| Diarrhea in weaned piglets is one of the most threatening diseases in the pig industry and a leading cause of piglet mortality.ETEC is the most common foodborne pathogen causing diarrhea in weaned piglets.After ETEC infection,enterotoxins can be released to stimulate the excessive secretion of intestinal fluids and electrolytes,leading to the occurrence of diarrhea.Moreover,the proliferation of ETEC in the intestine can also cause intestinal flora disorders and induce intestinal inflammation.Studies have shown that Xiangsha Liujunzi decoction has significant effects on gastrointestinal function regulation,intestinal barrier protection,and intestinal inflammation.To investigate the intervention effect of Xiangsha Liujunzi decoction on ETEC-induced diarrhea in weaned piglets and its regulatory mechanism,the following studies were carried out:1.The active ingredients of the Xiangsha Liujunzi decoction components and their targets were searched in the TCMSP database using the parameters"OB≥30%,DL≥0.18".Swiss Target Prediction was applied to analyze active ingredients’targets."ETEC diarrhea"was used as the keyword to screen the related targets of ETEC diarrhea in the OMIM,Gene Cards,Drug Bank,and Disgenet databases.A Venn diagram was used to find the common targets of ETEC-induced diarrhea and Xiangsha Liujunzi decoction treatment.The network of Xiangsha Liujunzi decoction-ETEC diarrhea-target genes was constructed by Cytoscape software,and the main active components were screened by topological analysis.The intersection targets’protein interaction networks were examined.GO functional enrichment analysis and KEGG enrichment analysis were performed.Finally,the binding ability of the main active ingredients to the target proteins was detected by molecular docking.The results were as follows:The Xiangsha Liujunzi decoction contains 169 active ingredients,and 340 targets were screened.It was found to act on ETEC-induced diarrhea via 116 active ingredients and 48 targets.The main active ingredients included quercetin,luteolin,kaempferol,formononetin,isorhamnetin,naringenin,nobiletin,and baicalein.The core targets in the protein interaction network analysis included tumor necrosis factor（TNF）,AKT/protein kinase（AKT1）,interleukin-6（IL-6）,vascular endothelial growth factor A（VEGFA）,epidermal growth factor receptor（EGFR）,signal transducer and activator of transcription 3（STAT3）,mitogen-activated protein kinase 1（MAPK1）,mitogen-activated protein kinase 8（MAPK8）,interleukin-8（IL-8）,and interleukin-1β（IL-1β）.GO enrichment showed that the biological processes mainly included the inflammatory response,the response to oxidative stress,the regulation of the MAPK cascade,etc.The cellular components included the vesicle lumen,the membrane rafts,the transcriptional regulatory complexes,etc.The molecular functions mainly involved cytokine receptor binding,protein kinase activity,kinase regulatory activity,etc.KEGG enrichment showed that the signaling pathways of Xiangsha Liujunzi decoction in the treatment of ETEC-induced diarrhea included TNF,Toll-like receptors,and MAPK signaling pathways.Molecular docking showed that the main active ingredients of Xiangsha Liujunzi decoction had strong binding abilities to the core targets.These results indicate that Xiangsha Liunjunzi Decoction can regulate TNF,Toll-like receptor,and MAPK signaling pathways by acting on TNF,AKT1,IL-6,VEGFA,EGFR,STAT3,MAPK1,MAPK8,IL-8,and IL-1βtargets through quercetin,luteolin,kaempferol,formononetin,isorhamnetin,naringenin,nobiletin,and baicalein and play a therapeutic role in ETEC-induced diarrhea.2.Twenty-four weaned piglets were randomly divided into three groups:the control group（CON）,the model group（MOD）,and the Xiangsha Liujunzi decoction group（XS）,with eight replicates in each group.After 3 days of adaptive feeding,the piglets in the XS group were given Xiangsha Liujunzi decoction（1 g/m L）at a dose of 1 m L/kg·bw for 14consecutive days,and the piglets in the CON and MOD groups were given the same amount of