能及其稳态对维持肠道功能，特别是肠道菌群，具有至关重要的作用。在本项目中我们拟采用母婴分离及避水应激两种IBS大鼠模型，通过实时定量PCR、免疫荧光、ELISA等分子生物学和形态学方法，从多角度、多层面明确应激对Paneth细胞的影响，及其带来的肠道菌群紊乱在IBS发病机制中的作用。本研究有助于对IBS发病机制的全面了解，为临床诊疗提供有效的靶标。

肠易激综合征(IBS)的发病机制尚未被完全阐明，应激在 IBS 的发病机制中发挥重要作用，应激不仅可增强免疫细胞活性，改变消化道运动和内脏感受阈值，亦可引起胃肠菌群显著改变，但具体机制不明。本课题建立模拟人类IBS大鼠内脏高敏感状态的母婴分离模型，并且进行双歧杆菌的干预研究。研究结果提示母婴分离可以引起大鼠肠道敏感性增加，回肠末端的绒毛隐窝高度比例发生变化，双歧杆菌治疗能够降低大鼠的内脏高敏感。通过免疫组化及qRT-PCR、western-blot方法检测显示母婴分离能够引起Paneth细胞数量的增加，同时使得其分泌的Defesin-5,lysozyme表达增多。本课题同时建立稳定的大鼠避水应激模型进行实验研究，应用双歧杆菌组进行干预。避水应激可以引起大鼠内脏敏感性增加，大鼠回肠末端绒毛隐窝高度比例发生变化，杯状细胞数量明显减少，通过免疫荧光、qRT-PCR、western-blot方法观察潘氏细胞的量和功能变化，研究发现避水应激导致Paneth细胞数量轻度增加，导致Defesin-5,lysozyme表达增加。而双歧杆菌治疗能够有效减少Paneth细胞的增多，减少Defesin-5,lysozyme表达。扫描电镜观察，双歧杆菌能能够有效定植于回肠末端粘膜上皮。同时通过16SrDNA检测发现避水应激可导致大鼠肠道微生态发生显著变化，α-多样性检测提示物种多样性显著减少，Beta 多样性分析显示避水应激造成大鼠肠道菌群发生明显变化。避水应激导致肠道菌群在门水平上拟杆菌丰度明显增加，厚壁菌丰度明显减少。在属水平，避水应激导致坦纳拟普雷沃菌丰度增加，乳酸菌及普雷沃菌属丰度减少，双歧杆菌治疗能够在门、属水平能够改善这一变化。因此，本研究认为应激可能导致了Paneth细胞的功能变化，而 Defesin-5,lysozyme这些抗菌肽的表达变化，使得肠道微生态发生了改变，双歧杆菌能够有效通过与肠道上皮作用，改变Paneth细胞的功能，进而调控肠道微生态。