The Role Of MAPK/ERK Signaling Pathway In Chicken Necrotic Enteritis Was Analyzed Based On Microbiomics And Metabolomics

Clostridium perfringens(C.perfringens)is a conditionally pathogenic bacterium that causes necrotic enteritis(NE)in chickens,mainly causing small intestinal lesions.The intestinal barrier of chicks infected with C.perfringens is damaged and the absorption of nutrients is blocked,resulting in slow growth of chicks and serious economic losses to the poultry industry.But the mechanism of intestinal damage caused by NE in chickens has not been elucidated.This study intends to analyze the changes of intestinal tissue structure,intestinal mucosal tight junction proteins and mucin in diseased chickens by constructing a model of NE in chickens,and to explore its mechanism by cecal microflora detection and serum metabolomics.1.Establishment of a model of NE in chickensThe experiment was divided into three groups.Blank control group(Group CON)fed the basic diet,fishmeal group(Group F)fed basic diet contented 50% fishmeal.The infection group(Group NE)was gavaged with C.perfringens on a basic diet supplemented with 50% fish meal.The changes of body weight,intestinal lesion score,intestinal tissue morphology,intestinal tight junction proteins and mucin in each group were examined.The results showed that compared with the blank control group,the weight of chicks in the fish meal group decreased,there was no significant difference in intestinal lesion score,the intestinal tissue structure was intact,the m RNA expression of the jejunal mucosal tight junction proteins Occludin and Claudin-1 was significantly increased(P<0.05)and the m RNA expression of the MUC-2 was increased(P>0.05).Compared with the blank control group,the body weight of the chickens in the infection group was significantly decreased(P<0.05),the intestinal lesion score was significantly increased(P<0.05),the epithelial cells of the jejunum mucosa were necrotic and exfoliated,the ileum was atrophied,and the m RNA expression of jejunum mucosa tight junction proteins Claudin-1 and ZO-1 was significantly decreased(P<0.05),and m RNA expression of MUC-2 was decreased(P>0.05).The results showed that feeding fish meal alone was not sufficient to cause NE.Infected chicks lost weight,the structural integrity of the jejunum and ileum was damaged,the expression of intestinal tight junction proteins and mucin were decreased,which showed the changes of subclinical NE.2.Analysis of intestinal flora and serum metabolomics in chickens with NEThe cecal microflora of chicks in the above groups was analyzed by high throughput sequencing technology.The results showed that the composition and structure of intestinal flora in the fish meal group changed significantly,and the diversity of intestinal flora in the infection group decreased significantly.The relative abundance of beneficial flora,such as Lactobacillus,Blautia,norank＿f＿＿Ruminococcaceae and Ruminococcus＿gauvreauii＿group,was significantly lower in the fishmeal group and infection group than in the blank control group(P<0.05).The relative abundance of conditionally pathogenic bacteria,such as norank＿f＿＿norank＿o＿＿Clostridia＿UCG-014,norank＿f＿＿norank＿o＿＿clostridia＿vadin BB60＿group and Escherichia-Shigella,was higher significantly in the fishmeal group than in the blank control group(P<0.05).The relative abundance of Clostridiaceae and Erysipelatoclostridium in the infection group was higher significantly than that in the blank control group(P<0.05).The results showed that fish meal changed the structure of the intestinal flora,resulting in beneficial flora decreased significantly and harmful flora increased significantly of chickens with NE.Metabolites in serum samples of chicks in each group were analyzed by metabolomics technology.The results showed that there were significant differences in the metabolic pathways of glycerophospholipid metabolism,arginine and proline metabolism in the fish meal group compared to the blank control group.The differences in metabolic pathways in the infection group compared to the blank control group were mainly in metabolic pathways related to cell proliferation,differentiation and migration such as the MAPK signalling pathway.Intestinal villus shortening and intestinal epithelial cells necrosis in the NE chickens suggested that MAPK/ERK pathway might play an important role in the development of NE in chickens.3.Validation of MAPK/ERK signaling pathway in jejunumThe expression of genes related to MAPK/ERK signaling pathway in jejunal mucosa was examined by q PCR and Western blot.Compared with the blank control,at the m RNA level,the expression of Raf1 was reduced of infection group,the expression of MEK and ERK was significantly reduced of infection group(P<0.01).At the protein level,the expression of Raf1,MEK and ERK was reduced of infection group,the expression of phosphorylated MEK and phosphorylated ERK was significantly reduced of infection group(P<0.05).To verify the inhibition of cell proliferation during the development of NE the expression of PCNA in the jejunum was detected using q PCR and IHC.It was found that the expression of PCNA of the infection group was significantly lower(P<0.05)compared to the blank control group and fish meal group.The above findings suggest that the MAPK/ERK pathway,which is associated with cell proliferation,is inhibited in the jejunum of chickens with NE,which is likely to be associated with intestinal damage and repair in chickens with NE.In conclusion,feeding fishmeal alone is not sufficient to induce chickens with NE,but it can alter the intestinal flora to facilitate the proliferation of Clostridium perfringens and induce NE.Intestinal epithelial barrier was damaged and activation of MAPK/ERK signaling pathway was inhibited of NE chickens,therefore inhibits the proliferation of intestinal epithelial cells and hinders the repair of the intestinal epithelial barrier.