Effect And Mechanism Of Zinc Laurate On Enterotoxigenic Escherichia Coli(ETEC) Enteritis

Enterotoxigenic Escherichia coli（ETEC）infection is an important factor leading to diarrhea in weaning piglets based on the high adhesion and virulence factors.Antibiotic were forbidden to apply in feeds in China.Zinc oxide was the important nutrition strategy to deal with ETEC infection,but the low absorption rate and high emission of zinc oxide would increase the pressure of environmental pollution.In recent years,more and more nutritionists were focused on lauric acid because of its inhibiting the proliferation of harmful bacteria,maintaining intestinal health and promoting animal growth.On the other hand,lauric acid has the ability of metal-chelating.Therefore,in this study,zinc Laurate（ZnLa）was used to study the mechanism of clarify the inhibitory effect by establishing the model of marginal zinc deficiency and ETEC infected weaned mice in vivo and in vitro,and to further reveal its regulatory effect and mechanism on intestinal injury in ETEC infected mice.The specific results are were follows:1.Mechanism of marginal zinc deficiency on intestinal inflammation and intestinal barrier function in ETEC infected mice.In the experiment,a total of 36 female and 3-weeks-old ICR mice were fed with zinc-deficient feed or normal zinc-level feed for 16 days to establish a zinc-deficient mouse model,and then randomly divided into 4 groups:zinc-deficiency group（dZn）,zinc-deficiency challenge group（dZn+ETEC）,normal zinc group（ZnSO₄）and normal zinc challenge group（ZnSO₄+ETEC）.The test period was 16 days.On day 14th,ETEC 1×10⁹ CFU or LB medium was administered into the stomach,and the animals were anesthetized and sampled after fasting for 8 hours.The results showed that marginal zinc deficiency aggravated weight loss,diarrhea rate,intestinal injury and intestinal inflammation in ETEC infected mice,mainly characterized by decrease of villus height and villus height/crypt depth,decrease of intestinal Occludin,Muc-2 and NHE3 gene expression,and significant increase of serum DAO,DLA and endotoxin,IL-1βand TNF-αconcentrations.Marginal zinc deficiency significantly decreased serum zinc level and jejunal zinc receptor GPR39 gene expression in non-ETEC infected mice,but did not significantly change serum zinc level and jejunal GPR39 gene expression in ETEC infected mice.Further studies showed that marginal zinc deficiency could significantly increase ETEC colonization in jejunum of ETEC-infected mice,and significantly up-regulate the expression levels of heat-labile enterotoxins（eltA and eltB）,heat-resistant enterotoxins（estB）,exercise（Mota）,cell adhesion（faeG）and biofilm（bssS）-related virulence genes in cecal chyme of ETEC-infected mice.Marginal zinc deficiency aggravates intestinal inflammation and intestinal barrier damage caused by ETEC infection,which is highly related to the enhancement of ETEC virulence factor gene expression by marginal zinc deficiency.2.Inhibitory effect and mechanism of ZnLa on ETEC and S.aureus in vitroThe minimal inhibitory concentration（MIC）,bacteriostatic growth curve and bacteriostatic effect of ZnLa were determined in vitro with ETEC and S.aureus,and the antibacterial mechanism of ZnLa was investigate based on the conductivity of the bacterial solution,the leakage of intracellular biomacromolecules and the morphology of the bacterial cells changes.The results showed that the MIC of ZnLa to ETEC and S.aureus were 1000μg/m L and 2000μg/m L,respectively,and the inhibition rates were more than 93.4%and 89.9%in 24 h respectively.In addition,the concentration and reaction time of ZnLa were positively correlated with the bacterial solution conductivity,protein and nucleic acid leakage of ETEC and S.aureus.ETEC and S.aureus cells were shrinkaged,collapsed and damaged at MIC of ZnLa.Therefore,under the experimental conditions,ZnLa inhibithted the bacteria growing by destroying the integrity of cell membrane and improving the permeability of cell membrane.3.Effect of ZnLa on intestinal damage in ETEC infected mice45 3-week-old ICR female mice were randomly divided into zinc deficiency group（dZn）,zinc deficiency challenge group（dZn+ETEC）,low,medium and high dose ZnLa challenge group（ZnLa LOW+ETEC,ZnLa LOW+ETEC,ZnLa MID+ETEC,andZnLa HIGH+ETEC）.On the day 16,all the challenge groups were intragastrically with ETEC 1×10⁹ CFU,and the control group were orally the equal dose of LB medium.The results showed that ZnLa decreased significantly could effectively alleviate the weight loss,intestinal barrier and permeability damage of mice with infected by ETEC.Compared with dZn+ETEC group,the body weight loss,diarrhea rate,liver,kidney and thymus index,and increased intestinal villus height and villus height/crypt depth ratio were decreased significantly in ETEC infected mice,and also significantly decreased the contents of serum DAO,DLA,endotoxin,IL-1β,IL-6 and TNF-αin mice.The relative expression of mRNA of TLR4,NF-κB,MyD88,IL-1β,IL-6,TNF-αand CFTR in jejunum were decreased,but the expression of GPR39,Occludin,ZO-1and NHE3 in jejunum were significantly increased,and the colonization of ETEC in jejunum and feces and the expression of eltA,eltB,estB,bssS and FaeG-related virulence factors in cecum were decreased.The intestinal injury of ETEC infectious was alleviated by ZnLa,which was closely related to the intestinal barrier function repair,inhibition of ETEC colonization and virulence gene expression,and the middle dose of ZnLa was the best for the inhibition of inflammation in our experiment.