Protective effects and mechanisms of omega-3 polyunsaturated fatty acid on intestinal injury and macrophage polarization in peritoneal dialysis rats

Abstract

Aim: This study was conducted to investigate the chronic injury of peritoneal glucose injection on the peritoneum and intestine and the protective effects of omega-3 polyunsaturated fatty acid (ω-3PUFA) during peritoneal dialysis (PD).

Methods: Peritoneal dialysis animal models were established by intraperitoneal injection of 4.25% glucose for 28 days. Protein expression in ileum and peritoneum was measured by immunofloresence and immunohistochemistry. Protein expression in macrophages was measured by Western blot. Fibrosis was analyzed by Masson staining.

Results: Peritoneal dialysis significantly increased the structural injury and decreased junction-related protein ZO-1 and occludin expression in ileum, the expression of proteins relating to the activation of M2 (Erg2, IRF4), but not M1 (CD38, IRF5) macrophages. PD significantly increased the expression of TGF-β1, VEGF and ALK5 protein in peritoneal tissues. PD significantly increased fibrosis (Masson staining) and the expression of fibroblast marker α-SMA in peritoneal tissues. Injection of macrophage clean reagent and ω-3PUFA significantly inhibited M2 activation, and decreased Masson staining, α-SMA, TGF-β1, VEGF and ALK5 protein expression in peritoneal tissues in PD treated rats. ω-3PUFA injection significantly decreased PD-induced injury in ileum and normalized the expression of ZO-1 and occludin in the ileum of PD rats.

Conclusion: Omega-3 fatty acids can provide a protective role on PD-induced peritoneal fibrosis and injury of the intestine.

Keywords: fibrosis; intestinal injury; macrophage polarization; omega-3 polyunsaturated fatty acid; peritoneal dialysis.

Figure 1

The morphological changes in ileum. (A) Representative H&E staining of ileum. Original magnification, 100×. PD significantly decreased the length of ileal villus (B), had no effect on the depth of crypt (C), and decreased the ratio of depth crypt/the length of ileal villus (D). NC, normal control; PD, peritoneal dialysis; ***, P < 0.001 versus NC and Sham; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 versus PD.

Figure 2

Immunofluoresence of ZO‐1 expression in the ileum. Original magnification, 400×. Changes in spatial expression of ZO‐1 protein were qualitative only. Same slide was stained with rabbit anti‐ZO‐1 antibody, followed by Alexa fluor 488 conjugated IgG (ZO‐1). Nuclei were further stained with DAPI (DAPI). The pictures of ZO‐1 and DAPI were merged (Merge). DAPI, 4′,6‐diamidino‐2‐phenylinodele; PD, peritoneal dialysis; ω‐3PUFA LD, ω‐3PUFA low dose; ω‐3PUFA HD, ω‐3PUFA high dose.

Figure 3

Immunofluoresence of occludin expression in the ileum. Original magnification, 400×. Changes in spatial expression of occludin protein were qualitative only. Same slide was stained with rabbit anti‐occludin antibody, followed by Alexa fluor 488 conjugated IgG (Occludin). Nuclei were further stained with DAPI (DAPI). The pictures of Occludin and DAPI were merged (Merge). DAPI, 4′,6‐diamidino‐2‐phenylinodele.

Figure 4

Western blot of M1/M2 polarization‐related protein expression. (A) Representative Western blots of CD38, EgR2, IRF5, IRF4, and β‐actin. (B) Semi‐quantitative analysis of CD38/Egr2 ratio. (C) Semi‐quantitative analysis of IRF5 expression. (D) Semi‐quantitative analysis of IRF4 expression. **, P < 0.01; ***, P < 0.001 versus normal control; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 versus PD.

Figure 5

Immunohistochemistry of TGF‐β1 expression in peritoneal tissues. (A) Representative immunohistochemical staining of TGF‐β1 protein expression. (B) The percentage of cells with positive TGF‐β1 staining.

Figure 6

Immunohistochemistry of VEGF expression in peritoneal tissues. (A) Representative immunohistochemical staining of VEGF protein expression. (B) The percentage of cells with positive VEGF staining.

Figure 7

Immunohistochemistry of ALK5 expression in peritoneal tissues. (A) Representative immunohistochemical staining of ALK5 protein expression. (B) The percentage of cells with positive ALK5 staining.

Figure 8

Masson staining in peritoneal tissues. Fibrosis is depicted with blue staining.

Figure 9

Immunohistochemistry of α‐SMA expression in the fibroblasts of peritoneal tissues. (A) Representative immunohistochemical staining of α‐SMA protein expression. (B) The percentage of fibroblasts with positive α‐SMA staining (red color).