高脂饮食诱导高脱氧胆酸通过内质网应激损害潘氏细胞功能导致肠道菌群紊乱的机制研究

High fat feeding (HF) leads to gut dysbiosis, mucosal inflammation and metabolic endotoxemia. It is unclear how HF induces gut dysbiosis. HF diets (HFD) lead to a increase of the secondary bile acid----deoxycholic acids (DCA) production. Our previous immunohistochemical studies of rat ileum showed G protein coupled bile acid receptor (TGR5) was expressed in lysozyme+ Paneth cells. Illumina MiSeq analysis of the ileal mocosa following 2 wk HFD showed a gut dysbiosis (significant decrease in Lactobacillaceae and increases in Streptococcaceae, Peptostreptococcacea, Erysipelotrichaceae and Streptococcaceae), accompanying with the reduction of α-defensin 5 and 6, which was restored by cholestyramine (bile-acid sequestrant). To make it clear how the elevated DCA impaire Paneth cell function, we will feed rats with HFD mixed with cholestyramine or diet with cholic acids for 2 wk, The inhibitor of endoplasmic reticulum stress ( ER stress) will be given as an intervention and the TGR5 knock out mice will also be applied. In vitro study, we plan to culture Enteroid (not affected by gut microbiota）from rat or human with the DCA or oleanolic acid (specific activator of TGR5) to detect the gene and protein expression of α-defensin peptides and interleukin 23 (IL-23), the markers of DNA damage (Caspase-3, PARP-1), autophagy (LC3, Atg16l1), and ER stress (XBP1, Bip). Then we will interrupt this pathway on different cellular or molecular levels by the treatment with antagonist of TGR5 or the inhibitors of ER stress. With the evidence from in vivo and in vitro studies, we aim to demonstrate that the increased DCA following HF damage the function of Paneth cell（inhibit production of anti-microbial peptides and /or increase the expression of IL-23）by upregulating the pathways of ER stress. These may lead to gut dysbiosis, mucosal inflammation and metabolic endotoxemia.

高脂饮食可诱导肠道菌群紊乱和脱氧胆酸(DCA)产生增加。我们发现大鼠回肠潘氏细胞表达胆汁酸受体TGR5，2周高脂饮食降低了潘氏细胞来源的防御素，伴随肠道菌群的紊乱，胆汁酸螯合剂考来烯胺则可改善之。但是，高浓度DCA如何影响潘氏细胞依然需要研究。为此，在体我们给高脂饮食大鼠予考来烯胺或内质网应激抑制剂干预、并采用胆酸饮食处理和TGR5基因敲除小鼠模型，研究末段回肠潘氏细胞的免疫调节功能（分泌防御素5和6、溶菌酶，及细胞因子--IL-23）及其内质网应激水平，检测胆汁酸的组成、浓度及肠道菌群结构。离体拟采用Enteroid（大鼠或人体）的培养技术，观察DCA直接对肠道干细胞分化而来的潘氏细胞及其内质网应激的作用；并从细胞、分子水平观察阻断DCA-TGR5途径或抑制内质网应激关键环节的效应。本研究旨在证实高浓度DCA通过诱导内质网应激破坏潘氏细胞功能，可能为高脂饮食诱发肠道菌群紊乱的机制之一。

背景:高脂饮食（HFD)导致肠道菌群失调的机制尚不清楚。最近我们发现在肠上皮细胞中存在G蛋白偶联胆汁酸受体(TGR5)。我们推测高脂饮食引起的高浓度胆汁酸可能作用于肠上皮细胞，降低潘氏细胞（Paneth cells）的抗菌肽分泌，导致肠道菌群失调。.方法：分别采用检测细菌的Illumina MiSeq法、免疫组化法和流式细胞术检测小鼠回肠隐窝细胞。应用Western blot和RT-qPCR检测抗微生物肽、自噬和内质网应激标志物的表达。.结果:高脂饮食导致末段回肠粘膜的细菌发生广泛的菌群分类学改变，其特征是乳酸菌（Lactobacillus）数量减少，Akkermansia muciniphila, Clostridium XIVa, Ruminococcaceae, Lachnospiraceae富集。这些变化可被胆汁酸螯合剂考来烯胺逆转。免疫组化研究显示，在喂食HFD或添加胆酸的正常饮食的情况下，潘氏细胞中TGR5表达上调。同时伴随着溶菌酶（lysozyme）+ 的Paneth细胞数目的减少和潘氏细胞分泌的α-defensin 5和6的表达的降低,同时伴随着内质网应激的标记（XBP-1）和自噬标记（ATG16L1）表达的增加。用内质网应激抑制剂4PBA或胆汁酸螯合剂考来烯胺预处理可逆转这些变化。在喂食高脂饲料或胆酸饲料的大鼠末段回肠的潘氏细胞中，透射电镜可观察到潘氏细胞的分泌颗粒的降低，以及含空泡的分泌颗粒比例的增加。离体回肠组织与脱氧胆酸的共培养，可观察到潘氏细胞分泌的α-defensin 5和6的显著降低，以及XBP-1增加，这些变化可被用TGR5抗体或内质网应激拮抗剂（4PBA）预处理逆转。.关键词:高脂饮食，胆汁酸，Paneth细胞，TGR5，内质网应激.结论:本研究首次证实了胆汁酸的膜受体TGR5在Paneth细胞中的表达。高脂饮食导致回肠粘膜的肠道菌群失调、TGR5的上调、内质网应激、自噬的增强，同时伴随着α-defensin 5和6的减少。这些变化被可被胆汁酸螯合剂考来烯胺或内质网应激抑制剂4PBA阻断。肠上皮细胞功能障碍可能是高脂饮食所致肠道菌群失调的重要原因之一。.项目研究成果在《胃肠病学》期刊发表论文1篇；在美国消化病周（DDW）消化界国际学术会议发言一次。