Abstract:

Objective This study aimed to investigate whether the intestinal mucosa of laying hens developed enhanced resistance to secondary injury after initial injury and to explore the underlying mechanisms， thereby promoting new perspectives for developing approaches to repair intestinal mucosal damage in laying hens. Method Seventy-eight 7-day-old Hyline White chicks were divided into 13 treatment groups （6 chicks per group）. Six chicks were radomly selected and received an intraperitoneal injection of PBS as the first negative control group （1^st NC）. The remaining 72 chicks were subjected to intestinal mucosal injury via intraperitoneal injection of lipopolysaccharide （LPS， 10 mg/kg BW）. Six chicks were radomly selected， euthanized and sampled at 1， 2， 4， 6， 8 and 24 hours post-injection （hpi）， designated as 1^st Xhpi groups （X represents the time of sampling after injection）. At 48 hours post-injection， six chicks from the LPS-injected group were radomly selected and injected with PBS as the second negative control group （2^nd NC， equivalent to the 1^st 48hpi group）， the remaining chicks received a second injection of the same dose of LPS. Six chicks were radomly selected， euthanized and sampled at 1， 2， 4， 6 and 8 hours post the second LPS challenge， designated as 2^nd Xhpi groups. Blood， duodenal tissues were collected， and intestinal crypts were isolated for analysis of barrier function， intestinal stem cells （ISCs） activity， and crypt niche changes. Result Following the first LPS challenge， inflammatory cell infiltration， decreased villus height/crypt depth， increased permeability， and decreased mucus layer thickness were observed in the intestinal mucosa of the 1^st 4hpi group. At 48 hours post injection， the degree of injury in the 2^nd NC group was reduced， and the barrier function was enhanced， which was manifested as the expression level of the tight junction Ocln gene， the density of goblet cells， the thickness of the mucus layer， and the expression level of the mucin Muc2 gene were all higher than those in the 1^st NC group. After the second LPS challenge， compared with 2^nd NC group，in the 2^nd 4hpi group， inflammatory cell infiltration and permeability did not significantly increase， but Claudin-1 and complement protein C5 gene levels were markedly upregulated （P<0.05）. The ISCs analysis results showed that the activity of active intestinal stem cells （aISCs） in the 1^st 4hpi group decreased and then gradually recovered， returning to normal at 48 hours post injury.The reserve ISCs （rISCs） in the 2^nd 4hpi group could be rapidly activated and maintain high activity. The analysis results of intestinal crypts showed that the Notch signal pathway of intestinal crypts was inhibited in both injury processes. Conclusion Following initial injury and repair， the intestinal mucosa exhibited enhanced barrier function and suppressed Notch signaling pathway in crypts. Secondary injury triggered rapid activation of rISCs and promoted differentiation of ISCs into secretory epithelial cells.

Key words: laying hens; intestinal mucosal repair; intestinal barrier function; intestinal stem cells