Faecalibacterium prausnitzii-derived microbial anti-inflammatory molecule regulates intestinal integrity in diabetes mellitus mice via modulating tight junction protein expression

Abstract

Background: Impaired intestinal barrier structure and function have been validated as an important pathogenic process in type 2 diabetes mellitus (T2DM). Gut dysbiosis is thought to be the critical factor in diabetic intestinal pathogenesis. As the most abundant commensal bacteria, Faecalibacterium prausnitzii (F. prausnitzii) play important roles in gut homeostasis. The microbial anti-inflammatory molecule (MAM), an F. prausnitzii metabolite, has anti-inflammatory potential in inflammatory bowel disease (IBD). Thus, we aimed to explore the function and mechanism of MAM on the diabetic intestinal epithelium.

Methods: 16S high-throughput sequencing was used to analyze the gut microbiota of db/db mice (T2DM mouse model). We transfected a FLAG-tagged MAM plasmid into human colonic cells to explore the protein-protein interactions and observe cell monolayer permeability. For in vivo experiments, db/db mice were supplemented with recombinant His-tagged MAM protein from E. coli BL21 (DE3).

Results: The abundance of F. prausnitzii was downregulated in the gut microbiota of db/db mice. Immunoprecipitation (IP) and mass spectroscopy (MS) analyses revealed that MAM potentially interacts with proteins in the tight junction pathway, including zona occludens 1 (ZO-1). FLAG-tagged MAM plasmid transfection stabilized the cell permeability and increased ZO-1 expression in NCM460, Caco2, and HT-29 cells. The db/db mice supplemented with recombinant His-tagged MAM protein showed restored intestinal barrier function and elevated ZO-1 expression.

Conclusions: Our study shows that MAM from F. prausnitzii can restore the intestinal barrier structure and function in DM conditions via the regulation of the tight junction pathway and ZO-1 expression.

Keywords: Faecalibacterium prausnitzii; diabetes mellitus; diabetic pathology; gut microbiota; intestinal barrier; 普拉梭菌; 糖尿病; 糖尿病病变; 肠道屏障; 肠道菌群.

Figure 1

Damaged intestinal barrier structure and function in diabetic mice. (A) The blood LPS concentration in the DM group was markedly elevated in contrast to those in the control group (n = 6, *P < 0.05). (B) The intestinal tight junction ultrastructure (arrows) was disrupted in the DM group compared to the control group (bars = 500 nm). (C‐E) Compared to the control group, the expression of ZO‐1 mRNA and protein were significantly decreased in the colonic epithelium of the DM group (n = 6, *P < 0.05). (F) IHC indicated the lower intestinal ZO‐1 expression in the DM group in contrast to the control group (bar = 20 μm). (DM group: db/db mice, control group: littermate mice)

Figure 2

DM‐induced gut microbiota dysbiosis. (A, B) The gut microbiota Chao1 and Shannon index in the DM group were significantly lower than those in the control group (*P < 0.05). (C) The Firmicutes to Bacteroidetes ratio (F/B) in the DM group continuously descended while F/B in the control group kept ascending during the observation (*P < 0.05). (D) The gut microbiota composition of DM and control groups at the phylum level. (DM group: db/db mice, control group: littermate mice)

Figure 3

F. prausnitzii dramatically decreased in DM mice. (A). Heatmap depicted the relative percentage of each bacterial genus in the DM and control groups. (bacterial genus on the Y‐axis, sample name on X‐axis). (B). The blood glucose in the DM group sustained at a high level (8 weeks: 32.25 ± 1.46 mmol/L; 10 weeks: 31.50 ± 1.78 mmol/L; 12 weeks: 31.12 ± 2.94 mmol/L). By comparison, the blood glucose of control group kept steady at a low level (8 weeks: 7.83 ± 0.43 mmol/L; 10 weeks: 7.43 ± 0.55 mmol/L; 12 weeks: 7.83 ± 0.31 mmol/L). (C) The qRT‐PCR indicated the drastically low abundance of F. prausnitzii in the DM gut microbiota (*P < 0.05). (D) The qRT‐PCR indicated the markedly low expression of MAM mRNA in the DM gut microbiota (*P < 0.05). (DM group: db/db mice, control group: littermate mice)

Figure 4

The establishment of MAM in vitro experiment model; the exploration of MAM downstream factors and modulation mechanisms. (A) The MAM expressions were successfully detected after transfection of FLAG‐MAM in NCM460, Caco2, and HT‐29 (*P < 0.05). (B) MAM‐interacted proteins were captured by IP. Prior to the MS analysis, proteins were separated by 10% SDS‐PAGE and stained with Coomassie blue. 293 T was taken as the transfection positive control. NCM460‐FLAG and 293 T‐FLAG: NCM460 and 293 T were incubated with FLAG antibody for IP. NCM460‐IgG and 293 T‐IgG: NCM460 and 293 T were incubated with nonimmune rabbit IgG polyclonal antibody for IP. (C) Gene ontology analysis on MAM‐interacted candidates reveals the potential functions of MAM in regulating intestinal epithelium biological processes. (D) KEGG analysis suggests MAM protein is potentially involved in the modulation of many signaling pathways. The regulation on the tight junctions is one of the most correlated pathways

Figure 5

Transfection of FLAG‐tagged MAM protein decreased cell permeability and upregulated ZO‐1 expression in different colonic epithelial cells after LPS treatment. (A) NMC460, Caco2, and HT‐29 were treated with 1 g/mL LPS for 48 hours to establish the impaired cell permeability model. Meanwhile, cells were transfected with FLAG‐tagged MAM or empty vector. FLAG‐tagged MAM decreased FITC‐D4 permeation in NMC460, Caco2, and HT‐29. (B–D) The abundance of ZO‐1 mRNA and protein increased after transfection of FLAG‐tagged MAM in the impaired cell permeability model (NMC460, Caco2, and HT‐29 with LPS treatment) (*P < 0.05)

Figure 6

The intervention of recombinant His‐tagged MAM protein repaired DM mice intestine barrier. (A) Profile of pET‐6xHis/TEV/MAM plasmid encoded the full length of MAM sequence (named His‐tagged MAM). (B) Western blot with anti‐His antibody for His‐tagged MAM protein verification in BL21(DE3) bacterial protein extraction after IPTG induction. (C) His‐tagged MAM supplementation reduced the blood LPS concentration in db/db mice (DM + MAM vs DM + PBS, *P < 0.05). (D) Constitutive tight junction and undamaged intercellular gaps (arrows) were observed in both DM + PBS and DM + MAM groups; restored tight junctions and narrower intervals (arrows) were observed among colon epithelium in the DM + MAM group compared to the DM + PBS group (bars = 500 nm)

Figure 7

The intervention of recombinant His‐MAM protein upregulated ZO‐1 expression in DM mice. (A) Intestinal ZO‐1 mRNA abundance was upregulated in the DM + MAM group compared to the DM + PBS group (*P < 0.05). (B, C) His‐tagged MAM protein intervention increased the colonic epithelial ZO‐1 protein expression in the DM + MAM group compared to the DM + PBS group (*P < 0.05). (D) IHC showed the ZO‐1 positive cell distributed evenly in colonic epithelium in the DM + MAM group whereas they mainly distributed in epithelial cells near the lumen in the DM + PBS group (bar = 20 μm)