normal saline.On the 15th day,piglets in the XS and MOD groups were given ETEC bacterial solution(1×1011 CFU/m L)at a dose of 1 m L/kg·bw for 3 consecutive days,piglets in the CON group were given the same amount of LB broth,and piglets in the XS group were given Xiangsha Liujunzi decoction.On the 18th day,the piglets were euthanized,and after measurement and localization,the small intestinal tissues were collected,fixed,and frozen,and the ileocecal contents were collected and frozen.The stool status of piglets in each group was observed,and the diarrhea score was recorded.The duodenum,jejunum,and ileum tissues were collected for paraffin sectioning and H&E staining for pathological examination,and the villus height and crypt depth of each intestinal segment tissue were measured.The results indicated that:Compared with the CON group,the diarrhea score of piglets in the MOD group was significantly increased（P<0.01）;The different degrees of damage was observed in the duodenum,jejunum,and ileum in the MOD group,mainly manifested as villus abscission and decreased distribution of crypts and glands;The MOD group had significantly lower villus height in the duodenum（P<0.01）,jejunum（P<0.05）,and ileum（P<0.05）compared to the CON group;The jejunal and ileal crypt depth in the MOD group was significantly increased compared to the CON group（P<0.01）;The V/C values of duodenum（P<0.05）,jejunum（P<0.05）,and ileum（P<0.01）in the MOD group was significantly decreased compared to the CON group.Compared with the MOD group,the diarrhea score of piglets in the XS group was significantly decreased（P<0.01）;The injury of the duodenum,jejunum,and ileum in the XS group was significantly alleviated,and the structure of the intestinal villi was intact.The villus height of jejunum（P<0.05）and ileum（P<0.01）in the XS group was significantly increased compared to the MOD group.The crypt depth of jejunum and ileum was significantly decreased in the XS group compared to the MOD group（P<0.01）.The V/C values of duodenum（P<0.05）,jejunum（P<0.01）,and ileum（P<0.01）in the XS group was significantly increased compared to the MOD group.Xiangsha Liujunzi decoction alleviates diarrhea caused by ETEC infection and inhibits inflammatory damage to intestinal tissue in weaned piglets.3.High-throughput sequencing of the 16S r DNA V3-V4 region of ileocecal orifice contents was performed on the Illumina platform.The raw data of sequencing were subjected to quality control,denoising,splicing,and de-chimerization,in turn,using QIIME2 software and the DADA2 method,and the ASV/OTU table was drawn.Alpha diversity index analysis（Chao1 index,observed＿species index,Shannon index,Simpson index,etc.）was used to evaluate the richness and evenness of intestinal microorganisms.Non-metric multidimensional scaling analysis（NMDS）was used to evaluate beta diversity among different treatment groups.Statistical analyses were performed on the abundance of taxa at the phylum and genus levels.LEf Se analysis was used to reveal the differences in gut microbiota among groups and identify potential marker species.Based on the KEGG database,PICRUSt was used to predict and analyze microbial function.The results showed that:There was no significant difference in Alpha diversity between the MOD group and the CON group.NMDS analysis showed that the MOD group was significantly clustered and deviated from the CON group（Stress<0.2）.Compared with the CON group,proteobacteria were significantly increased and firmicutes were significantly decreased in the MOD group at the phylum level（P<0.01）;Lactobacillus was decreased and Shigella was increased in the MOD group at the genus level;In the prediction of microbiota function,glycolysis/gluconeogenesis（P<0.01）,bile acid synthesis（P<0.01）,and galactose metabolism-related processes（P<0.05）in the MOD group were significantly decreased compared to the CON group;Lipopolysaccharide synthesis,peroxisome,C5-branched dicarboxylic acid metabolism,and biotin metabolism in the MOD group were significantly increased compared