| Post-weaning diarrhea of piglet is a common disease in animal husbandry,which often results in the decline of growth performance and even death of piglets,Enterotoxigenic Escherichiacol（ETEC）is the main pathogen causing this disease.The infected piglets showed frequent excretion of watery or serous feces,severe cases showed mental depression and growth retardation,pathological anatomy showed damage of intestinal morphology and inflammation.Xiangsha Liujunzi decoction comes from“Ancient and modern famous medical Prescriptions”,it is mainly used for spleen deficiency,dampness and phlegm stagnation.Xiangsha Liujunzi decoction has beneficial effects of invigorating qi and spleen,dryness and phlegm reduction.It has been shown to have a good effect on post-weaning diarrhea of piglet.Glycyrrhiza is one of the medicines that make up Xiangsha Liujunzi decoction,glycyrrhiza polysaccharide（GP）is the main active ingredient of glycyrrhiza.Our study showed that GP can effectively relieve ETEC induced intestinal epithelial cell and ileal tissue inflammation of piglets.However,the mechanism of Xiangsha Liujunzi decoction and GP on intestinal injury induced by ETEC remains unclear.In this study,transcriptomic sequencing technology was used to research the effects of ETEC infection on the transcriptomic changes of related genes in the ileum of weaned diarrhea piglets,and the intervention effect of traditional Chinese medicine was discussed.RT-q PCR and Western-blot were used to verify the transcriptome levels of related genes and the expression levels of signaling pathway proteins.1.Transcriptomic analysis of ileal tissuePiglets weaned at 21 days of age were randomly divided into control group（CON）,model group（MOD）,Xiangsha Liujunzi decoction group（XS）and GP group（GP）.After 3 days of adaptive feeding,piglets in XS/GP were given XSLJZT and GP solution for 17 days,and piglets in CON and MOD were given the same amount of normal saline.piglets in MOD,XS and GP were given ETEC bacterial solution with a concentration of 1×1011CFU/m L at 1 m L/kg·BW for consecutive 3d from 15 to 17days,and piglets in CON were given an equal amount of LB medium.On the 18th day,all piglets were sacrificed after anesthesia.We collected ileum tissues and stored them at-80℃.Three ileal tissue samples from each group were frozen in liquid nitrogen for transcriptomic sequencing analysis.DEseq found that the 353 differentially expressed genes（DEGs）between the CON and the MOD included 190 up-regulated genes and163 down-regulated genes.Among 110 DEGs in MOD and XS,22 genes were up-regulated and 88 genes were down-regulated.Among 445 DEGs between the MOD and GP,201 genes were up-regulated and 244 genes were down-regulated.GO enrichment analysis showed that CON and MOD DEGs were mainly enriched in cell components and negative regulation of lymphocyte proliferation and activation,negative regulation of mononuclear cell proliferation,negative regulation of leukocyte proliferation,negative regulation of cell activation,synaptic assembly,brush border and so on.MOD and XS were mainly concentrated in cell components,defense response to bacteria and humoral immune response.MOD and GP mainly enriched in immune system process and immune response,cell surface receptor signaling pathway,defense response.KEGG enrichment analysis showed that CON and MOD were enriched in various amino acid and triglyceride metabolism,calcium signaling pathway,NF-κB signaling pathway.MOD and XS were mainly enriched in PPAR signaling pathway,IL-17 signaling pathway,TRP channel regulation of inflammatory mediators,cytokine-cytokine receptor interaction and MAPK signaling pathway.MOD and GP were mainly enriched in cytokine-cytokine receptor interaction,chemokine signaling pathway,IL-17 signaling pathway,B cell receptor signaling pathway,NF-κB signaling pathway and Jak-STAT signaling pathway.It was speculated that ETEC induced ileal inflammatory response mainly through calcium signaling pathway and NF-κB regulating downstream signaling pathway,and Xiangsha Liujunzi decoction and GP alleviated IL17 signaling pathway,cytokine-cytokine receptor interaction,chemokine pathway,MAPK signaling pathway and NF-κB signaling pathway.2.RT-q PCR verification of differential genesBased on the results of KEGG enrichment analysis,the selected inflammatory related differential genes were verified by Real time fluorescence quantification PCR（RT-q PCR）.The results showed that the m RNA expression abundance differences and significance of TRAF5,ERBB2,LBP,CXCL2,CCL28,MMP3,FABP6 and IL21R were consistent with the sequencing results,which indicated that the transcriptomic sequencing results were reliable and valuable for reference.The above key genes and their signaling pathways are closely related to MAPK signaling pathway,which suggests that MAPK signaling pathway may be a key signaling pathway in the ileum inflammation induced by ETEC and regulation of ileal inflammation by drugs.3.Detection of key proteins of MAPK signaling pathwayThe expression levels of ERK,p-ERK,p38,p-p38,JNK and p-JNK key proteins of MAPK signaling pathway in ileum of piglets in each group were detected by Western Blot.The P-P38/p38 ratio of MOD was significantly higher than that of CON（P<0.05）,the p-JNK/JNK ratio of MOD group was extremely significantly higher than that of CON（P<0.01）,and the p-ERK/ERK ratio of MOD was slightly higher than that of CON,but there was no significant difference（P>0.05）.Xiangsha Liujunzi decoction significantly inhibited the phosphorylation of p38 and ERK induced by ETEC（P<0.01）,GP significantly inhibited the phosphorylation of p38（P<0.01）,and significantly inhibited the phosphorylation of ERK and JNK（P<0.05）.In summary,RT-q PCR confirmed that the transcriptome sequencing results were accurate and reliable,and WB results showed that Xiangsha Liujunzi decoction and GP inhibited the phosphorylation of key proteins of MAPK signaling pathway through various signaling pathways,alleviating the ileum inflammatory response induced by ETEC. |
