Profile of Pancreatic and Ileal Microbiota in Experimental Acute Pancreatitis

Abstract

Acute pancreatitis (AP) is accompanied by gut microbiota dysbiosis. However, the composition of the pancreatic and ileal microbiota associated with AP is still unknown. This study aims to examine the alterations in the microbial composition of the pancreas and ileum in the context of experimental acute pancreatitis, as well as explore the potential interplay between these two regions. Methods: Caerulein (CAE), caerulein+lipopolysaccharide (CAE+LPS), and L-arginine (ARG) were used to induce AP in mice. The pancreas and ileum were collected for histological study and bacterial 16S rRNA gene sequencing. The results showed microbial structural segregation between the AP and control groups and between ARG and the two CAE groups (CAE, CAE+LPS) in the pancreas and ileum. Taxonomic analysis at the genus level and linear discriminant analysis effect size (LEfSe) at the operational taxonomic units (OTUs) level illustrated that AP mice exhibited a marked increase in the relative abundance of Muribaculaceae and a decrease in that of Dietzia both in the pancreas and ileum, and a reduction in Bifidobacterium only in the ileum; in addition, Roseburia was enriched in the two CAE groups in the pancreas and/or ileum, while Escherichia-Shigella expanded in the pancreas of the ARG group. Spearman correlation analysis between pancreatic and ileal microbiota revealed that the abundance of Muribaculaceae and Dietzia in the pancreas was related to that in the ileum. These findings demonstrated that caerulein and L-arginine differentially disturbed the pancreatic and ileal microbiota when inducing AP. Furthermore, these findings provide preliminary support for an association between the microbiota of the pancreas and ileum, which could be caused by AP-induced microbial translocation.

Keywords: Dietzia; Muribaculaceae; acute pancreatitis; ileal microbiota; pancreatic microbiota.

Figure 1

Phenotypes of three AP mouse models induced by Caerulein, Caerulein+LPS, and L-Arginine. (A) Histopathological changes in pancreatic samples were observed by HE staining (×200). Scale bar = 100 μm. Pancreatic histopathological scores were evaluated by Schmidt criteria. (B,C) Serum levels of amylase and lipase. (D,E) Serum levels of TNF-α and IL-1β. (F) Histopathological changes of ileal samples observed by HE staining (×200). Scale bar = 100 μm. (G) The protein levels of claudin-1 in the ileum were analyzed by Western blotting. Gels and blots were cropped. Symbol * means p < 0.05, ** means p < 0.01, *** means p < 0.001, **** means p < 0.001, ns means p > 0.05, Student’s t-test.

Figure 2

Alpha-diversity in AP and control group both in pancreas and ileum. (A–D) The estimators of ACE, chao1, OTUs and Shannon of pancreatic and ileal microbiota in each group. Symbol * means p < 0.05, ** means p < 0.01, *** means p < 0.001, **** means p < 0.0001, ns means p > 0.05, Student’s t-test.

Figure 3

The pancreatic microbiota was changed by AP. (A) Principal coordinates analysis plots based on the Bray–Curtis distances showed the differences in the bacterial microbiota between the pancreas of the AP and control groups. The box plots comparing the coordinate positions of the four sets of samples on PC1 and PC2 are shown on the lower left and right, respectively. (B) Nonmetric multidimensional scaling analysis based on the Bray–Curtis distances showed the differences among the pancreas of the four groups. Each symbol represents one sample. The box plots comparing the coordinate positions of the four sets of samples on PC1 and PC2 are shown on the lower left and right, respectively. (C) Microbiota composition at the phylum level in the pancreas. (D) Microbiota composition at the genus level in the pancreas.

Figure 4

The maximum difference in microbial structures in the pancreas between the CON and AP groups. A heatmap analysis of the microbiomes in the pancreas. Symbol * means p < 0.05, ** means p < 0.01, *** means p < 0.001.

Figure 5

The ileal microbiota was changed by AP. (A) Principal coordinates analysis plots based on the Bray–Curtis distances showed the differences in the bacterial microbiota between the ileum of the AP and control groups. The box plots comparing the coordinate positions of the four sets of samples on PC1 and PC2 are shown on the lower left and right, respectively. (B) Nonmetric multidimensional scaling analysis based on the Bray–Curtis distances showed the differences among the ileum of the four groups. Each symbol represents one sample. The box plots comparing the coordinate positions of the four sets of samples on PC1 and PC2 are shown on the lower left and right, respectively. (C) Microbiota composition at the phylum level in the ileum. (D) Microbiota composition at the genus level in the ileum.

Figure 6

The maximum difference in microbial structures in the ileum between the CON and AP groups. A heatmap analysis of the microbiomes in the ileum. Symbol * means p < 0.05, ** means p < 0.01, *** means p < 0.001.

Figure 7

Differences in bacterial composition among the three AP groups in the pancreas and ileum. (A) Differences in bacterial composition in the pancreas between the ARG and CAE groups. (B) Differences in bacterial composition in the pancreas between the ARG and CAE+LPS groups. (C) Differences in bacterial composition in the pancreas between the CAE and CAE+LPS groups. (D) Differences in bacterial composition in the ileum between the ARG and CAE groups. (E) Differences in bacterial composition in the ileum between the CAE and CAE+LPS groups. (F) Differences in bacterial composition in the ileum between the ARG and CAE+LPS groups. Symbol * means p < 0.05, ** means p < 0.01, Mann–Whitney U tests.

Figure 8

Correlation analysis of changed microbiota in the pancreas and ileum. A Spearman correlation heatmap reflected the correlation between pancreatic microbiota and ileal microbiota. Symbol * means p < 0.05, ** means p < 0.01, *** means p < 0.001, Spearman test.