Effects Of Total Glucosides Of Paeony And Paeoniflorin On Expression Of Inflammatory Damage Of Porcine Intestinal Epithelial Cells Induced By ETEC

Enterotoxigentic Escherichaia(ETEC)is a common intestinal pathogen of pigs,which can cause severe diarrhea in piglets,delay the growth and development of weaned piglets,reduce feed conversion rate,and cause serious harm to the pig breeding industry.Once a piglet is infected with ETEC,it will cause a series of host immune responses and severe intestinal inflammatory reactions,resulting in a series of clinical symptoms,and in severe cases,high mortality.Traditional Chinese medicinal materials have a long history of clinical application in China.Having the advantages of wide antibacterial activity,good curative effect,resistance to drug resistance and low residual toxicity.Used in the prevention and treatment of livestock and poultry diarrhea and other diseases widely.Paeonia lactiflora is the traditional Chinese medicine,commonly used in the treatment of dysentery.The total glucosides of paeony are one of the main components of peony,which has a series of pharmacological effects such as anti-inflammatory,analgesic,anti-tumor and neurological health protection.Paeoniflorin is the main effective component of total glucosides of paeony and has the effect of promoting immune regulation.Therefore,in order to explore the in vitro antibacterial effect of total glucosides of paeony and paeoniflorin on ETEC’s in vitro antibacterial effect and the intervention effect and mechanism on the immune function of ETEC infected pig intestinal epithelial cells,this paper constructed a model of ETEC infection of pig intestinal epithelial cells.the results are as follows:1.MTT method detects the IC50 of ETEC acting on porcine intestinal epithelial cells,confirms that the infection concentration are MOI=1:100,and the infection time is 2h.RT-PCR was used to detect the mRNA expression of receptors NOD1,NOD2,TLR2,TLR4 and cytoinflammatory factors IL-6,IL-8 and TNF-α when ETEC infected porcine intestinal epithelial cells for 30,60,105,and 120 minutes.And by adding NOD1/2 mixed inhibitor NOD-IN-1 and TLR2/4 mixed inhibitor AMG-9810 to confirm whether ETEC induces inflammation by activating various receptors.The results showed that after 2 hours of ETEC infection,the mRNA expression of NOD2 and TLR2 in porcine intestinal epithelial cells reached a peak at 30 minutes(P<0.01),and the mRNA expression of NOD1 and TLR4 reached a peak at 120minutes(P<0.01).The expression of IL-6,IL-8 and TNF-α mRNA in porcine intestinal epithelial cells increased significantly with the infection time(P<0.01).After the addition of signal pathway inhibitors,the mRNA expression of NOD1/2 and TLR2/4 in porcine intestinal epithelial cells was significantly reduced,but the expression increased after infection with ETEC,and the downstream inflammatory cytokines IL-6,IL-8 and TNF-α mRNA expression decreased compared with the model group.2.The MIC of total glucosides of paeony and paeoniflorin on enterotoxin-producing E.coli are both 25mg/m L.Detect the effect of MIC,1/2MIC,1/4MIC,1/8MIC,1/16 MIC total glucosides of paeony and paeoniflorin on the growth of bacteria every hour when ETEC is used for 6 hours.The results showed that different concentrations of total glucosides of paeony and paeoniflorin were negatively correlated with the growth inhibition of ETEC.The higher the drug concentration,the slower the growth of the bacteria,and total glucosides of paeony had better ETEC growth inhibition rate than paeoniflorin.3.Detect the cytotoxicity of total glucosides of paeony and paeoniflorin to porcine intestinal epithelial cells in the concentration range of 1μg/m L、2μg/m L、3μg/m L、4μg/m L、5μg/m L、6.25μg/m L、12.5μg/m L、25μg/m L、50μg/m L by MTT method,and determine 5μg/m L,3μg/m L,and 1μg/m L as the total glucosides of paeony and paeoniflorin high,medium and low concentration.Flow cytometry detection of total glucosides of paeony and paeoniflorin and ETEC co-acting on the apoptosis of porcine intestinal epithelial cells for 2 hours.The RT-PCR test was carried out to detect the changes in the mRNA expression of NOD1,NOD2,TLR2,TLR4 and pro-inflammatory cytokine IL-6,IL-8,TNF-α mRNA in the porcine intestinal epithelial cell when different concentrations of total glucosides of paeony and paeoniflorin interact with ETEC for 30 min,60min,105 min,120min.The results showed that: High,medium and low concentrations of total glucosides of paeony and paeoniflorin can increase cell viability and inhibit cell apoptosis caused by ETEC infection.High,medium and low concentrations of total glucosides of paeony and paeoniflorin can extremely significantly or significantly reduce the mRNA expression of cell receptors NOD1,NOD2,TLR2 and TLR4,and reduce the pro-inflammatory cytokines IL-6 and IL-8,TNF-α mRNA expression(P<0.05 或 P<0.01).In summary,ETEC can activate TLR2,TLR4,NOD1,NOD2 receptors in porcine intestinal epithelial cells,and activate downstream inflammatory cytokines.Total glucosides of paeony and paeoniflorin can inhibit bacterial growth in vitro.Total glucosides of paeony and paeoniflorin can inhibit cell apoptosis caused by ETEC infection,and improve cell viability.And by inhibiting the activation and expression of NOD1/2 and TLR2/4 cell receptors in porcine intestinal epithelial cells infected by ETEC,thereby inhibiting the mRNA expression of downstream inflammatory cytokines IL-6,IL-8,and TNF-α.To achieve the effect of regulating the inflammatory response of porcine intestinal epithelial cells induced by ETEC,and to explain the regulation effect and molecular mechanism of total glucosides of paeony and paeoniflorin on the inflammatory response of porcine intestinal tract induced by ETEC.