Diabetes exacerbated sepsis-induced intestinal injury by promoting M1 macrophage polarization via miR-3061/Snail1 signaling

Abstract

Background: Macrophages play important roles in diabetes and sepsis-related intestinal injury. Accumulating evidence suggests that microRNAs (miRNAs) act as the fundamental link between macrophage polarization and tissue injury. However, the underlying mechanisms of miRNAs in regulating macrophage polarization-related intestinal injury under diabetes and sepsis conditions remain unclear.

Methods: The cecal ligation and puncture (CLP)-induced sepsis models were established in male wild-type (WT) and diabetic mice. Clodronate liposome was used to deplete macrophage. H&E staining, inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6], and intestinal mucosal barrier function markers [occludin, ZO-1, lipopolysaccharide (LPS), and intestinal fatty acid binding protein (iFABP)] were used to assess elevated intestinal damage. miRNA array, RNA-seq, and bioinformatic analysis were performed to detect the miRNA and messenger RNA (mRNA) expression and the potential regulation mechanism. In vitro, RAW264.7 cells were cultured in the absence or presence of high glucose and LPS, miR-3061 mimics, and Snail small interfering RNA stimulation, respectively, for further mechanism studies. Luciferase reporter assay was used to confirm the interplay between miRNA and its target genes.

Results: Compared with WT CLP mice, the diabetic CLP mice showed severe intestinal damage characterized by significant increases in Chui's scores, expression of inflammatory cytokines (TNF-α, IL-1β, and IL-6), serum LPS and iFABP concentration, and significant reductions in tight junction protein occludin and ZO-1 levels. Macrophage depletion reversed the intestinal damage caused by CLP. The bioinformatic analysis revealed that miR-3061/Snail1 might be a potential regulation axis of macrophage polarization. Furthermore, high glucose and LPS stimulation increased M1 macrophage and reduced the levels of miR-3061, which was negatively associated with Snail1 in RAW264.7 cells. Mechanistic studies demonstrated that miR-3061 regulated macrophage polarization by targeting the Snail1 mRNA 3'-untranslated region. Moreover, miR-3061 overexpression suppressed Snail1 expression and inhibited M1 macrophage and inflammatory cytokines.

Conclusion: This study elucidated that diabetes exacerbated sepsis-induced intestinal injury by promoting M1 macrophage polarization and further demonstrated that the miR-3061/Sani1 axis may be the potential target of macrophage polarization.

Keywords: Snail1; diabetes; intestinal injury; macrophage polarization; miR-3061; sepsis.