The MRNA Expression Of Toll-Like Receptors And Cytokines In IPEC-J2Cells Induced By Rota Virus Infection And Bacillus Subtilis Pretreatment

Probiotics have the ability of curing enteritis, diarrhea, improving animal growth performance and regulating the immune responses. Probiotics are attracted widespread attention for these beneficial effects, but the mechanism of probiotics antiviral is little known. Rotavirus (RV), a major pathogen that causes infants and piglets diarrhea, primarily infects mature epithelial cells of the small intestine villi, seriously impacting people’s health and the development of aquaculture industry. In order to investigate the interaction among probiotics-host-virus, Bacillus subtilis that was isolated and had the ability of antirotavirus was used in this experiment. We hypothesized Bacilus subtilis regulated the immune responses whether by activating the expression of TLRs and increasing the secretion of cytokines thereby initiating antivirus responses. The main research were as follows:59probiotics were separated from the gastrointestinal tract of pigs by our laboratory. The MTT method was used to screen the probiotics which had the ability of inhibiting RV. The probiotic had the highest resistance of RV was Bacillus subtilis according to the results of biochemical and16sRNA indentification.Since porcine intestinal epithelial cells are the major target cells of RV infection, we choose porcine intestinal epithelial cells (IPEC-J2) as the cell model of RV infection in vitro. We detected TCID50of virus at different time points when RV infected IPEC-J2cells and on the pretreatment of Bacillus subtilis. The growth curve of RV was figured. The results showed that RV was slowly proliferation at first24h. RV rapidly proliferated from24h to48h and the virus titer reached the peak. After48h later, the titer of RV began to drop. The growth curve of RV on the cells pretreatment with Bacillus subtilis had the similar trends, but always had a lower virus titer.The confluent monolayer IPEC-J2cells were divided into five groups:normal control group (control), RV infection group (RV), RV infection with Bacillus subtilis pretreatment group (BS+RV), only Bacillus subtilis treatment group (BS) and only E. coli treatment group (E.coli). The cell samples were collected at1h,3h,6h,12h,24h,48h. The mRNA expression of IL-6, IL-8, IFN-y, TLR2, TLR3, TLR9was measured at different time points by Real-time PCR. The data was statistical analysis. All of RV group, BS group and BS+RV group could stimulate the mRNA expression of IL-6, IL-8, IFN-y, TLR2, TLR3, TLR9in the early time. The mRNA expression of cytokines and TLRs in most treatment groups returned to the normal levels6h later. The mRNA expression of TLR9and cytokines IL-6, IL-8, IFN-y in E.coli were increased in the early time.In summary, Bacillus subtilis can inhibit RV replication and proliferation in IPEC-J2cells. The incubated period of RV infection is about24h, then begin to rapidly proliferate. Bacillus subtilis and RV stimulating IPEC-J2cells short time can increase the mRNA expression of IL-6, IL-8, IFN-y, TLR2, TLR3, TLR9, activate the host innate immune response, resist to virus infection. IPEC-J2cells pretreated with Bacillus subtilis then infected virus can also stimulate the expression of these genes, and show additive effect. We assum that Bacillus subtilis stimulate the expression of TLRs in IPEC-J2cells to activate NF-κB pathway, increasing the secretion of some Thl cytokines, regulating the innate immunity and enhancing the resistance to virus.