Study On Probiotic Enterococcuus Faecium HDRsEf1 Anti ETEC Intestinal Infection

Enterotoxigenic Escherichia coli(ETEC) were essential pathogenic bacteria which cause human and procine diarrhea, and had bad effect on Livestock and poultry breeding. Enterococcus faecium was regarded as a kind of probictic bacteria in intestinal flora, In this research we study on the Enterococcus faecium HDRsEf1 which was isolated from an healthy piglet, and expore its positive effect on ETEC infection, to lay the foundation of HDRsEf1 applied in controling Escherichia coli infection.Fistly, ETEC mouse infected model was established to assessed the effect of probiotic HDRsEf1 anti ETEC infection. Forty fifth-week ICR mouse were divided into four groups. Control group: from day 1 to day 21, 200μL of saline solution were gavaged to each mouse. Prevent group: each mouse in the Prevent group were gaven a daily dose of 109 viable HDRsEf1 cells, from day 1 to day 10, and on day 11 each mouse received a dose of 109 viable ETEC cells, then from day 12 to day 21 200μL of saline solution were provide to each mouse. Model group: on day 11 each mouse received a dose of 109 viable ETEC cells. From day 1 to day 10 and from day 12 to day 21, these mice also received 200μL of saline solution. Treatment group: from day 1 to day 10, 200μL of saline solution were gavaged to the animals in this group. A single dose of 109 viable ETEC on day 11 was provided to each mouse. From day 12 to day 21, a daily dose of HDRsEf1 cells was provided to each mouse. Then the ETEC infect MODE-K cell experiment was inplemented to expoler the molecular mechanism of HDRsEf1 regulate OCLN expression. Experimental results and conclusions are as follows:Diarrhea rate and body weight:Three days post ETEC challenge, the diarrhea rate was 66.7%, body weight was 24.30±0.18 g in model group which was significantly decreased compared with control group 25.2±0.49 g(P<0.05). The diarrhea rate was 33.3% in prevent group which was lower than model group. Ten days post ETEC challenge, the mouse in prevent group was 27.77±0.25 g signifigantly higher than 25.57±0.57 g(P<0.01)in model group. the treatment group diarrhea rate was 16.7% lower than model group, body weight was 27.50±0.26 g signifigantly higher than model group(P<0.01).Intestinal inflammatory response: One day after ETEC challenge, The Rating score of model group was 2.08±0.38 which was higher than 2, and then we would regard it as moderate ETEC infection, the prevent group was 1.50±0.25 lower than 2. Ten days post ETEC challenge, prevent rating score was 1.50±0.43 lower than model group 2.50±0.43(P < 0.05), the treatment group was 2.0±0.25. One day post ETEC challenge, the jejunum IL-1β 、TNF-α、IFN-γ expression increased but IL-10 expression decreased in the model group mouse(P < 0.05), prevent group downregulate IL-1β 、TNF-α、IFN-γ expression P < 0.05) and up-regulate IL10 expression(P < 0.05). Ten days post ETEC challenge, treatment group downregulate TNF-α、IFN-γ expression and up-regulate IL-10 expression.Intestinal chemical barrier: One day after ETEC infection, the number of jejunum IgA positive B cell and MUC-2、LYZ-2 mRNA lever decreased in model group, but they were much higher in prevent group. Ten days after ETEC infection, prevent group upregulate the number of jejunum IgA positive B cell and MUC-2、LYZ-2 mRNA lever, in treatment group the number of jejunum IgA positive B cell and MUC-2 mRNA was up-regulated.Intestinal tight junction protein expression: we analyze the effects of HDRsEf1 on mouse jejunum OCLN and ZO-1 expression at mRNA and protein level after ETEC challenge. One day after ETEC infection, OCLN expression decreased but not ZO-1 in model group. Ten day after ETEC infection, prevent and treatment group upregulate OCLN expression.The molecular mechanism of HDRsEf1 regulating OCLN expression: The invitro experiment found that HDRsEf1 could protect MODE-K from OCLN decrease caused by ETEC, which was similar with the vivo result. We transfer the reporter plasmid pGL3-Ocd-Luc into MODE-K cell and found that HDRsEf1 could protect occludin promoter activity from ETEC, and the RNA interference experiment reveal that HDRsEf1 regulate OCLN expression through active TLR-2 and PI3 K related signal pathway.In conclusion, probiotic HDRsEf1 could enhance intestinal barrier function to defense ETEC infection, including decrease diarrhea rate、body weight lose and alleviate intestinal inflamation; HDRsEf1 could regulate OCLN expression at transcriptinal lever and TLR-2 and PI3 K signal pathway play an role in HDRsEf1 increase OCLN expression.