Ghrelin improves small intestinal barrier damage in sepsis by promoting miR-143/ATG2B-mediated autophagy

Abstract

Intestinal barrier damage is crucial for the development of sepsis. Ghrelin (GHS) can restore intestinal barrier function. However, the mechanisms of GHS on intestinal barrier damage in sepsis remain unclear. We aimed to explore the mechanisms of GHS against intestinal barrier damage in sepsis. Septic models were established by cecal ligation and puncture surgery for rats and lipopolysaccharides exposure for IEC-6 cells. Furthermore, these septic models were overexpressed miR-143 and treated with GHS. In vivo, small intestinal pathological injury and D-lactic acid level were detected. Tight junction protein (Claudin-1, Occludin and ZO-1) expressions and autophagosome number were evaluated. In vitro, cell viability, autolysosome number, and relationship between miR-143 and ATG2B were determined. miR-143, ATG2B and autophagy-related protein (Beclin-1, p62 and LC3I/LC3II) levels were evaluated in rats and cells. GHS mitigated small intestinal pathological injury and decreased D-lactic acid level for septic rats. Additionally, GHS elevated tight junction protein expressions, ATG2B, Beclin-1 and LC3I/LC3II levels, and autophagosome number, but reduced miR-143 and p62 levels for septic rats. However, miR-143 overexpression presented the opposite results. Consistently, cellular experiments found that GHS increased cell viability, autolysosome number, and presented similar results for miR-143, ATG2B and autophagy-related protein levels for lipopolysaccharides-exposed cells. Additionally, ATG2B directly targeted miR-143 in IEC-6 cells. Both animal and cellular experiments found the effects of GHS on sepsis-induced small intestinal barrier damage were reversed by miR-143 overexpression. GHS may improve small intestinal barrier damage in sepsis through miR-143/ATG2B-mediated autophagy, indicating miR-143/ATG2B was an underlying therapeutic target for sepsis.

Fig 1. Ghrelin (GHS) improved barrier damage, modulated miR-143/ATG2B expression, and promoted autophagy in the small intestine for rats with sepsis.

(A) The pathological injury in the small intestine were evaluated by hematoxylin-eosin (H&E) staining. Magnification×200, scale bar = 100 μm; Magnification×400, scale bar = 500 μm. Red arrows: the intestinal mucosa exhibited partial shedding; Yellow arrows: the inflammatory cell infiltration. (B) The expressions of the main tight junction proteins (including Claudin-1, Occludin and ZO-1) in the small intestine were detected by Western blot analysis. n = 3. (C) The serum D-lactic acid (D-LA) level was detected in each group. n = 5. (D) Quantitative real time polymerase chain reaction (qPCR) was used to test the expressions of miR-143 and ATG2B mRNA in the small intestine. n = 3. (E) The expressions of ATG2B and autophagy-related protein (Beclin-1, p62 and LC3I/LC3II) in the small intestine were quantified by Western blot analysis. n = 3. *P < 0.05; **P < 0.01. Results were presented as mean±standard deviation (SD).

Fig 2. miR-143 overexpression worsened barrier damage and reduced ATG2B level in the small intestine for septic rats treated with GHS.

(A) Small intestinal pathological injury of each group were measured by H&E staining. Magnification×200, scale bar = 100 μm; Magnification×400, scale bar = 500 μm. Red arrows: the intestinal mucosa exhibited partial shedding; yellow arrows: the inflammatory cell infiltration. (B) Claudin-1, Occludin and ZO-1 protein levels in small intestine tissues of each group were detected by Western blot analysis. n = 3. (C) The D-LA level in the serum of each group was detected. n = 5. (D) The levels of miR-143 and ATG2B mRNA in small intestine tissues in each group were quantified by qPCR. n = 3. *P < 0.05; **P < 0.01. Results were presented as mean±SD.

Fig 3. miR-143 overexpression suppressed autophagy in the small intestine for sepsis rats treated with GHS.

(A) Western blot analysis of ATG2B and autophagy-related protein (Beclin-1, p62 and LC3I/LC3II) expressions in the small intestine of the rats. n = 3. (B) Transmission electron microscopy was applied to test the number of autophagosomes in the small intestine of the rats. Magnification×800, scale bar = 1 μm. n = 3. *P < 0.05; **P < 0.01. Results were presented as mean±SD.

Fig 4. GSH improved cellular models of sepsis by promoting autophagy through the targeting of miR-143/ATG2B.

(A) qPCR was conducted to verify miR-143 overexpression. n = 3. (B) The cell viability of each group was detected by cell titer-blue assay. n = 3. (C) The expression levels of miR-143 and ATG2B in each group were assessed by qPCR. n = 3. (D) The expressions of ATG2B and autophagy-related proteins (including Beclin-1, p62 and LC3I/LC3II) in the cells were tested by Western blot analysis. n = 3. (E) The number of autophagosomes in each group was detected using AdPlus-mCherry-GFP-LC3B adenovirus. Magnification×630, scale bar = 10 μm. n = 3. (F) The target relationship between miR-143 and ATG2B in IEC-6 cells was determined by dual-luciferase reporter assay. n = 3. *P < 0.05; **P < 0.01. Results were presented as mean±SD.