Study On The Funtion Of TLR3and NF-κB Passway On Immune Response In Porcine Rotavirus Infection Of Intestinal Epithelial Cells In Vitro

Poreine Rotavirus(PRV) belongs to the family reoviridae,species rotavirus.It is one of themajor pathogens that cause life-threatening diarrhea in Piglets. Intestinal epithelial cells as atarget cell PRV infection by toll-like receptors (TLRs) identification of pathogen associatedmolecular patterns (PAMs) activation of host innate immune system. As rotavirus is anonenveloped,11-segmented dsRNA virus。We hypothesized that epithelial sensing of rotavirusmight target dsRNA, which can be detected by TLR3，and then trigger the transcription ofNF-κB which can mediated a variety of cytokines.This study we used PRV DN30209and Poly(I: C) to infection IPEC-J2cell line in vitro, to evaluate the effect before and after infection forTLR3, NF-κB and the expression of a variety of cytokines expression, discuss PRV infectionfor IPEC-J2cell molecular pathogenic mechanism. The main steps of this method are asfollows (1)The IPEC-J2cells wes divided into co-culture with PRV (100TCID50),co-culturewith Poly(I:C)(25μg/mL)，IPEC-J2and culture supernatant were selected respectively at0h,1h,2h,4h,6h,12h,24h,48h.The changes of TLR3，NF-κB,IL-1β,IL-6,IL-10,TNF-α,IFN-β inmRNA level were analyzad by Qrt-PCR.IL-1β,IL-6,TNF-α in supernatant were analyzed byELISA and compared with control group.(2) cells in experimental groups were divided intoinfected with PRV （1TCID50、100TCID50、105TCID50） and Poly(I:C)(5μg/mL,25μg/mL,50μg/mL,100μg/mL).After cultured4h, supernatant from each groupwere collected, respectively. Detection of IL-1β、IL-6、TNF-α expression by ELISA.(3)Theprotein levels of NF-κB/p65in nucleus was detected by Western blot.The result of Qrt-PCRshowed that The TLR-3mRNA and NF-κB mRNA expression level of co-culture withPRV(100TCID50) were continuously strengthen with prolonged infection. Co-culture withPoly(I: C) group of TLR3and NF-κB mRNA level also reached the highest expression in1hand2h. Other cytokine mRNA level to the highest levels of raised at different time pointswhich was extremely notably compared with the control group (0h)(P <0.05).ELISA to detectthe results showed that the supernatant of PRV infection group of IL-1β and IL-6protein levelsreached the maximum at6h and4h, respectively, while the expression of TNF-α and timedependence to the viral infection, Poly (I: C) infection in the group of IL-1β and IL-6proteinlevels were the highest possible level of secretion in6h, TNF-α and Poly(I:C) infection in same time dependence. In addition, the inflammatory cytokines IL-1β, IL-6and TNF-α co-culturewith PRV and Poly (I: C) the amount of infection is dose dependent and the express level ofprotein were extremely signifieantly increased compared with control group(P <0.05). Westernblot results showed that PRV can activate the NF-κB/p65.Our finding suggest that TLR3andNF-κB is involved in the immune responses of IPEC-J2cells after exposure to PRV andPoly(I:C) and has influence the expression of a variety of inflammatory cytokines.