Exploration Of The Effect And Mechanism Of Glycyrrhiza Polysaccharide On ETEC Induced Intestinal Inflammatory Injury In Piglets

Piglet diarrhea is a difficult problem in the pig industry.Postweaning diarrhea often occurs in early weaned piglets,and the main pathogenic bacteria is Enterotoxigenic Escherichia coli.Diarrhea after weaning caused growth retardation and feed utilization rate decline,which caused serious economic losses in pig industry.Many studies have shown that glycyrrhiza polysaccharide is one of the main effective components of glycyrrhiza and has a variety of biological activities,such as anti-tumor,anti-bacterial,anti-virus,anti-oxidation and so on.However,there are few researches on the application of glycyrrhiza polysaccharide as a Chinese medicine additive in pig industry.In order to explore the prevention effect and control mechanism of glycyrrhiza polysaccharide on ETEC-induced diarrhea in piglets,the following studies were carried out:1.Twenty-four 21-day-old weaned piglets were randomly divided into control group（Con）,model group（Mod）,glycyrrhiza polysaccharide group（GP）with 8piglets in each group.During the first 14 days of the experiment,GP group was given glycyrrhiza polysaccharide solution（1 g/m L）daily at the dose of 1 g/kg·bw.The Mod group and GP group were given 10¹¹ CFU/m L ETEC bacterial solution at the dose of 1 m L/kg·bw for 3 consecutive days.Meanwhile,GP group continued to receive glycyrrhiza polysaccharide.On day 18,intestinal tissue samples were collected from each group.During the whole experiment,the diarrhea of piglets in each group was observed daily,and the stool score was recorded.The sections of small intestine of each group were stained by H-E staining.And the villus height and crypts depth of each segment of small intestine were measured,and the villus height/crypts depth of small intestine（V/C）were calculated.Results:No diarrhea was observed in each group before challenge,and almost no diarrhea occurred in GP groups after challenge,while diarrhea score in Mod group was significantly higher than that in Con and GP groups（P<0.01）.Compared with Con group,Mod group had inflammatory exudate,villus abscission and mucosal epithelial defect,among which jejunum and ileum had the most obvious inflammatory injury,while GP group had less inflammatory injury than Mod group.The results showed that the V/C values of each segment of small intestine in Con group and GP group had no significant difference（P>0.05）.The V/C value of duodenum in Mod group was very significantly lower than that in Con group（P<0.01）,and the V/C value of jejunum and ileum in Mod group was significantly lower than that in Con group（P<0.01）.The V/C value of duodenum and jejunum in GP group was significantly higher than that in Mod group（P<0.01）,and the V/C value of ileum in GP group was significantly lower than that in Mod group（P<0.05）.2.The m RNA expression of inflammatory factors in duodenum,jejunum and ileum was detected by q PCR.The expression of the jejunal mucosal tight junction protein Occludin was detected by Western Blot method.The results showed:Compared with Con group,the m RNA transcription levels of TNF-α,IL-10 and IL-8 in duodenal tissue of piglets in Mod group after ETEC challenge were significantly increased（P<0.05）,and the m RNA transcription level of IL-1βwas extremely significantly increased（P<0.01）.The transcription levels of IL-1β,IL-6 and IL-8m RNA in jejunum were significantly increased（P<0.05）,and the transcription levels of IL-1β,IL-6 and IL-8 m RNA in ileum were very significantly increased（P<0.01）.Compared with Mod group,the transcription levels of the above inflammatory cytokines in each section of small intestine of piglets in GP group were significantly decreased（P<0.05 or P<0.01）.Compared with Con group,the Occludin expression level in Mod group was significantly increased（P<0.05）,and the Occludin expression level in GP group was extremely significantly increased（P<0.01）,while there was no significant difference between Mod group and GP group（P>0.05）.3.Glycyrrhiza polysaccharide with concentrations of 100,25,6.25μg/m L were co-cultured with IPEC-J2 for 24 hours,then ETEC infected cells with a MOI index of1:100 for 120min.Cell necrosis and apoptosis were detected by flow cytometry.The results showed:The apoptosis rate of model group was significantly higher than that of control group（P<0.05）,while there was no significant difference between high,medium and low concentration drug groups and control group（P>0.05）.After ETEC infection,the cell necrosis rate of model group,high concentration group,medium concentration group and low concentration group was significantly higher than that of control group（P<0.01）,compared with model group,cell necrosis rate in high concentration group was significantly decreased（P<0.01）.4.Glycyrrhiza polysaccharide with concentrations of 100,25,6.25μg/m L were co-cultured with IPEC-J2 for 24 hours,then ETEC infected cells with a MOI index of1:100 for 60min and 120min.Western Blot method was used to detect the expression levels of p-ERK/ERK,p-JNK/JNK,p-p38/p38,p-IκBα/IκBα,p-p65/p65 in MAPK/NF-κB signaling pathway and tight junction protein Occludin and Claudin-1.The results indicated that:ETEC infection of IPEC-J2 cells for 60 min induced the expression of MAPK signaling pathway protein p-ERK/ERK and the expression of p-p38/p38 in IPEC-J2 cells increased（P<0.05）,however,there were no significant changes in p-JNK/JNK expression and phosphorylation（P>0.05）.Meanwhile,the expression levels of tight junction proteins Occludin and Claudin-1 showed an increasing trend.ETEC induced p-IκBα/IκBαof NF-κB signaling pathway in IPEC-J2cells after 120 min of ETEC infection,and the expression of p-p65/p65 was significantly increased（P<0.05）,while the expression of Occludin was significantly decreased（P<0.05）.There was no significant difference in Claudin-1 protein expression level（P>0.05）.The expression and phosphorylation of p38 and ERK1/2 in IPEC-J2 cells induced by ETEC were significantly inhibited by Glycyrrhiza polysaccharides after ETEC infection for 60 min（P<0.05）.Glycyrrhiza polysaccharides significantly inhibited the phosphorylation of ERK1/2 and p38induced by ETEC in IPEC-J2 cells after ETEC infection for 60 min（P<0.05）.When ETEC was infected for 120 min,glycyrrhiza polysaccharides significantly inhibited the phosphorylation of p65 and IκBα（P<0.05）.Compared with Con and Mod groups,glycyrrhiza polysaccharides（25μg/m L）significantly increased the expression of tight junction protein Occludin（P<0.01）.For Claudin-1 protein,there was no significant difference between the three drug groups and the control group and the model group（P>0.05）.In summary,glycyrrhiza polysaccharides can effectively relieve diarrhea of weaned piglets caused by ETEC infection,alleviate intestinal inflammatory response caused by ETEC,and inhibit transcription of pro-inflammatory factors such as IL-1β,IL-8,IL-6 and TNF-αin duodenum,jejunum and ileum.By inhibiting the phosphorylation of key proteins in the MAPK/NF-κB signaling pathway,such as ERK1/2,p38,p65 and IκBα,glycyrrhizal polysaccharides inhibited the expression of downstream inflammatory cytokines induced by ETEC in porcine intestinal epithelial cells,thus regulating the ETEC induced inflammatory response in porcine intestinal epithelial cells.The effect and mechanism of glycyrrhiza polysaccharide on ETEC induced intestinal inflammation in piglets were explained.