Epigenetic Modulation Of Folic Acid And Methionine On Pro-inflammatory Cytokines Gene Transcription In Broiler PBMC

Inflammation triggers immunological stress response, affects body energy and protein metabolism, finally may decrease feed intake and growth performance. The gene expression of inflammatory cytokines is a crucial control during inflammatory response. Epigenetic modification can regulate the expression of special genes. The study is composed of three trails, starting with the construction of lipopolysaccharide(LPS)-induced inflammatory model in broiler peripheral blood mononuclear cells(PBMC), analyzing the epigenetic modify of active pro-inflammatory cytokine genes, investigating the epigenetic regulation of folic acid(FA) and methionine(Met) on the transcription of pro-inflammatory cytokine genes, and aiming to provide a theoretical basis for the nutri-epigenetic regulation of broiler inflammation. Experiment 1 Effects of LPS stimulation on gene expression of pro-inflammatory cytokines in broiler PBMCThe present experiment was conducted to establish LPS inflammation model by investigating the effects of different concentrations(0, 5, 10, 15, 20 mg/L) and durations(0, 3, 6 h) of LPS stimulation on pro-inflammatory cytokine gene expression of broiler PBMC. The results showed that an interaction existed between LPS gradient and durations in PBMC pro-inflammatory cytokine gene expression(P<0.001). Treatment duration 3 h significantly increased pro-inflammatory cytokines expressions(P<0.001). All the treatment concentrations(5, 10, 15, 20 mg/L) significantly increased pro-inflammatory cytokine genes expression(P<0.001), IL-1? expression was significantly higher in 5 mg/L than other groups; IL-6 expression was significantly higher in 15 mg/L and 20 mg/L than other groups. LPS 5 mg/L and 3 h significantly increased the expression of IL-1?; LPS 20 mg/L and 3 h significantly increased the expression of IL-6; LPS 10 mg/L and 3 h significantly increased the expression of TNF-?. Consequently, we establish LPS inflammation model with stimulating concentration at 10 mg/L and stimulating duration at 3 h. Experiment 2 The epigenetic modification analysis of active pro-inflammatory cytokine genes induced by LPS in broiler PBMCThe present experiment was to determine the relation of pro-inflammatory cytokine genes expression and epigenetic modification including DNA methylation and chromation structure. We detected DNA methylation and chromation structure of gene promoter using BSP method and Dnase I-qPCR method, and the effects of 5-aza-2'-deoxycytidine(AZA) and trichostatin A(TSA) treatment on pro-inflammatory cytokines expressions in broiler PBMC. The expression of DNMT1 showed down regulation in respond to LPS stimulation(P<0.05). LPS treatment repressed the expression of HDAC2(P<0.05). Several HATs showed higher relative expression and this expression was significant for both PCAF and Tip60 after LPS treatment(P<0.05). The demethylation of IL-6 gene-302 and-264 cytosine-guanine(CpG) sites occurred after LPS treatment(P<0.05), as well as TNF-? gene-371 CpG site(P<0.05), whereas methylation in the IL-1? gene promoter region was not affected. Otherwise, LPS stimulation relaxed chromatin structure of IL-1? and IL-6 promoter(P<0.05).LPS decreased the expression of DNMT1, DNMT3 a and DNMT3b(P<0.05), but AZA addition had no effects. LPS increased the expression of IL-1?, IL-6 and TNF-?(P<0.05), and the expression of IL-6 and TNF-? was higher in AZA/LPS group than LPS group(P<0.05).TSA decreased the expression of HDAC2, HDAC3, HDAC8 and HDAC9(P<0.05). LPS decreased the expression of HDAC2, HDAC3, HDAC7, HDAC8, HDAC9 and HDAC10(P<0.05). TSA/LPS decreased the expression of HDAC2, HDAC3, HDAC8, HDAC9 and HDAC10(P<0.05). Compared with LPS group, TSA/LPS group decreased the expression of IL-1?, IL-6 and TNF-?(P<0.05). IL-1? expression is related to an accessible chromatin structure. IL-6 expression is related to open chromatin conformation and DNA hypomethylation. TNF-? expression is related to promoter demethylation. HDACi could be potential anti-inflammation angent. Experiment 3 Epigenetic modulations of folic acid and methionine on pro-inflammatory cytokines gene transcription in broiler PBMCThe present experiment studied the epigenetic modulation of folic acid and methionine on pro-inflammatory cytokine genes transcription, by analyzing cell proliferation, genes expression and promoter DNA menthylation of pro-inflammatory cytokine in broiler PBMC.The results showed that an interaction existed between FA and Met in PBMC proliferation(P<0.001) and DNMTs expressions(P<0.001). Low-Met and Low-FA suppressed cell proliferation(P<0.001), High-Met and High-FA promoted cell proliferation(P<0.001). Low-Met significantly increased the expression of DNMT1 and DNMT3 a, and High-Met significantly decreased the expression of DNMT3 a. Low-FA significantly increased the expression of DNMT1 and DNMT3 a, and High-FA significantly increased the expression of DNMT1. Compre to Low-Met, High-Met significantly decreased the expression of IL-6(P<0.05), and Medium-Met significantly decreased the expression of TNF-?(P<0.05). There is no effect of FA on the expression of pro-inflammatoy cytokines. High-Met significantly increased DNA methylation levels of IL-6 promoter-191 CpG site(P<0.001). Medium-Met significantly increased DNA methylation levels of TNF-? promoter-419 CpG site(P<0.05). Met can reduce inflammation by descreasing gene expression of pro-inflammatory cytokines through effecting DNA methylation of special sites.In summary, the expressions of pro-inflammatory cytokines induced by LPS stimulations are related to their promoter epigenetic modification in broiler PBMC. Met can reduce inflammation by descreasing gene expression of pro-inflammatory cytokines through increasing their promoter DNA methylation. There is no effect of FA on the expression of pro-inflammatoy cytokines.