Chronic Exposure to Cigarette Smoke Affects the Ileum and Colon of Guinea Pigs Differently. Relaxin (RLX-2, Serelaxin) Prevents Most Local Damage

Abstract

Cigarette smoking (CS) is the cause of several organ and apparatus diseases. The effects of smoke in the gut are partially known. Accumulating evidence has shown a relationship between smoking and inflammatory bowel disease, prompting us to investigate the mechanisms of action of smoking in animal models. Despite the role played by neuropeptides in gut inflammation, there are no reports on their role in animal models of smoking exposure. The hormone relaxin has shown anti-inflammatory properties in the intestine, and it might represent a putative therapy to prevent gut damage caused by smoking. Presently, we investigate the effects of chronic smoke exposure on inflammation, mucosal secretion, and vasoactive intestinal peptide (VIP) and substance P (SP) expressions in the ileum and colon of guinea pigs. We also verify the ability of relaxin to counter the smoke-induced effects. Smoke impacted plasma carbon monoxide (CO). In the ileum, it induced inflammatory infiltrates, fibrosis, and acidic mucin production; reduced the blood vessel area; decreased c-kit-positive mast cells and VIP-positive neurons; and increased the SP-positive nerve fibers. In the colon, it reduced the blood vessel area and the goblet cell area and decreased c-kit-positive mast cells, VIP-positive neurons, and SP-positive nerve fibers. Relaxin prevented most of the smoking-induced changes in the ileum, while it was less effective in the colon. This study shows the diverse sensitivity to CS between the ileum and the colon and demonstrates that both VIP and SP are affected by smoking. The efficacy of relaxin proposes this hormone as a potential anti-inflammatory therapeutic to counteract gut damage in humans affected by inflammatory bowel diseases.

Keywords: blood vessels; inflammation; mast cells; mucins; relaxin hormone; substance P; vasoactive intestinal peptide.

FIGURE 1

Hematoxylin–eosin (H&E) and Toluidine blue (TB) staining and cell infiltrates. (A–C) Ileum. (A) Full-thickness section showing the integrity of the enteric wall. (B) Numerous eosinophil granulocytes (arrows) in the mucosa. (C) TB staining pointing out several plasma cells (arrows) in the mucosa. (D) Colon. Full-thickness section showing the integrity of the enteric wall. Bars, 100 μm (A,D) and 25 μm (B,C).

FIGURE 2

Hematoxylin–eosin (H&E) staining and blood vessel area changes. (A,B) Ileum. (D,E) Colon. Representative images of the submucosa and the mucosa of control (A,D) and cigarette smoke (CS)-exposed (B, E) guinea pigs showing several blood vessels containing erythrocytes (arrows). Quantitation of the blood vessel area in the ileum (C) and colon (F) demonstrated a significant decrease in CS-exposed animals. Relaxin-2 (RLX-2), chronically administered in CS-exposed animals, prevented this decrease (C,F). Bar, 25 μm (A,B,D,E). *p < 0.05.

FIGURE 3

Periodic acid–Schiff (PAS) reaction and Toluidine blue (TB) staining and mucin changes in the cigarette smoke (CS) group. (A–H) Ileum. Representative images of the mucosa stained with PAS (A–C) and TB (E–G). Both dyes stained the goblet cells. Quantitation of the stained cells showed no differences among the groups of animals using the PAS reaction (D), whereas a significant increase in the number of goblet cells stained with TB was detected in CS-exposed animals (H). This increase was prevented by relaxin-2 (RLX-2) treatment (H). (I–P) Colon. Representative images of the mucosa stained with PAS (I–K) and TB (M–O). Quantitation of the stained areas, with each dye showing a significant decrease in CS-exposed animals (L, P). RLX-2 did not prevent the decreases (L, P). Bar, 50 μm (A–C, E–G, I–K, M–O). *p < 0.05; ***p < 0.0001.

FIGURE 4

c-Kit-positive mast cells decreased in the cigarette smoke (CS) group. (A–C) Ileum. c-Kit-positive cells corresponding to mast cells are present in the mucosa surrounding the glandular fundus. (E) Quantitation of the number of c-kit-positive mast cells showed a significant decrease in CS-exposed animals. (D) Negative control. This decrease was prevented by relaxin-2 (RLX-2). Bar, 25 μm (A–D). *p < 0.05.

FIGURE 5

A–I Vasoactive intestinal peptide (VIP)-immunoreactive (IR) submucosal neurons in the ileum (A–D) and colon (F–I) of the cigarette smoke (CS) group and effects of relaxin-2 (RLX-2). Full-thickness sections. In both regions and in all groups of animals, labeling was detected only inside the ganglia. IR had a granular aspect and was located in the somata and in the first tracts of the processes. (E) In the ileum, the number of VIP-IR neurons was significantly decreased in CS-exposed animals, and RLX-2 administration prevented this decrease. (D,I): negative controls. (J) In the colon, the number of VIP-IR neurons was significantly decreased in CS-exposed animals, but RLX-2 did not prevent this decrease. CML, circular muscle layer; SM, submucosa; MM, muscularis mucosae; GF, glandular fundus. Bar, 25 μm (A–D; F–I). *p < 0.05; **p < 0.005.

FIGURE 6

Increase in substance P (SP)-immunoreactive (IR) nerve fibers in the submucosa and the mucosa in the ileum of the cigarette smoke (CS) group and effects of relaxin-2 (RLX-2). (A, C) Laminae, submucosa. SP-IR was located in numerous nerve fibers forming nerve bundles (NB) and in the nerve strands (NS). (B–E) Full-thickness section, mucosa. Thin SP-IR nerve fibers ran along the axes of the villi (V) mostly lining the epithelium (E). (E) Negative control. (F) SP-IR nerve fibers significantly increased in the mucosa and submucosa of CS-exposed animals. This increase was insensitive to RLX-2 treatment. CML, circular muscle layer; LP, lamina propria. Bar, 50 μm (A–E). *p < 0.05.

FIGURE 7

Decrease in substance P (SP)-immunoreactive (IR) nerve fibers in the submucosa in the colon of the cigarette smoke (CS) group and effects of relaxin-2 (RLX-2). (A,B) Laminae, submucosa. SP-IR was located in numerous nerve fibers that formed nerve bundles (NB) and in the nerve strands (NS). (C) The SP-IR nerve fibers significantly increased in CS-exposed animals. This increase was insensitive to RLX-2 treatment. Bar, 50 μm (A,B). **p < 0.005.