Dysbiosis of intestinal microbiota and decrease in paneth cell antimicrobial peptide level during acute necrotizing pancreatitis in rats.
Dysbiosis of intestinal microbiota and decrease in paneth cell antimicrobial peptide level during acute necrotizing pancreatitis in rats.
复制标题
急性坏死性胰腺炎大鼠肠道菌群失调及潘氏细胞抗菌肽水平下降
DOI：
10.1371/journal.pone.0176583
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Zeng Y
中科院分区：
文献类型：
--
作者：
Chen J;Huang C;Wang J;Zhou H;Lu Y;Lou L;Zheng J;Tian L;Wang X;Cao Z;Zeng Y
目的肠屏障功能障碍在急性坏死性胰腺炎(ANP)发病中起重要作用，肠道微生物区系失调参与肠屏障功能衰竭。潘氏细胞保护肠道屏障，与肠道微生物区系有关。在这里，我们研究了ANP期间肠道微生物区系和回肠Paneth细胞抗菌肽的变化。方法用3.5%牛磺胆酸钠逆行胆胰管注射建立大鼠ANP模型，分别于24小时和48小时处死。用组织病理学评分评价胰腺和回肠远端的损伤。用血浆二胺氧化酶活性(DAO)和D-乳酸测定肠屏障功能。分别以肿瘤坏死因子α、白介素1β和白介素17A为指标进行全身炎症反应和肠道炎症反应的检测。采用粪便16S rRNA高通量测序方法，观察ANP组48h后肠道微生物区系的变化。用实时荧光定量聚合酶链式反应、免疫印迹和免疫荧光法检测溶菌酶和α-防御素-5。结果与假手术(SO)组比较，ANP大鼠胰腺和回肠远端组织病理损伤严重，肠屏障受损，血浆和回肠远端组织中肿瘤坏死因子α、IL-1β和IL-17A表达增加。主成分分析(PCA)显示SO和ANP组之间存在结构分离。ANP组的操作分类单位(OTU)数和ACE指数显示微生物区系多样性降低。分类学分析显示肠道微生物区系结构失调。在门水平上，糖化菌和发酵剂显著减少。属水平上，志贺氏菌和链球菌显著增加，假丝酵母菌_蔗糖单胞菌、普氏杆菌_UCG-001、乳螺科_UCG-001、瘤胃球菌_UCG-008、瘤胃球菌科_UCG-008显著下降。ANP组48h时溶菌酶和α-Defensin5mRNA表达水平显著降低。ANP组24、48h溶菌酶蛋白表达降低。同时，大肠杆菌-志贺氏菌的相对丰度与溶菌酶的减少呈负相关。结论ANP时肠道微生物区系紊乱和Paneth细胞抗菌肽减少可能参与了ANP时肠屏障功能障碍的发病机制。
Objectives Intestinal barrier dysfunction plays an important role in acute necrotizing pancreatitis (ANP) and intestinal microbiota dysbiosis was involved in intestinal barrier failure. Paneth cells protect intestinal barrier and are associated with intestinal microbiota. Here, we investigated changes in intestinal microbiota and antimicrobial peptides of Paneth cells in ileum during ANP. Methods Rats with ANP were established by retrograde injection of 3.5% sodium taurocholate into biliopancreatic duct and sacrificed at 24h and 48h, respectively. Injuries of pancreas and distal ileum were evaluated by histopathological score. Intestinal barrier function was assessed by plasma diamine oxidase activity (DAO) and D-lactate. Systemic and intestinal inflammation was evaluated by TNFα, IL-1β and IL-17A concentration by ELISA, respectively. 16S rRNA high throughput sequencing on fecal samples was used to investigate the changes in intestinal microbiota in the ANP group at 48h. Lysozyme and α-defensin5 were measured by real-time PCR, western blot and immunofluoresence. Results ANP rats had more severe histopathological injuries in pancreas and distal ileum, injured intestinal barrier and increased expression of TNFα, IL-1β and IL-17A in plasma and distal ileum compared with those of the sham-operated (SO) group. Principal component analysis (PCA) showed structural segregation between the SO and ANP groups. Operational taxonomic unit (OTU) number and ACE index revealed decreased microbiota diversity in the ANP group. Taxonomic analysis showed dysbiosis of intestinal microbiota structure. At phyla level, Saccharibacteria and Tenericutes decreased significantly. At genus level, Escherichia-Shigella and Phascolarctobacterium increased significantly, while Candidatus_Saccharimonas, Prevotellaceae_UCG-001, Lachnospiraceae_UCG-001, Ruminiclostridium_5 and Ruminococcaceae_UCG-008 decreased significantly. Lysozyme and α-defensin5 mRNA expression levels decreased significantly in ANP group at 48h. Protein expression of lysozyme decreased in ANP groups at 24h and 48h. Meanwhile, the relative abundance of Escherichia-Shigella correlated inversely with the decrease in lysozyme. Conclusion The disorder in intestinal microbiota and decreases of Paneth cell antimicrobial peptides might participate in the pathogenesis of intestinal barrier dysfunction during ANP.