Effect Of Jinying Huanggui Decoction On Cytokines Mediated By E.coli LPS Extracted From Swine

Escherichia coli is a conditional pathogen of human and animal gut,which is a class of multiple,lethal intestinal infectious diseases.Endotoxin is the lipopolysaccharide(LPS)in the outer membrane of Gram-negative bacteria cell wall.It is one of the most important pathogenic factors in the pathogen-induced damage to the host.It mainly stimulates the expression of TNF-?,IL-6 and IL-1? by activating the TLR4 signal transduction pathway and produced a large number of other inflammatory factors,causing the host inflammatory response.The preliminary study of the laboratory shows that Jinying Huang Gui Tang has a good antibacterial effect on Staphylococcus aureus.At the same time,it has a clinical effect on dairy cows with recessive mastitis.In this study,1 strain of Escherichia coli was isolated from clinical isolates,and the LPS of Escherichia coli was extracted,purified and analyzed.The mouse mammary epithelial cells(MECs)were used in vitro cell models to study the effect of Jinyinghuanggui decoction on LPS-mediated cytokines(TNF-?,IL-1? and IL-6).The main research contents are as follows:1.Isolation and identification of Escherichia coli from pigs and JYHGT of antibacterial test in vitro:Pathological specimen were collected from clinic disease piglet.The bacterium was isolated,purified,observed morphology and pathogenicity.They were also identified with 16 SrDNA.Test tube and plate methods were adopted to measure the minimal inhibitory concentration(MIC)and minimal bacterium concentration(MBC).The results showed that culture features and biochemical assays indicated that the bacterium isolated from clinic was E.coli.The amplification for 16 S rDNA PCR results showed that the specific fragment was 1466 bp,which as coincident with the expect result.The blast result showed that it was homologous similarity and were up to 99%.The MIC and MBC of Jingying Huanggui decoction acting on E.coli were 31.25 mg/m L and 62.5 mg/m L respectively.2.Extraction,Purification and Activity Analysis of Lipopolysaccharide from Escherichia coli Isolated from Swine:Hot phenol water method was used for LPS isolation.DNase?,RNase A and protease K were applied for purificatiotn.The contents for polysaccharides,protein and nucleic acid of LPS was weighed by Phenolsulfuric acid,Bradford and UV-spectrophotometry methods.Quantitatives limulus reagent method was used for detecting its activity.The median mice lethal dose was tested by acute toxicity experiment.The results showed that The average production rate of isolation and purification for LPS of E.coli was 3.74%.The content of polysaccharides was 13.4%,protein was 0.48%,nucleic acid was 0.06%.The extracted LPS belongs to the smooth LPS(S-LPS).The active value was 1.21 ×107 EU/mg.LD50 was 31.86mg/kg.3.Effects of JYHGT on TNF-?,IL-6,IL-1? in mice mediated by LPS:The primary MECs cells were obtained by enzyme digestion method,and the optimal concentration of LPS was measured by MTT assay in mice MECs cells in vitro.ELISA was used for detecting the impact that Jingying Huanggui decoction induce MECs to secret pro-inflammatory factor.The results showed that MECs cells with high purity were obtained successfully,The optimal concentration of LPS stimulated mouse MECs cells was 8 ?g/m L.Jingying Huanggui decoction could inhibited the secretion of IL-1??IL-6?TNF-? prominently.Conclusion:A pig derived Escherichia coli was isolated successfully in this experiment,Jin Ying Huang Gui decoction has a good antibacterial effect on it.The LPS was extracted from this strain of Escherichia coli.It was highly pure,highly yield as well as strong pathogenicity.A large number of mammary epithelial cells can be isolated by mixed enzyme digestion in a short time.And the purity of the cells reached 98%.Jin Ying Huang Gui Decoction significantly inhibited MECs secretion of TNF-?,IL-6,IL-1? by LPS in mice.