Ghrelin/GHSR Axis Induced M2 Macrophage and Alleviated Intestinal Barrier Dysfunction in a Sepsis Rat Model by Inactivating E2F1/NF- κ B Signaling

Abstract

Sepsis is an inflammatory reaction disorder state that is induced by infection. The activation and regulation of the immune system play an essential role in the development of sepsis. Our previous studies have shown that ghrelin ameliorates intestinal dysfunction in sepsis. Very little is known about the mechanism of ghrelin and its receptor (GHSR) on the intestinal barrier and the immune function of macrophage regulation. Our research is to investigate the regulatory effect and molecular mechanism of the ghrelin/GHSR axis on intestinal dysfunction and macrophage polarization in septic rats. A rat model of sepsis was established by cecal ligation and puncture (CLP) operation. Then, the sepsis rats were treated with a ghrelin receptor agonist (TZP-101) or ghrelin inhibitor (obestatin). The results suggested that TZP-101 further enhanced ghrelin and GHSR expressions in the colon and spleen of septic rats and obestatin showed the opposite results. Ghrelin/GHSR axis ameliorated colonic structural destruction and intestinal epithelial tight junction injury in septic rats. In addition, the ghrelin/GHSR axis promoted M2-type polarization of macrophages, which was characterized by the decreases of IL-1β, IL-6, and TNF-α, as well as the increase of IL-10. Mechanistically, the ghrelin/GHSR axis promoted E2F2 expression and suppressed the activation of the NF-κB signaling pathway in septic rats. Collectively, targeting ghrelin/GHSR during sepsis may represent a novel therapeutic approach for the treatment of intestinal barrier injury.

Figure 1

Ghrelin/GHSR axis ameliorated colonic structural destruction in septic rats. (a–d) The expression of ghrelin and GHSR in the colon and spleen tissues of septic rats was determined by Western blot. (e) Colon histopathological changes were analyzed by H&E stain (200x and 400x magnification). (f) Representative photomicrographs of PAS-stained colon tissues (400x magnification). (g) The goblet cell count of the colonic tissue. ^∗∗P < 0.01 (vs sham group), ^#P < 0.05 (vs CLP group), ^##P < 0.01 (vs CLP group), ^&P < 0.05 (vs ghrelin group), and ^&&P < 0.01 (vs ghrelin group).

Figure 2

Ghrelin/GHSR axis attenuated intestinal epithelial tight junction injury in septic rats. (a) FITC-dextran serum levels were measured 4 hours after gastric administration to determine intestinal permeability. Higher serum FITC-dextran levels indicated more intestinal permeability. Serum LPS (b) and colon LPS (c) were assayed by ELISA. (d–g) Immunofluorescence (IF) analysis determining the expression and distribution of claudin-5 and ZO-1 in colon tissues of septic rats (100x magnification). ^∗∗P < 0.01 (vs sham group), ^#P < 0.05 (vs CLP group), ^&P < 0.05 (vs ghrelin group), and ^&&P < 0.01 (vs ghrelin group).

Figure 3

Ghrelin/GHSR axis inhibited colonic inflammation in septic rats. (a–d) The mRNA levels of IL-1β, IL-6, IL-10, and TNF-α in colon tissues of septic rats were tested by RT-qPCR. (e–i) Protein levels of IL-1β, IL-6, TNF-α, and IL-10 were assayed by Western blot analysis. ^∗∗P < 0.01 (vs sham group), ^#P < 0.05 (vs CLP group), ^##P < 0.01 (vs CLP group), ^&P < 0.05 (vs ghrelin group), and ^&&P < 0.01 (vs ghrelin group).

Figure 4

Ghrelin/GHSR axis promoted M2-type polarization of macrophages. (a–d) The colon samples of septic rats were subjected to an immunohistochemical (IHC) stain with a primary antibody against CD86 and CD206 (200x and 400x magnification). (e–h) The spleen tissues were stained with anti-CD86 and anti-CD206 and analyzed by IHC stain (200x and 400x magnification). ^∗∗P < 0.01 (vs sham group), ^#P < 0.05 (vs CLP group), ^##P < 0.01 (vs CLP group), ^&P < 0.05 (vs ghrelin group), and ^&&P < 0.01 (vs ghrelin group).

Figure 5

Ghrelin/GHSR axis activated E2F1 expression and suppressed the activation of the NF-κB signaling pathway in septic rats. (a–c) The mRNA levels of E2F1, IκB, and p65 in colon tissues of septic rats were detected by RT-qPCR. (d–f) Proteins in cytoplasmic or nuclear extracts were determined using Western blot. ((g), (h)) Nuclear translocation of p65 was determined using immunofluorescence. The scale labels shown are 50 µm and 20 µm. The white arrows indicate the nuclear translocation of p65. ^∗∗P < 0.01 (vs sham group), ^#P < 0.05 (vs CLP group), ^##P < 0.01 (vs CLP group), ^&P < 0.05 (vs ghrelin group), and ^&&P < 0.01 (vs ghrelin group).