Effects Of Enterotoxigenic Escherichia Coli F4ac On Intestinal Injury In Piglets

Enterotoxigenic Escherichia coli(ETEC)is an important pathogen causing diarrhea of newborn and post-weaned piglets,which has caused huge economic losses to pig industry.To investigate the pathogenic mechanism of ETEC F4ac,this study used ETEC F4ac to infect mice and piglets with intestinal epithelial cells IPEC-J2,respectively,to investigate the mechanism of ETEC F4ac’s effect on intestinal damage in piglets by simulating in vivo and in vitro infection.The main contents are as follows:(1)The diarrhea model of ETEC F4ac infected mice was established.Forty C57BL/6j mice(half male and half female)aged 18 g to 22 g were randomly divided into four groups.Mice were treated with different concentrations of ETEC(control group,1×10~9,3×10~9and5×10~9CFU/m L)by oral gavage at 0.03 m L/g,and diarrhea was detected after 7 days of continuous gavage.The results showed that diarrhea occurred in mice of different concentration of attack bacteria,and the diarrhea rate increased with the increase of attack bacteria concentration.The congestion phenomenon of duodenum and jejunum was found in the examination,which proved that the diarrhea model of mice was successfully constructed.When the concentration of ETEC was 5×10~9CFU/m L,the diarrhea rate was 100%and the mortality rate was 30%,which was the optimal concentration for constructing the diarrhea model of mice.(2)Ten C57BL/6j mice were gavaged with ETEC F4ac for seven consecutive days using the attack model constructed above,while another 10 mice of the same age were gavaged with the same dose of saline as a control group.It was found that the mice in the test group had reduced body weight and impaired intestinal barrier function.Specifically,the intestinal villi tissue was loosened,the villi were shed and broken,and the intestinal crypt was severely damaged.The level of DAO(Diamine oxidase)in serum was significantly higher than that in control mice(P<0.05).The relative m RNA expression of intestinal tissue tight junction proteins Occludin,claudin-1,ZO-1 and MUC1,MUC4,MUC13,MUC20 were significantly decreased(P<0.05).(3)Further exploration of the intestinal inflammatory response triggered by ETEC F4ac attacking bacteria showed that the serum levels of MPO(myeloperoxidase)were significantly higher in the test mice than in the control mice(P<0.01).The levels of TNF-α,IL-6,and IL-βin the duodenum,jejunum and ileum were significantly higher than those in the control group(P<0.05),and the levels of IL-6 and IL-βin the jejunum were extremely significantly higher than those in the control group(P<0.01).Quantitative real-time fluorescence(q RT-PCR)results showed that the relative m RNA expressions of inflammatory factors TNF-α,IL-6,IL-β,LIF,IFN-γ,and TGF-βwere significantly increased in the duodenum,jejunum and ileum of mice in the test group compared with the control group(P<0.05).The results show that ETEC F4ac infection leads to overexpression of intestinal inflammatory factors.(4)Further validation of ETEC F4ac infection-induced intestinal damage in piglets using porcine small intestinal epithelial cells.The results showed a significant decrease in cell activity(P<0.05),a significant increase in apoptosis(P<0.01),and a significant increase in c GMP and c AMP levels in the attacking group(P<0.05),which were closely related to the water and electrolyte balance in the cells.q RT-PCR results further showed that the relative m RNA expression of inflammatory factor-related genes such as TNF-α,IL-β,and IL-6 were significantly higher in the attacking group compared with the control group(P<0.01).(5)To investigate the mechanism of action of ETEC F4ac-induced intestinal injury based on the p38-MAPK signaling pathway.Protein blotting(Western Blot)was used to detect changes in the expression of ETEC F4ac attacking bacteria on the p38-MAPK signaling pathway and the pro-apoptotic protein Bax and the inhibitory protein Bcl-2.The results showed that p38-MAPK signaling pathway and Bax protein expression were increased(P<0.05)and Bcl-2 protein expression was decreased(P<0.01)in the jejunum of mice in the tapping group,suggesting that ETEC F4ac may induce intestinal injury through the p38-MAPK signaling pathway.Further validation of this mechanism using piglet intestinal epithelial cells confirmed that ETEC infection also resulted in increased of p-p38-MAPK,p38-MAPK and Bax protein expression(P<0.05)and decreased of Bcl-2 protein expression(P<0.05)in piglet small intestinal epithelial cells.Compared with the control group,the ETEC group showed significantly lower cellular activity within 12 h after attacking the bacteria,while SB203580(p38-MAPK signaling pathway inhibitor)alleviated the cellular damage caused by ETEC F4ac and rescued the changes in m RNA expression of p38-MAPK signaling pathway proteins and pro-inflammatory factors triggered by ETEC F4ac infection(P<0.05).In summary,the p38-MAPK signaling pathway may mediate ETEC to promote intestinal epithelial cell apoptosis and inflammatory injury,impairing intestinal barrier function and thus disrupting the integrity of the intestinal mucosa.