Experimental Study Of Punicalagin In Improving Intestinal Barrier Through NOD2/TLR4 Signaling Pathway

Objective:The Intestinal barrier is an important line of defense for the body.The integrity of the barrier is positively related to the occurrence and progress of various diseases.Punicalagin（PUN）is a phenolic compound extracted from pomegranate peel and has various pharmacological activities,such as antioxidant,anti-inflammatory.The purpose of this study is to study the effect of PUN on intestinal flora and its role in protecting intestinal barrier by interfering with TLR4/NOD2 signal pathway,so as to provide experimental basis for the prevention and treatment of diseases related to intestinal barrier.Methods:After 45 BALB/c mice were gavaged with pathogenic Escherichia coli O₁₀₁to establish Bacterial enteritis model,they were randomly divided into model group（Model）,PUN administration group and cefixime（CEF）administration group.Another 15 normal BALB/c mice were taken as control group（Control）,and the administration group was gavaged for 7 days.During the administration,the physical signs of mice in each group were observed and the DAI and body weight change rate were calculated.After the administration,the intestinal tissue was taken and the intestinal contents were separated,16S r DNA high-throughput sequencing was used to detect the diversity and abundance of mouse intestinal flora,and analyze the changes of its structure and function;The pathological changes of intestinal tissues were observed by HE staining;The expression of occludin and ZO-1 was detected by immunohistochemistry;MPO activity was used to detect the degree of neutrophil infiltration in the intestinal mucosal barrier;The expression levels of LPS,IL-6 and TNF-αin serum were detected by ELISA.The m RNA expressions of NOD2,TLR4,NF-κB P65,TNF-αand IL-6 were detected by q PCR.The expression levels of NOD2,TLR4,NF-κB P65,TNF-αand IL-6 in intestinal wall were detected by WB.The intersection target of Pun and Bacterial enteritis was screened by using the specific database,then import targets into the STRING database to retrieve the PPI and screen the key targets.The DAVID database was used for the enrichment analysis.Finally,molecular docking was performed using the specific software to predict the binding degree between the active components of Pun and the key targets.Lipopolysaccharide（LPS）、Muramyl Dipeptide（MPD）and Caco2 cells were incubated in groups,and the concentration of IL-6 in cell supernatant was detected by ELISA;The lentivirus vector silenced by NOD2 gene was constructed.After transfection into Caco2 cells,PCR and WB were used to verify that the gene expression was reduced,and then the cells and ligands were co-incubated again to detect the concentration of IL-6 in the cell supernatant.Results:1.Compared with the model group,the DAI score of the model group was significantly lower than that of the model group.HE staining showed that the congestion of small intestinal mucosa in the model group was serious,while the edema of small intestinal tissue in the drug group was significantly reduced.2.Immunohistochemical method showed that the expression of Occludin and ZO-1 in the small intestinal mucosa of the drug group was higher than that of the model group.3.Pun can inhibit MPO activity in intestinal mucosal barrier,LPS,IL-6 and TNF-αin serum Level.4.Pun can inhibit expression of the intestinal inflammation related factors NOD2,TLR4,NF-κB p65,TNF-α,IL-6 m RNA and protein.5.16s r DNA high-throughput sequencing showed that pun could improve the diversity,structural composition and functional changes of intestinal flora in enteritis mice.6.130 intersection targets of Pun and disease were screened by network pharmacology.Enrichment analysis showed that cross genes were closely related to cancer regulation and TNF signaling pathway.At the same time,Molecular docking model shows that the active components of Pun have certain binding activity with the core targets such as TNF and IL-6.7.in vitro experiment,1μg/ml LPS and 0.3μg/ml MDP incubation for24h was the best safe dose and time for Caco2 cells.The concentration of IL-6 in cell supernatant was significantly lower after NOD2 gene silencing than before.Conclusion:1.PUN can significantly reduce the clinical manifestations and pathological damage of enteritis mice,and the therapeutic effect of pun was similar to that of antibiotics.2.Effect of pun treatment on improving intestinal permeability in mice with enteritis.3.PUN treatment can improve the intestinal epithelial injury of enteritis mice.4.PUN can inhibit the activation of NOD2/TLR4 signal to alleviate inflammatory injury.5.PUN can improve the structure and function disorder of intestinal flora.6.Vitro experiment showed that there was a synergistic relationship between NOD2/TLR4.