Abstract:

Objectives To discuss the inhibitory effect of Lactobacillus reuteri on the replication of rotavirus （RV） strain SA11 in vivo and in vitro， and to clarify its effect on the expression of related immune factors. Methods For in vitro experiments， Lactobacillus reuteri was cultured and identified， and the standard curve and growth curve were plotted to screen the optimal time and concentration for Lactobacillus reuteri cultivation. The cells were infected with Lactobacillus reuteri at the concentrations of 5×10⁸， 10×10⁸， 50×10⁸， 100×10⁸， 200×10⁸， and 500×10⁸ CFU·mL^-1， and the surival rates of Caco-2 cells were detected by trypan blue staining method. Various concentrations of Lactobacillus reuteri were co-incubated with RV in vitro and applied to the Caco-2 cells. The cells were divided into negative control group （NC group）， positive control group （PC group）， and 10⁷， 10⁸， 10⁹， and 10¹⁰ CFU·mL^-1Lactobacillus reuteri groups. Immunofluorescence focus method was used to detect the viral titers in the Caco-2 cells after treated with Lactobacillus reuteri and real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the copy numbers of RV VP6 gene in the Caco-2 cells after treated with various concentrations of Lactobacillus reuteri. In in vivo experiments， 25 litters of SPF suckling mice were divided into control group， RV group （infected with SA11 strain）， Ab-NC group （treated with antibiotic to deplete gut microbiota）， Ab-RV group （depleting gut microbiota and then infected with SA11 strain）， and Ab-Lac-RV group （depleting gut microbiota， treated with Lactobacillus reuteri， and then infected with SA11 strain）. The fecal samples were collected on days 2， 4， 6， 8， and 10 gavage， colon tissue sample were collected on day 4 of and RT-qPCR method was used to detect the copy numbers of RV VP6 gene in feces and the mRNA expression levels of interleukin （IL）-1β， IL-8， IL-10， interferon-γ （IFN-γ）， and tumor necrosis factor-α （TNF-α） in colon tissue of the suckling mice in vartious groups. Results The Lactobacillus reuteri grew well， with round， smooth， and milky white convex colonies and neat edges. After Gram staining， the bacteria appeared purple， irregular， and square-shaped rods. 16SrDNA sequencing showed 99% sequence homology， indicating successful activation of Lactobacillus reuteri. The number of live Lactobacillus reuteri was linearly related to the absorbance （A） value， and the standard curve for regression analysis was Y=0.437 5X+0.000 6， R²=0.999 4. During the 0-2 h cultivation period， the bacteria were at the logarithmic growth phase with slow growth； from 2-14 h， the bacteria grew rapidly and stabilized at 14-16 h， reaching the growth rate peak at 16 h， after which they entered the decline phase. Infection with Lactobacillus reuteri at concentrations of 5×10⁸， 10×10⁸， 50×10⁸， 100×10⁸， and 200×10⁸ CFU·mL^-1 resulted in the survival rates of Caco-2 cells were all >90%， so these concentrations were selected for the further experiments. Compared with PC group， the copy numbers of RV VP6 gene in the Caco-2 cells in 5×10⁸， 10×10⁸， 50×10⁸， 100×10⁸， and 200×10⁸ CFU·mL^-1Lactobacillus reuteri groups were significantly decreased （P<0.01）. Compared with PC group， the viral titers in the Caco-2 cells in 10⁷， 10⁸， 10⁹， and 10¹⁰ CFU·mL^-1Lactobacillus reuteri groups were significantly decreased （P<0.01）. Compared with control group， the numbers of gut microbiota colonies in Ab-NC， Ab-RV， and Ab-Lac-RV groups were significantly decreased， indicating successful depletion of gut microbiota in the suckling mice. On days 2 and 4 after gavage， the RV VP6 gene copy number in the feces in Ab-RV group was significantly lower than that in RV group （P<0.05）. On days 4， 6， 8， and 10 after gavage， the RV VP6 gene copy number in the feces in Ab-Lac-RV group was significantly lower than that in Ab-RV group （P<0.05）. Compared with control group， the expression levels of IL-1β， IL-10， IFN-γ， and TNF-α mRNA in colon tissue in Ab-RV and Ab-Lac-RV groups were significantly increased （P<0.05 or P<0.01）， while the expression level of IL-8 mRNA was significantly decreased （P<0.05），and the expression level of IL-10 mRNA in colon tissue in Ab-LAC-RV group was significantly increased （P<0.01）. Conclusion Lactobacillus reuteri may inhibit the RV replication by upregulating the expressions of IL-1β， IL-10， IFN-γ， and TNF-α mRNA and downregulating the expression of IL-8 mRNA.

Key words: Lactobacillus reuteri, Rotavirus, Suckling mice, Immune factor, Standard curve