| Enterotoxigenic Escherichia coli(ETEC)is the main cause of white and yellow scour,edema and post-weaning diarrhea in newborn piglets,which results in the growth retardation of piglets and the decrease of feed utilization rate,causing the pig breeding industry to suffer serious economic losses.Infected with ETEC causes intestinal epithelial damage accompanying a severe inflammatory response in weaned piglets,which can lead to local or systemic infections,such as diarrhea,urethritis,mastitis,encephalitis and septicemia,and other clinical symptoms.In severe cases,ETEC can lead pig to death.At present,antibiotics are commonly used to treat piglet infected by ETEC.Due to the serotypes of ETEC are various and the antibiotic resistance strains are easy to emerge.The prevention and treatment of ETEC disease by using vaccination and antibiotics are little effective.Chinese herbal compound prescriptions are often used to prevent and cure ETEC intestinal diseases in piglets,and glycyrrhiza is one of the commonly used Chinese medicine in the formulae.Some studies have shown that glycyrrhiza polysaccharide is one of the main pharmacodynamic components of glycyrrhiza uralensis,which has the functions of enhancing immunity,bacteriostasis,antiviral and anti-tumor.Therefore,in order to explore the effect of ETEC infection on the immune function of pig intestinal epithelial cells and the regulatory effect of glycyrrhizin polysaccharides,the following studies were conducted:1.Cell viability of porcine intestinal epithelial cells infected with different concentrations of ETEC was determined by CCK-8 method.The results show that the inhibition of cell proliferation was most obvious when 1×10~8 CFU/mL ETEC was treated for 2h(P<0.05).The porcine intestinal epithelial cells were infected with ETEC at a concentration of 1×10~8 CFU/mL for 15,30,45,60,75,90,105 min and 120 min,respectively.The mRNA expressions of TLR2,TLR4,TLR9,IL-1,IL-4,IL-6,IL-8,IL-10 and TNF-αin intestinal epithelial cells were detected by real-time PCR.The results show that after ETEC infected porcine intestinal epithelial cells with a concentration of 1×10~8 CFU/mL at different time periods,the expressions of TLR2mRNA were significantly higher than that of the control group at 60,90min and 105min(P<0.05).The expressions of TLR4 and IL-4 mRNA were significantly higher than that of the control group at 105min(P<0.05).The expressions of TLR9 mRNA were significantly higher than that of the control group at 75min and 105 min(P<0.05),and significantly lower than that of the control group at other time periods(P<0.05).The expressions of IL-1βmRNA were significantly higher than that of the control group at15,30min and 45 min(P<0.05).The expressions of IL-6 mRNA were significantly lower than that of the control group at 15min and 30min(P<0.05),but significantly higher than that of the control group at 90min and 105min(P<0.05).The expressions of IL-8 and TNF-αmRNA were significantly higher than that of the control group from45min to 120min(P<0.05).The expressions of IL-10 mRNA were significantly higher than that of the control group from 15min to 105min(P<0.05).ETEC infection can cause proinflammatory and anti-inflammatory cytokines expression in intestinal epithelial cells,leading to inflammatory damage.2.After co-cultured with porcine intestinal epithelial cells for 48 hours by different concentrations of glycyrrhiza polysaccharides(3.125,6.25,12.5,25,50,100,200,400μg/mL).The cell viability of porcine intestinal epithelial cells was detected by CCK-8 method.The results show that 125,6.25,12.5,25,50,100μg/mL glycyrrhiza polysaccharide had no significant effect on the cell viability of pig intestinal epithelial cells(P>0.05).They have no cytotoxic effect on pig intestinal epithelial cells.Glycyrrhiza polysaccharides with concentrations of 3.125,6.25,12.5,25,50,100μg/mL were co-cultured with pig intestinal epithelial cells for 48 hours,then ETEC infected cells with a MOI index of 1:100 for 2 hours.The cell viability of porcine intestinal epithelial cells was detected by CCK-8 method.The results show that glycyrrhiza polysaccharides can effectively maintain normal cell viability of ETEC infected pig intestinal epithelial cells at concentrations of 3.125,6.25,12.5,25,50,100μg/mL.3.Different concentrations of glycyrrhiza polysaccharides(3.125,6.25,12.5,25,50,100μg/mL)were co-cultured with porcine intestinal epithelial cells for 48 hours,then ETEC infected cells with a MOI index of 1:100 for 15,30,45,60,75,90,105min and 120min.Real-time PCR was used to detect the expressions of TLR2,TLR4,TLR9,IL-1β,IL-6,IL-8,and TNF-αmRNA in intestinal epithelial cells.The results show that glycyrrhiza polysaccharides was co-cultured with pig intestinal epithelial cells for 48h.After 105 minutes of ETEC infection,the expression of TLR2 receptor was decreased by 3.125μg/mL-100μg/mL glycyrrhiza polysaccharide(P<0.05).The expression of TLR4 receptor was decreased by 6.25μg/mL-100μg/mL glycyrrhiza polysaccharide(P<0.05).TLR9 receptor expression was decreased by 12.5μg/mL and 25μg/mL glycyrrhiza polysaccharide(P<0.05).After 15 minutes of ETEC infection,3.125,25,50,100μg/mL glycyrrhizin polysaccharide can inhibit the increase of IL-1βmRNA expression in ETEC infected cells(P<0.05).12.5μg/mL-100μg/mL glycyrrhiza polysaccharide can inhibit the increasing expression of IL-6 mRNA in ETEC infected cells(P<0.05).After 105 minutes of ETEC infection,6.25μg/mL-100μg/mL glycyrrhiza polysaccharide can inhibit the increase of IL-6 mRNA expression in ETEC infected cells(P<0.05).12.5μg/mL-100μg/mL glycyrrhiza polysaccharide can inhibit the expression of IL-8 mRNA in ETEC infected cells(P<0.05).50μg/mL and 100μg/mL glycyrrhizin polysaccharide can inhibit the expression of TNF-αmRNA in ETEC infected cells(P<0.05),which reduced the inflammatory damage of porcine intestinal epithelial cells induced by ETEC.In conclusion,glycyrrhizin polysaccharide can inhibit the inflammatory response of porcine intestinal epithelial cells caused by ETEC infection by inhibiting the expressions of TLR2,TLR4,TLR9 and proinflammatory cytokines IL-1,IL-6,IL-8,TNF-αmRNA,so as to protect the normal immune function of porcine intestinal cells under ETEC infection,which has clinical application prospect. |
PDF Full Text Request