The Expression Of TLR3 And Some Cytokines In St Cells Induced By Tgev Infection And Lactobacillus Acidophilus Exopolysaccharides Pretreatment

Probiotics have a natural, safe, non-toxic, no residue and other characteristics, so probiotics has widespread concern in animal husbandry industry. For now there are many relevant reports of probiotics about antibacterial, immune regulation, less antiviral are reported. Porcine transmissible gastroenteritis virus(TGEV) can cause porcine transmissible gastroenteritis, the disease is a serious threat to the development of aquaculture, it is important for the disease prevention. Therefore, this study choose a strain of Lactobacillus acidophilus which has antiviral activity, by extracting the extracellular polysaccharide(EPS), then treated swine testicular cells(ST cells) with extracellular polysaccharide. MTT assay was used to verify the extracellular polysaccharide anti-TGEV effect. According to this, we carried out this study about the expression of TLR3, IFN-γ, IL-6, IL-8 and cell signaling pathway transcription factor NF-κB in ST cells induced by TGEV infection and lactobacillus acidophilus extracellular polysaccharide pretreatment to explore cellular mechanisms of probiotic extracellular polysaccharide against viral infection.ST cells covered with a single layer were divided into four groups: control group, TGEV group, EPS with TGEV group, EPS group. We collected the cell supernatants and cells respectively at 2h, 4h, 6h, 12 h, 24 h after cells were treated differently. Using real-time PCR method to detect TLR3, IFN-γ, IL-6, IL-8 m RNA expression levels in each group cells, the method is based on TLR3, IFN-γ, IL-6, IL-8 and housekeeping gene(β-actin) sequences in Gen Bank and primer sequence in references to design and synthesize fluorescent primers, establish real-time PCR detection method; Detected IFN-γ, IL-6, IL-8 protein expression levels in each group supernatant by ELISA; Detected cell signaling pathway transcription factor NF-κB expression levels by Western blot.The statistical analysis of experimental results data showed that EPS group, TGEV group and EPS+TGEV group TLR3, IFN-γ, IL-6, IL-8 m RNA expression are relatively more than the control group, with the extension of time, the m RNA expression of TLR3, IFN-γ, IL-6, IL-8 all show a tendency of that first increase then decrease and return to normal levels. Most treated group TLR3, IFN-γ, IL-6, IL-8 m RNA expression levels close to normal levels after 6h. EPS+TGEV group the m RNA expression of IL-8 reached the maximum at 2h, the m RNA expression of IFN-γ, IL-6 reached the maximum at 4h, it expressed significant differences between the controls and the treated group. The overall trend by ELISA is the same with the test results by real-time PCR. Western blot studies showed that Lactobacillus acidophilus extracellular polysaccharides and TGEV can stimulate the expression of cell signaling pathway nuclear transcription factor NF-κB protein. The nuclear expression of NF-κB in EPS+TGEV group, TGEV group, EPS group were higher than those in the control group, and the nuclear expression of NF-κB in EPS+TGEV group were higher than those in TGEV group, EPS group.The results above show that Lactobacillus Acidophilus exopolysaccharides and TGEV can induce the expression of cell surface receptor of TLR3, initiate the downstream NF-κB pathway and induce secretion of cytokine IFN-γ, IL-6, IL-8, and stimulate cell innate immune responses, thereby enhance cellular immunity against the virus. It produced the same effect with EPS pretreatment then infected TGEV, and TLR3, IFN-γ, IL-6, IL-8 expression level appeared superimposed effect. The study provides new evidence for antiviral mechanism of probiotic in terms of probiotic extracellular polysaccharides induce the expression of cytokines, involved in resistance to viral infection.