Mechanism of macrophage activation in Hirschsprung-associated enterocolitis

Abstract

Objective To explore the role of macrophage activation in a murine model of Hirschsprung-associated enterocolitis. Methods The Ednrb knockout mice were bred and fed at a SFP level laboratory. Experimental group: 21-day-old EdnrB-/- mice were selected(n=6) and comparable-age wild-type mice as control group(n=6). Intestinal samples were collected. The experimental colons were divided into narrow, transitional and expansion sections according to their morphological changes. The control colons were divided into distal, middle and proximal sections. Hematoxylin & eosin(HE) staining was used for assessing the degree of bowel inflammation injury. The expressions of CD68 and tumor necrosis factor-alpha(TNF-α) were detected by immunofluorescent staining for assessing the activation level of intestinal macrophage. And the expression of TNF-α protein was measured by Western blot. Results HE staining showed that dilated bowel of EdnrB-/- mice had varying degrees of inflammatory injury. Immunofluorescent staining showed that the numbers of CD68 positive cells in narrow, transitional and expansion sections were(10.81±2.38)%,(8.33±1.78)% and(4.28±1.25)% respectively. However those numbers in distal, middle and proximal sections of control group were(2.15±1.17)%,(1.89±0.98)% and(1.97±1.32)% respectively. The TNF-αpositive cells in narrow, transitional and expansion sections were(6.81±2.35)%,(5.00±1.41)% and(2.80±0.98)% respectively. In control groups, those numbers were(1.21±0.56)%,(0.89±0.46)% and(1.07±0.32)% respectively. Compared with wild-type murine intestines and narrow and transitional sections, the positive cells of CD68 and TNF-α were more intensely stained in dilated colonic segments(P<0.05). Western blot showed that the expression of TNF-α significantly increased in dilated colonic segments(P<0.05). However, the level of TNF-α was lower in transitional section than that in expansion section(P<0.05). However, it was still higher than that in narrow section in EdnrB-/- and wide-type mice(P<0.05). The control group showed no significant expression of TNF-α. Conclusions The injury of expansion section of bowel tissue is serious in a murine model of Hirschsprung's disease. And it may be associated with intestinal macrophage activation and secretion of proinflammatory cytokines. Key words: macrophage activation; Hirschsprung disease; Enterocolitis