Dysbiosis of Intestinal Microbiota and Decreased Antimicrobial Peptide Level in Paneth Cells during Hypertriglyceridemia-Related Acute Necrotizing Pancreatitis in Rats

Abstract

Hypertriglyceridemia (HTG) aggravates the course of acute pancreatitis (AP). Intestinal barrier dysfunction is implicated in the pathogenesis of AP during which dysbiosis of intestinal microbiota contributes to the dysfunction in intestinal barrier. However, few studies focus on the changes in intestine during HTG-related acute necrotizing pancreatitis (ANP). Here, we investigated the changes in intestinal microbiota and Paneth cell antimicrobial peptides (AMPs) in HTG-related ANP (HANP) in rats. Rats fed a high-fat diet to induce HTG and ANP was induced by retrograde injection of 3.5% sodium taurocholate into biliopancreatic duct. Rats were sacrificed at 24 and 48 h, respectively. Pancreatic and ileal injuries were evaluated by histological scores. Intestinal barrier function was assessed by plasma diamine oxidase activity and D-lactate level. Systemic and intestinal inflammation was evaluated by tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, and IL-17A expression. 16S rRNA high throughput sequencing was used to investigate changes in intestinal microbiota diversity and structure. AMPs (α-defensin5 and lysozyme) expression was measured by real-time polymerase chain reaction (PCR) and immunofluorescence. The results showed that compared with those of normal-lipid ANP (NANP) groups, the HANP groups had more severe histopathological injuries in pancreas and distal ileum, aggravated intestinal barrier dysfunction and increased TNFα, IL-1β, and IL-17A expression in plasma and distal ileum. Principal component analysis showed structural segregation between the HANP and NANP group. α-Diversity estimators in the HANP group revealed decreased microbiota diversity compared with that in NANP group. Taxonomic analysis showed dysbiosis of intestinal microbiota structure. In the HANP group, at phyla level, Candidatus_Saccharibacteria and Tenericutes decreased significantly, whereas Actinobacteria increased. At genus level, Allobaculum, Bifidobacterium, and Parasutterella increased significantly, while Alloprevotella, Anaerotruncus, Candidatus_Saccharimonas, Christensenellaceae_R-7_group, Rikenellaceae_RC9_gut_group, Ruminiclostridium_5, Ruminococcaceae_UCG-005, and Ruminococcaceae_UCG-014 decreased. Compared with those in the NANP rats, mRNA expression of lysozyme and α-defensin5 and protein expression of lysozyme decreased significantly in the HANP rats. Moreover, in the NANP rats and the HANP rats, Allobaculum abundance was inversely correlated with lysozyme expression, while Anaerotruncus abundance was positively correlated with it by Spearman test. In conclusion, intestinal microbiota dysbiosis and decreased AMPs of Paneth cells might participate in the pathogenesis of intestinal barrier dysfunction in HANP.

Keywords: Paneth cell; acute necrotizing pancreatitis; antimicrobial peptides; hypertriglyceridemia; intestinal microbiota.

FIGURE 1

Study design. SO, sham-operated; ANP, acute necrotizing pancreatitis; SO24h, normal lipid + sham-operated + sacrificed at 24 h; SO48h, normal lipid + sham-operated + sacrificed at 48 h; NANP24h, normal lipid + ANP + sacrificed at 24 h; NANP48h, normal lipid + ANP + sacrificed at 48 h; HSO24h, HTG + sham-operated + sacrificed at 24 h; HSO48h, HTG + sham-operated + sacrificed at 48 h; HANP24h, HTG + ANP + sacrificed at 24 h; HANP48h, HTG + ANP + sacrificed at 48 h.

FIGURE 2

HTG aggravated pathological changes in the pancreas and the distal ileum in ANP. (A) Serum TG and TC levels in normal lipid and HTG groups. (B) Histopathological changes in the pancreas and the distal ileum of rats [hematoxylin-eosin (HE), ×100]. (C) Histopathological scores of the pancreas and the distal ileum. ^∗p < 0.05. Student’s t-test.

FIGURE 3

HTG aggravated changes in intestinal barrier permeability and the expression of inflammation cytokines in plasma and the distal ileum in ANP. (A) Measurement of plasma diamine oxidase (DAO) activity and D-lactate at 24 and 48 h after ANP induction. (B) Plasma inflammation cytokines TNFα, IL-1β, and IL-17A expression levels in rats 24 and 48 h after ANP induction. (C) Intestinal inflammatory cytokines TNFα, IL-1β, and IL-17A expression levels at 24 and 48 h after ANP induction. ^∗p < 0.05. Student’s t-test.

FIGURE 4

Rarefaction analysis and Shannon diversity of all samples.

FIGURE 5

Changes in intestinal microbiota diversity in HTG-related ANP. (A–D) β-diversity reflected by PCA among the four groups. (E) The estimators of α-diversity of intestinal microbiota in each group. ^∗p < 0.05. Student’s t-test.

FIGURE 6

(A) Relative abundance of phyla in intestinal microbiota of the four groups. (B) Relative abundance of genera in intestinal microbiota of the four groups. (C) The heatmap of bacterial phyla among the four groups.

FIGURE 7

Relative abundance of significantly different phyla and genera among the four groups. (A) The significantly different phyla among the four groups. (B) The significantly different genera among the four groups. ^∗p < 0.05. Mann–Whitney test.

FIGURE 8

Antimicrobial peptides expression decreased in HTG-related ANP. (A) Antimicrobial peptides (lysozyme and α-defensin5) mRNA expression by real-time PCR in the distal ileum at 24 and 48 h after ANP induction. (B) Quantification of the Paneth cell numbers among the eight groups. (C) Lysozyme (red) protein expression produced by Paneth cells in the distal ileum by immunofluorescence. Nuclei were counterstained blue with DAPI (original magnification, ×200). (D) The abundance of Allobaculum (r = –0.661, p < 0.05) and Anaerotruncus (r = 0.793, p < 0.05) significantly correlated with lysozyme expression in the NANP48h and HANP48h groups. ^∗p < 0.05. Student’s t-test and Spearman test.