Study On The Mechanism Of Polygonum Hydropiper Neutral Polysaccharide Against Escherichia Coli-induced Diarrhea And Its Powder Preparation

In the modern animal husbandry,diarrhea caused by pathogenic Escherichia coli is a common disease in veterinary clinic,and it is also one of the diseases with high mortality rate.E.coli-induced diarrhea can occur throughout the year especially in young animals,which seriously affects the development of livestock and poultry breeding.Polygonum hydropiper L.has the efficacy of clearing dampness,relieving diarrhea,dispersing blood stasis and alleviating pain,dispelling wind and killing insects.The decoction of P.hydropiper is widely used in veterinary clinic to treat diarrhea of calves,equine animals and piglets with remarkable curative effect.In our previous study,a stable and reliable model of diarrhea caused by E.coli was established,and the therapeutic effect of P.hydropiper neutral polysaccharide on mice with E.coli-induced diarrhea was also confirmed.In this paper,the mechanism of P.hydropiper neutral polysaccharide against diarrhea caused by E.coli was explored based on transcriptomics.Meanwhile,the compound anti-diarrhea powder preparation for veterinary use was prepared from P.hydropiper neutral polysaccharide and the quality standard of the preparation was preliminarily established.This study is helpful to elucidate the mechanism of P.hydropiper neutral polysaccharide against E.coli-induced diarrhea,and also provide the scientific basis for the development and utilization of P.hydropiper resources.The main research contents and results are as follows:1.BALB/c mice were randomly divided into five groups:the normal group,model group,enrofloxacin group（5 mg/kg）,low-dose group（40 mg/kg）and high-dose group（80 mg/kg）of P.hydropiper neutral polysaccharide.Except for the normal group,mice in the other groups were injected intraperitoneally with pathogenic E.coli to establish the diarrhea model.The mice in the treatment groups were given drugs intragastrically 2 days before modeling,twice a day for 10 consecutive days.Mice in the normal group and model group were given the same amount of normal saline intragastrically.The duodenal tissues were dissected on the 2^nd and 9^th day after modeling.The transcriptome sequencing was performed on duodenal tissue samples of normal group,model group and high-dose group of P.hydropiper neutral polysaccharide using Illumina Hi Seq TM platform.The accuracy of the transcriptome sequencing results were verified by real-time fluorescence quantitative polymerase chain reaction（q RT-PCR）.Transcriptome analysis results showed that there were 1020 different genes between the normal group and model group.Compared with the normal group,587 differentially expressed genes（DEGs）were up-regulated and 433DEGs were down-regulated in the model group.The results of KEGG enrichment analysis showed that the DEGs were mainly involved in cytokine-cytokine receptor interaction,toll-like receptor signaling pathway,NF-κB signaling pathway and chemokine signaling pathway etc.Compared with the model group,there were 960 DEGs in the high-dose group of P.hydropiper neutral polysaccharide,385 of which were up-regulated and 575 down-regulated.KEGG enrichment analysis showed that the DEGs were mainly involved in cytoplasmic DNA sensing pathway,Toll-like receptor signaling pathway,NF-κB signaling pathway,TNF signaling pathway,chemokine signaling pathway,etc.Eight DEGs,including CCL7,CXCR2,CXCL5,CXCL10,CCL22,CXCL1,TLR2,and CCL2,were randomly selected.The relative m RNA expression levels of these genes were detected by q RT-PCR.The results of q RT-PCR were consistent with those of transcriptome sequencing,which proved that the transcriptome data were accurate and reliable.2.The m RNA and protein expressions of CXCL12,CXCR4,TLR4,My D88,NF-κB p65 and IκBαwere detected by q RT-PCR,enzyme-linked immunosorbent assay（ELISA）and western blotting（Western Blot）.The results of q RT-PCR showed that the expression levels of CXCL12,CXCR4,TLR4,My D88,NF-κB p65 and IκBαm RNA in the duodenum of the model group were significantly higher than those in normal group（P<0.01）;compared with the model group,the m RNA expression levels of CXCL12,CXCR4,TLR4,My D88,NF-κB p65 and IκBαin the duodenum of P.hydropiper neutral polysaccharide groups were significantly reduced（P<0.01,P<0.05）.ELISA results showed that the protein content of CXCL12 and CXCR4 in the model group was significantly higher than those in normal group（P<0.01）;compared with the model group,the protein content of CXCL12 and CXCR4 in the duodenum of P.hydropiper neutral polysaccharide groups were significantly reduced（P<0.01,P<0.05）.The results of Western blot showed that the protein expression levels of CXCL12,TLR4,My D88,NF-κB p65 and p-NF-κB p65 in the model group were significantly higher than those in normal group（P<0.01）;the protein expression levels of CXCL12,TLR4,My D88,NF-κB p65and p-NF-κB p65（P<0.01,P<0.05）could be significantly down-regulated by P.Hydropiper neutral polysaccharides.In summary,P.hydropiper neutral polysaccharide can play an anti-E.coli diarrhea effect by regulating the CXCL12-CXCR4 axis and the TLR4/My D88/NF-κB signaling pathway.3.The prescription of the anti-diarrhea compound powder is as follows:P.hydropiper neutral polysaccharide 50 mg,Panax ginseng 2.5 g,Dioscotea opposite 5 g and Dolichos lablab 5 g.The powder was uniform in color,without pattern and color spot.The average proportion of the powder passing the NO.6sieve was 98.2%.The average water content of the powder was 5.14%.All the results were in accordance with Chinese Pharmacopoeia（2020 edition）.Panax ginseng,Dioscotea opposita and stir-fried Dolichos lablab in compound powder were qualitatively identified by microscopic identification method.The polysaccharide,ginsenoside Rg1,Chinese yam and fried white lentil were identified by TLC.The content of total sugar and total saponin in compound powder was determined by ultraviolet spectrophotometry.The sensitive,stable and reliable qualitative and quantitative methods for compound powder were established,meanwhile,the quality standard of P.hydropiper neutral polysaccharide compound powder was drafted.