to the CON group（P<0.01）.There was no significant difference in Alpha diversity between the XS group and the MOD group.NMDS analysis showed that the XS group was significantly clustered and deviated from the MOD group（Stress<0.2）.Compared with the MOD group,firmicutes were significantly increased and proteobacteria were significantly decreased in the XS group at the phylum level（P<0.01）;Lactobacillus was increased and Shigella was decreased in the XS group at the genus level;In the prediction of microbiota function,glycolysis/gluconeogenesis（P<0.01）and galactose metabolism-related processes（P<0.05）were significantly increased in the XS group compared with the MOD group;Lipopolysaccharide synthesis（P<0.01）,peroxisome（P<0.01）,C5-branched dicarboxylic acid metabolism（P<0.05）,and biotin metabolism（P<0.05）in the XS group were significantly decreased compared to the MOD group.LEf Se analysis showed that the marker species of each treatment group were Lactobacillaceae in the CON group,Shigella in the MOD group,and Deinococcus and Eubacterium in the XS group.Xiangsha Liujunzi decoction reduces the abundance of harmful bacteria such as Proteobacteria and Shigella and increases the abundance of beneficial bacteria such as Firmicutes and Lactobacillus to alleviate diarrhea caused by ETEC in weaned piglets.4.The mRNA expression levels of TNF-α,IL-1β,IL-6,and IL-8 in the duodenum,jejunum,and ileum were detected by real-time PCR.Western-blot was used to detect the expression of MAPK signaling pathway-related proteins in jejunal tissue.The results showed that:Compared with the CON group,the m RNA levels of TNF-α（P<0.05）,IL-1β（P<0.01）and IL-8（P<0.05）in the duodenum of the MOD group were significantly;The m RNA levels of IL-1β,IL-6,and IL-8 in the jejunum were significantly increased in the MOD group compared to the CON group（P<0.05）;The m RNA levels of IL-1β,IL-6,and IL-8 in the ileum were significantly increased in the MOD group compared to the CON group（P<0.01）;The p38 MAPK and p-JNK/JNK of the MOD group were significantly increased in the jejunum tissue compared to the CON group（P<0.05）.Compared with the MOD group,the m RNA levels of TNF-α（P<0.05）,IL-1β（P<0.05）and IL-8（P<0.01）in the duodenum of the XS group were significantly decreased;The m RNA levels of IL-1β（P<0.01）,IL-6（P<0.05）,and IL-8（P<0.01）in the jejunum were significantly decreased in the XS group compared to the MOD group;The m RNA levels of IL-1β（P<0.05）,IL-6（P<0.05）,and IL-8（P<0.01）in the ileum were significantly decreased in the XS group compared to the MOD group;The p38 MAPK（P<0.01）,JNK（P<0.05）,p-JNK（P<0.01）,p-JNK/JNK（P<0.01）,p-ERK1/2（P<0.01）,and p-ERK/ERK（P<0.01）of the XS group were significantly decreased in the jejunum tissue compared with the MOD group.Xiangsha Liujunzi decoction inhibits the activation of JNK and ERK1/2 and the expression of downstream inflammatory factors TNF-α,IL-1β,IL-6,and IL-8,thereby alleviating intestinal inflammation.In summary,Xiangsha Liujunzi decoction plays a therapeutic role in ETEC-induced diarrhea in weanling piglets through multiple components and targets.Network pharmacology found that the main active ingredients of Xiangsha Liujunzi decoction in the treatment of ETEC diarrhea included quercetin,luteolin,kaempferol,formononetin,isorhamnetin,naringin,nobiletin,and baicalein,etc.The core targets were TNF,AKT1,IL-6,VEGFA,EGFR,STAT3,MAPK1,MAPK8,IL-8,IL-1β,etc.The involved signaling pathways included TNF,Toll-like receptors,and MAPK signaling pathways.Xiangsha Liujunzi decoction can effectively alleviate ETEC-induced diarrhea in weaned piglets,inhibit intestinal tissue damage,reverse the changes in the structure of intestinal flora induced by ETEC,increase the abundance of beneficial bacteria in intestinal flora,and reduce the abundance of harmful bacteria.It also can alleviate intestinal inflammation by inhibiting the activation of JNK and ERK1/2 and the expression of downstream inflammatory factors TNF-α,IL-1β,IL-6,and IL-8. |
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