Study On Aptamer Interfering With ETEC K88 Adhesion To Porcine Intestinal Cell Line IOEC-J2 And Relieving Its Inflammatory Reaction

Weaning stress leads to piglets being susceptible to pathogen infection,which causes diarrhea and even mortality.Enterotoxigenic Escherichia coli(ETEC)which contains K88,K99,987 P and F4 is one of typical strains associated with post-weaning diarrhea.Thus,prevention and inhibition of ETEC infection are of great concern.Aptamer is a kind of ssDNA or RNA sequence that can bind to the target with high affinity and specificity.In this paper,by using intestinal porcine epithelial cell line IPEC-J2 as a model in vitro,we have found that aptamers,K88-Apt37(fimbrial-aptamer against ETEC K88)and K88-Apt A04(cell-aptamer against ETEC K88),can positively prevent bacteria from adhering to intestinal epithelial cells,resulting in decrease of subsequent inflammatory reaction,when there was pathogenic bacteria ETEC K88 strain.In all,this article includes the following points:(1)Firstly,IPEC-J2 was used as a model in vitro to evaluate the protecting effects of aptamers on the adhesion and cytotoxicity of cells attacked by ETEC K88.The concentration rate and action time of ETEC K88-IPEC-J2 interaction model were determined.And under these conditions,the effects of the three modes of adherence inhibition,adherence prevention and treatment on the interaction model weremeasured.The results showed that the aptamer could inhibit the adhesion of ETEC K88 to IPEC-J2 cells under three interaction models.(2)Secondly,in order to better observe the effect of aptamers on the interaction model which we constructed before,the fluorescent plasmid was constructed and then transfected into ETEC K88 to express EGFP protein successfully,however,the fluorescence intensity was 1-2 orders of magnitude lower than that of CFDA-SE staining.The effect of aptamer on reducing the number of bacterial adhered to cells in different models was further analyzed by flow cytometry and observed by fluorescence microscopy.It was found that as a drug to reduce infection of intestinal epithelial cells by pathogenic bacteria,a low concentration of K88-Apt 37 or a high concentration of K88-Apt A04 should be suitable due to higher affinity of the former and less cytotoxicity of the latter.Based on the effective tendency of reducing cytotoxicity and improving adhesion inhibition rate: adherence inhibition> adherence prevention> treatment,we recommended that it was better for aptamer as a preventive drug to administer before pathogenic infection.(3)Thirdly,the ELISA kit was utilized to measure the protein expression of typical inflammatory factors IL-8 and TNF-α,and the mRNA expression was observed by PCR and QPCR for exploring the molecular mechanism of aptamers help IPEC-J2 cells survive from ETEC K88 attacking.The results showed that the presence of aptamer could down-regulate the expression of TNF-α in IPEC-J2 cells which was challenged by ETEC K88 as well as down-regulate the mRNA expression of PRRs and pro-inflammatory cytokines,indicating that aptamers could reduce the pro-inflammatory cytokines secretion caused by the pathogenic bacteria attacking.In conclusion,the aptamer plays a protective role by interacting with bacteria and interfering bacterial adhesion to intestinal epithelial cells,thereby reducing the cytotoxicity and inflammation caused by bacteria.In this paper,the mechanism of protection we mentioned before was studied.Thus,our work plays an important role in the prevention and treatment of diarrhea diseases caused by ETEC K88 and provides a new method for the treatment of diseases caused by other pathogenic microorganisms.