Regulation Of Total Glucosides Of Paeony On Intestinal Transcriptome Changes And Inflammatory Injury In Diarrhea Piglets Induced By ETEC

Piglet weaning is a key period in pig production.However,piglets will have diarrhea due to many reasons after early weaning.Enterotoxigenic Escherichia coli（ETEC）is a common intestinal pathogen,which can lead to serious intestinal inflammatory reaction,cause diarrhea in weaned piglets,affect growth and development and even lead to death,and bring huge losses to the breeding industry.Traditional Chinese medicine Paeonia Alba has the effect of treating dysentery.Total glucosides of Paeonia（TGP）is the total glycosides of Paeonia Alba extract,which has many pharmacological effects,such as anti-inflammatory,analgesic,protecting nervous system and anti-tumor.In order to explore the preventive and therapeutic effect and mechanism of TGP on ETEC weaning diarrhea piglets,the following studies were carried out in this paper:1.Piglets were selected as experimental subjects.The effects of TGP on diarrhea and intestinal injury of weaned piglets induced by ETEC infection were analyzed by stool score and pathological section.Twenty-four 21 days old weaned piglets were randomly divided into control group（CON）,model group（MOD）and total glucosides of paeony treatment group（TGP）.The piglets in TGP group were given total glucosides solution（10 mg/m L）daily according to 1 m L/kg body weight for 14 days.On the 15th day of the test,the MOD group and TGP group piglets were given 10¹¹CFU/m L ETEC bacterial solution according to 1 m L/kg body weight for 3 days.At the same time,TGP group continued to be given total glucosides of paeony solution,and CON group piglets were given the same amount of normal saline.On the 18th day,piglets in each group were sacrificed and tissue samples of small intestine were collected.During the experiment,the daily diarrhea of piglets in each group was scored and recorded.Paraffin sections of duodenum,jejunum and ileum of piglets in each group were made,and H-E staining was used for microscopic examination to observe the effects of ETEC infection and TGP prevention and treatment on intestinal histopathological changes of piglets.The results showed that:prophylactic administration of TGP significantly reduced the fecal score of diarrhea piglets（P<0.01）,and could effectively alleviate the diarrhea of weaned piglets caused by ETEC infection.Observing the pathological sections stained with H-E,it showed that ETEC will damage the morphological structure of small intestine of piglets,make the villi of small intestine epithelium deform and fall off,deepen the crypts,and there is mucus exudation in the intestinal cavity,TGP can reduce the deformation and fall off of villi and prevent the deepening of crypts and mucus exudation.The villi height,crypts depth and villus height/crypt depth（V/C）ratio of each intestinal segment were counted and analyzed.The results showed that compared with CON group,the V/C of duodenum,ileum and jejunum in MOD group decreased significantly or very significantly（P<0.05 or P<0.01）,and injury of ileum segment was the most significant.Compared with MOD group,the villi height of duodenum in TGP group increased significantly（P<0.01）,the villi length,crypts depth and V/C value of jejunum increased significantly（P<0.01）,and the crypts depth of ileum decreased significantly（P<0.05）,indicating that TGP has a better protective effect on the morphological structure of small intestine of piglets infected with ETEC.2.The ileal tissue of piglets was selected as the research object.The gene expression changes of ileal tissue of weaned diarrhea piglets induced by ETEC infection and the intervention effect of total glucosides of paeony were analyzed by deep sequencing technology.The differentially expressed genes（EDGs）were screened by analyzing the gene expression differences between CON group and MOD group,MOD group and TGP group by DESeq technology.The screened significantly different genes were enriched and analyzed by Gene Ontology（GO）by top GO,and the Kyoto Encyclopedia of genes and genomes（KEGG）by cluster profiler;q RT-PCR was used to verify whether the results of transcriptome sequencing analysis were reliable.The results showed that:Compared with CON group,there were 353differential genes in ileum in MOD group,including 190 up genes and 163 down genes.GO analysis showed that the differential genes were significantly enriched in molecular functions such as extracellular region and brush border,biological processes such as amino acid metabolism and negative regulation of cell activation,and cellular components such as 2-acylglycerol o-acyltransferase activity and L-threonine ammonia lyase activity（P<0.05）.KEGG analysis showed that multiple differential genes were enriched in Ins P3/Ca²⁺signaling pathway（P<0.05）and NF-κB signal transduction pathway（P=0.0743）.At the same time,compared with MOD group,TGP group had 598 differential genes in ileum,including 214 up genes and 384 down genes.GO analysis showed that differential genes were significantly enriched in molecular functions such as extracellular region and brush border,biological processes such as bacterial defense response,humoral immunity,leukocyte chemotaxis and inflammatory response（P<0.05）.KEGG enrichment analysis showed that,Multiple differential genes were enriched in IL-17 signaling pathway,chemokine signaling pathway（P<0.05）and NF-κB signal transduction pathway（P=0.0627）.The results of q RT-PCR are consistent with those of transcriptomics,indicating that the sequencing analysis of transcriptomics is reliable.Therefore,it is preliminarily clarified that the pathogenic mechanism of ETEC infected weaned piglets and the action mechanism of TGP on piglet colibacillosis are related to the immune response and inflammatory response in animals,which need further verification and analysis.3.The expression levels of NF-κB pathway proteins p65 and p-p65 in duodenum,jejunum and ileum are detected by Western-Blot,and the m RNA transcription levels of proinflammatory cytokines IL-1β,IL-6 and IL-8 in duodenum,jejunum and ileum are detected by q RT-PCR.The results showed that:Compared with CON group,the expression levels of p65 and p-p65 in duodenum,the expression level of p-p65 and the ratio of p-p65/p65 jejunum in MOD group were significantly or extremely significantly higher（P<0.05 or P<0.01）;Compared with MOD group,the expression levels of p65 and p-p65 in duodenum,jejunum and ileum in TGP group decreased significantly（P<0.05 or P<0.01）,and the ratio of p-p65/p65 in jejunum decreased significantly（P<0.01）.Compared with CON group,the transcription levels of IL-1β,IL-6 and IL-8 m RNA in duodenum,jejunum and ileum in MOD group were significantly higher（P<0.05 or P<0.01）.Compared with MOD group,the transcription levels of IL-1β,IL-6 and IL-8 m RNA in duodenum,jejunum and ileum in TGP group were significantly lower（P<0.01）.These suggests that ETEC infection activates NF-κB pathway and induce the secretion of proinflammatory factors,resulting in the body’s inflammatory response.The preventive treatment of TGP can inhibit the phosphorylation of housekeeping protein p65 in NF-κB pathway and the secretion of downstream proinflammatory cytokines,and has the best effect on the jejunum tissue.In conclusion,TGP can effectively alleviate diarrhea caused by ETEC infection in piglets,reduce the damage of intestinal tissue morphology caused by ETEC and protect the intestinal epithelial barrier.Transcriptome analysis showed that the prevention and treatment of ETEC infection in weaned piglets by TGP is related to Ins P3/Ca²⁺pathway,IL-17 signaling pathway,chemokine signaling pathway and NF-κB signaling pathway.TGP can inhibit the phosphorylation of the key protein p65of intestinal NF-κB signaling pathway and the transcription of downstream proinflammatory cytokines IL-1β,IL-6 and IL-8,so as to regulate the inflammatory response in the intestinal tract of weaned piglets infected with ETEC,reduce the intestinal injury of piglets,and explain the protective mechanism of TGP on the intestinal injury of weaned piglets with diarrhea caused by ETEC infection and the regulation of intestinal inflammatory response.