Effect Of LCN2 On The Ability Of Defense Against Bacterial Infection In Mice

In pig production,E.coli infection can cause diarrhea and even death in piglets,which greatly increases the economic losses of farmers and enterprises.A large number of studies have reported that Lipocalin-2(LCN2)is an acute reactive protein,and its expression will be significantly up-regulated in body injury and inflammation.LCN2 can chelate the siderophore of bacteria to inhibit bacterial growth.But it is unclear whether it has antibacterial effects on host.In this study,C57BL/6J mice were used as the research object to construct a mouse model of Escherichia coli O157 infection.We studied the role of LCN2 in the process of anti-infection in mice and explored the anti-inflammatory effect of LCN2 in the process of infection and its regulation on intestinal barrier and iron metabolism.This study provides a theoretical basis for the industrial application of LCN2 and a novel idea for the prevention and treatment of Escherichia coli infection.In this study,30 male C57BL/6J mice with similar physical condition were divided into four groups:LCN2+/+group(n=5),LCN2+/++E.coli group(n=10),LCN2-/-group(n=5),LCN2-/-+ E.coli group(n=10).LCN2+/+ indicated wild-type(WT)mice and LCN2-/-indicated LCN2-knockout mice.Each of the LCN2+/+group and LCN2-/-group was intragastrically administrated with 200 μL sterile PBS solution,each of the LCN2+/+ E.coli group and LCN2-/-+E.coli group was intragastrically administrated with bacterial suspension containing about 2×108 CFU E.coli O157.The samples were sacrificed on the sixth day after intragastric administration for subsequent analysis.1.Effects of LCN2 on body weight and bacterial content in infected mice:1.Under normal conditions,there was no significant difference in body weight gain between the two genotypes of mice.After infection,the body weight gain of LCN2-/-mice was significantly lower than WT mice(P<0.01).2.Compared with infected WT mice,the content of harmful bacteria in liver of infected LCN2-/-mice was significantly increased(P<0.05),and the number of Escherichia coli in cecum content of infected LCN2-/-mice was significantly increased(P<0.01).These results indicated that LCN2 deficiency significantly reduced body weight gain and increased the growth and reproduction of E.coli in liver and intestine after infection with E.coli 0157,suggesting that LCN2 may be related to the immune response to bacterial infection.2.Effect of LCN2 on organ index of infected mice:under normal conditions,there was no significant difference in organ index between WT and LCN2-/-mice.Compared with infected WT mice,the liver index of infected mice was significantly increased(P<0.05),and the kidney index of infected LCN2-/-mice was significantly decreased(P<0.05).These results indicated that LCN2-/-mice were more seriously injured,occurring liver and kidney metabolic disorders.3.Effect of LCN2 on inflammation in infected mice:1.H&E staining showed that the jejunum of WT and LCN2-/-mice developed well under normal conditions.After bacterial treatment,the intestinal villi of the jejunum of infected WT mice were slightly damaged,while infected LCN2-/-mice were severely damaged.2.RT-qPCR results showed that,pro-inflammatory cytokines IL-6 and IL-1β in liver of infected LCN2-/-mice are significantly higher than infected WT mice(P<0.01).Pro-inflammatory cytokines IL-6 and TNF-α in spleen of infected LCN2-/-mice are significantly higher than infected WT mice(P<0.01).Anti-inflammatory cytokines IL-10 and TGF-β1 in spleen of infected LCN2-/-mice are significantly higher than infected WT mice(P<0.05).3.ELISA showed that the levels of IL-6 and IL-1β in serum of LCN2-/-mice were significantly higher than WT mice(P<0.05).These results indicated that the LCN2 deficiency aggravated the damage of E.coli to intestinal villi in mice,promoted the secretion and expression of pro-inflammatory cytokines IL-6 and IL-1β in mice,indicating that LCN2 could alleviate the inflammatory damage caused by E.coli O157 infection in mice.4.Effects of LCN2 on intestinal barrier of infected mice:1.Western Blot results showed that compared with WT mice,the level of tight junction protein Occludin in LCN2-/-mice was significantly decreased(P<0.05),and the level of Claudin-1 in LCN2-/-mice had no significant change(P>0.05).After infection,the protein levels of Occludin and Claudin-1 in infected LCN2-/-mice were significantly lower than infected WT mice(P<0.01).2.PAS staining results showed that under normal conditions,the goblet cells in the colon of WT and LCN2-/-mice were abundant and evenly distributed.In the case of infection,the number of goblet cells in the colon of infected WT mice was slightly reduced,while in infected LCN2-/-mice was significantly reduced and unevenly distributed.3.MPO immunohistochemical results showed that under normal conditions,there was no significant difference in MPO content in the ileum of WT and LCN2"/-mice.In the case of infection,compared with infected WT mice,the MPO content in ileum tissue of infected LCN2-/-mice was significantly increased.RT-qPCR results showed that compared with infected WT mice,the expressions of IL-6,IL-1β,TNF-α,IL-10 and TGF-β1 in ileum of infected LCN2-/-mice were significantly increased(P<0.01).4 The results of intestinal flora analysis showed that the richness index(Chao 1)and diversity index(Shannon)of intestinal flora in LCN2-/-mice were higher than those in WT mice,and the difference in Chao1 index was not significant(P>0.05),while the Shannon index showed a significant trend(P=0.052).At the phylum level,compared with WT mice,the abundance of Proteobacteria and Deferricobacter in LCN2-/-mice was significantly decreased(P<0.01),and the abundance of Patescibacteria and Actinomycete increased significantly(P<0.01).These results indicated that LCN2 deficiency significantly decreased the tight junction protein level,reduced the number of goblet cells in colon,increased the infiltration of neutrophils in ileum,and changed the intestinal flora of infected mice,which revealed that LCN2 could improve the intestinal barrier function of mice infected with Escherichia coli O157.5.Effect of LCN2 on iron metabolism in infected mice:1.Compared with uninfected mice,the relative mRNA expression of Hepcidin in liver and duodenum of infected WT and LCN2-/-mice increased significantly(P<0.01),but there was no significant difference between two genotypes(P>0.05).2.The relative mRNA expression of FPN in liver of WT and LCN2-/-mice was significantly decreased(P<0.01),but there was no significant difference between two genotypes(P>0.05).After infection,the expression of FPN in the duodenum of the two genotypes did not change significantly.But compared with WT mice,the expression of FPN in the duodenum of LCN2-/-mice showed a downward trend(P=0.055).3.The relative expression levels of FtH in liver and duodenum of infected WT mice were significantly decreased(P<0.01),while there was no significant change in FtH of infected LCN2-/-mice(P>0.05).Moreover,FtH in liver of LCN2-/-mice was significantly higher than WT mice(P<0.01).In duodenum,FtH of WT mice without infection was significantly higher than LCN2-/-mice(P<0.01).These results suggest that LCN2 deficiency has no significant effect on iron metabolism in infected mice.In summary,LCN2 can alleviate the inflammatory injury caused by E.coli O157 infection in mice and improve the intestinal barrier function of infected mice.