The effectiveness of probiotics in up or down regulation of the inflammatory genes was examined by comparison between treated HT-29 cells with probiotics versus control cells (not exposed HT-29 cells as negative control) and HT-29 cells exposed to sonicated pathogen as positive control. Also, the statistical relatedness between all treatments were examined (figure 2-4). The result of probiotic treatments in JAK/STAT and NF-kB pathway were summarized in figure 1.
The results of stat genes expression
The data of stat genes expression were showed in figure 1. Comparative analysis of stat1 gene expression between sonicated pathogens and negative control showed that SP-ETEC and SP-ST could increase the gene expression significantly after 48 hours (p < 0.0001). Probiotic treatments (6 hours before SP tratment) in the first 24 hours decrease significantly the expression level of stat1 gene, while probiotics significantly up-regulated stat1 gene expression 48 hours after the treatments. There was no significant difference between Lactobacillus spp. alone, Bifidobacterium spp. alone, and Lac/Bif in decreasing or increasing the mRNA level of gene expression.
Using sonictaed pathogens showed the significant increase level in stat2 expression level after 48 hours (p < 0.01). Lactobacillus spp. and Bifidobacterium spp. could significantly decrease the expression level (p < 0.001), in comparison with positive control (SP48) after 24 hours of treatment, while Lactobacillus spp. (LP48) had the most significant effects on increasing the expression level (p < 0.0001).
Comparative analysis of stat3 gene expression between sonicated pathogens and negative control showed that SP-ETEC and SP-ST could increase the gene expression, significantly (p < 0.0001). Using probiotic treatments (6 hours before SP treatment) showed that all the versions could increase the gene expression level, except for the treatment of Lactobacillus spp. after 24 hours (LP24). The most effective treatment in increasing the level of gene expression was Lactobacillus spp. after 48 hours of treatment (LP48)(p < 0.0001). In all formats of probiotic-SP treatments, increasing the gene expression level was more significant after 48 hours of treatments (p < 0.0001 and p < 0.001).
Treatments with sonicated pathogens showed the increased level of stat4 expression level; although the results of probiotic treatments were variable. Using Lactobacillus spp-SP. ,either after 24 or 48 hours, could increase the gene expression, significantly (p < 0.0001). Vice versa, Bifidobacterium spp-SP. decreased the stat4 expression level both after 24 and 48 hours of treatments (p < 0.0001). Using Lac/Bif-SP could decrease the expression level in the first 24 hours of treatment (p < 0.0001).
Comparative analysis of stat5 gene expression between sonicated pathogens and negative control showed that SP-ETEC and SP-ST could increase the gene expression, significantly, specifically after 24 hours. All the probiotic treatments (6 hours before SP treatment) decreased stat5 expression level (p < 0.0001), except for Lactobacillus spp. after 48 hours of treatment (LP48), that increase the mRNA level (p < 0.0001). Lactobacillus spp. had more significant effects on decreasing the gene expression in the first 24 hours of treatment (LP24).
After using sonicated pathogene, the expression level of stat6 increased, significantly after 48 hours of treatments (p < 0.0001). All the versions of probiotic treatmnets (6 hours before SP treatment) decreased the expression level, in comparison with positive control (SP48). Bifidobacterium spp. and Lac/Bif in the first 24 hours of treatment had the most significant effects on decreasing the expression level Fig 1.
The results of jak genes expression
The data of jak esxpressions was showed in figure 2. The results of jak expression level showed more homogenous outcomes. The results of both jak1 and jak3 showed that the treatment of sonicated pathogen could increase the expression level after 24 hours of treatment (p < 0.0001). All the versions of probiotic treatment decrease the mRNA level of gene expression. There was no significant difference between probiotic treatment.
Comparative analysis of jak2 gene expression indicated that sonicated pathogens increased the gene expression. All the probiotic treatments (6 hours before SP treatment) decrease the mRNA level of gene expression. Lac/Bif treatment had the lowest effect on decreasing the gene expression.
After using SP in the first 24 hours of treatment, the expression level of tyk2 was increased (p < 0.0001). All the probiotic treatments (6 hours before SP treatment), except for Lactobacillus spp. after 48 hours of treatment (LP48), could decrease the gene expression and Bifidobacterium spp. had the most significant effect Fig 2.
The results of inflammatory genes expression
The data of inflammatory gene esxpressions was showed in figure 3. The analysis of inflammatory genes including tirap, irak4, rip, and nemo resulted in up-regulation following treatment of SP-ETEC and SP-ST. In contrast, probiotics treatments down-regulated the inflammatory genes. Lactobacillus spp. and Bifidobacterium spp. had more significant effects on decreasing nemo expression level in comparison with Lac/Bif treatments. For other inflammatory genes, there was no significant difference between probiotic treatments Fig 3.
The result of pro-inflammatory cytokines production
The production of cytokines was significantly higher after SP treatments. however, probiotic treatments (6 hours before SP treatment) significantly could decreased cytokine production. No significant difference between Lactobacillus spp, Bifidobacterium spp, and Lac/Bif was seen at any time after treatment Fig 4.