Citric Acid Mediate The Protective Effects Of Hermetia Illucens Larvae Powder In ETEC Induced Piglets Intestinal Barrier Damage And Inflammatory Response

Due to changes in the environment and feed,newborn piglets are prone to hypoimmunity and intestinal barrier damage after weaning.Enterotoxigenic Escherichia coli（ETEC）K88 strain is a typical pathogen causing piglet sickness at this stage.Hermetia illucens larvae powder are widely used in livestock and poultry production due to their high nutritional value and immunomodulatory effects,which can enhance the immune homeostasis of the colonic mucosa of finishing pigs by altering bacterial composition and its metabolites.However,there are few reports on the protective mechanism of Hermetia illucens larvae powder on the weaning process of piglets,especially the protective mechanism of weaned piglets under the invasion of pathogenic bacteria.Therefore,this paper uses ETEC K88 strain to infect weaned piglets,and explores the effect and specific mechanism of adding Hermetia illucens larvae powder to the early feed on ETEC-infected piglets.The experiment selected 12 weaned piglets with an initial average body weight of7.68×0.26 kg,and were randomly divided into 3 groups with 4 pigs in each group.The experiment was divided into 3 treatment groups,C group（ETEC K88 group）,T1 group（4%Hermetia illucens larvae powder+ETEC K88 group）and T2 group（8%Hermetia illucens larvae powder+ETEC K88 group）.Piglets were fed with 0%,4%,and 8%Hermetia illucens larvae powder（group C,group T1,group T2）in their diet for 28 days.On day 29,ETEC K88(5×10¹⁰ CFU/m L)was instilled into piglets using a gavage catheter,and on day 32,blood was collected from the piglet’s anterior vena cava for metabolite detection.An appropriate amount of ileal tissue was taken and placed in PBS buffer to wash off the contents,and a part was placed in a 4%formaldehyde solution,followed by making slices;the other part was scraped on the ice plate to scrape the intestinal mucosa for related protein detection.The results showed that the levels of immunoglobulins Ig A and Ig G in the serum of the two groups of piglets added with Hermetia illucens larvae powder were significantly increased under the same infection with ETEC K88,and the ileum developmental morphology was more complete and the expression of tight junction proteins was higher,the expression levels of pro-inflammatory factors and the percentage of apoptosis were significantly decreased.Therefore,the addition of Hermetia illucens larvae powder improved the immune performance of piglets and inhibited the apoptosis and inflammatory response of ileal epithelial cells in ETEC K88 infected piglets.Serum metabolomic analysis found that the metabolite citric acid（CA）was significantly positively correlated with the added dose of Hermetia illucens larvae powder.The results show that CA can stimulate mouse peritoneal macrophages（RAW264.7）to secrete cytokines BAFF,IL-10 and other related gene expression related to B lymphocyte activation,and increase the proportion of Ig G⁺,Ig A⁺cells in CD138⁺CD19⁺cells in mouse spleen.Therefore,the addition of Hermetia illucens larvae powder can effectively increase the content of CA in serum,which plays an immunoregulatory role,this result also consistent with the elevation of Ig A and Ig G in piglet serum after the addition of Hermetia illucens larvae powder.The findings further suggest that CA can inhibit the inflammatory response and apoptosis in porcine ileal epithelial cells（IPI-2I）.The same results were also validated in the ETEC K88 infected mouse model.Therefore,CA plays an active role in maintaining the integrity of the intestinal barrier,which fully explains why the addition of Hermetia illucens larvae powder protects the intestinal barrier of piglets from damage.Combined analysis of stool 16S sequencing results and serum metabolomics revealed that CA was significantly positively correlated with intestinal Massilia.And we found that Massilia fermentation products contain a lot of CA.This gives us a hint that the increase of CA in piglets may be related to the intestinal bacteria Massilia.In conclusion,the protective effect of Hermetia illucens larvae powder on ETEC K88 induced piglet injury was mainly by effectively increasing the content of serum CA,which has the properties of promoting B lymphocyte differentiation,inhibits inflammatory response and has anti-epithelial cell apoptosis properties.The results provide new ideas for the treatment of piglets infected with other pathogenic microorganisms.