Absorption Of Phosphorus Across Normal And Gene-silenced Primary Cultured Duodenal Epithelial Cells Of Broiler Embryos

Based on the research results of in vivo with naturally feeding intact broilers and in vitro with primary cultured duodenal epithelial cells of broiler embryos from our laboratory,the following 3 experiments were conducted to further study the absorption of phosphorus across normal and gene-silenced primary cultured duodenal epithelial cells of broiler embryos and gene expressions of related transporters,so as to preliminarily reveal the molecular mechanism of phosphorus absorption in duodenum of broilers.Experiment 1 was conducted to study the phosphorus absorption and expressions of related phosphorus transporters in primary cultured duodenal epithelial cells of broiler embryos at different phosphorus concentrations.A single-factor completely randomized design was used in this experiment.The cells were randomly assigned to 1 of 5 treatments with 6 replicates per treatment,and treated with 0,0.5,1.0,2.0 and 4.0 mmol/L phosphorus for 87 min(determined by the previous results of in vitro cell experiment from our laboratory),respectively.The results showed that the phosphorus absorption percentages were significantly affected by different phosphorus incubation levels(P < 0.05),and the cellular phosphorus absorption percentage increased with the increase of phosphorus concentrations.Compared with the control group,the phosphorus incubation level of 4 mmol/L significantly decreased the mRNA expression of type IIb sodium-phosphate cotransporter(NaP-IIb)and inorganic phosphate transporter1(PiT-1)(P < 0.05),while mRNA expression of NaP-IIb,PiT-1 and PiT-2 were not significantly changed at 0.5,1 and 2 mmol/L(P > 0.05).The levels of phosphorus incubation had no significant effects on the protein expressions of NaP-IIb,PiT-1 and PiT-2(P > 0.05).The above results indicated that the phosphorus incubation levels(0.5-4 mmol/L)were too high for the expressions of phosphorus transporter,so the phosphorus incubation level should be further reduced in the experiment 3.Experiment 2 was conducted to study the silencing efficiency of different siRNA sequences for NaP-IIb and PiT-2,so as to obtain the siRNA sequence with the highest silencing efficiency,respectively.A completely randomized design was used in this experiment for siRNA sequences of NaP-IIb and PiT-2,respectively.The cells were randomly assigned to 1 of 4 treatments with 3 replicates per treatment for screening NaP-IIb-siRNA,and 4 treatments were blank control group(untransfected siRNA),negative control group(transfected negative siRNA)and 2 NaP-IIb-siRNA treatment groups(transfected NaP-Ⅱb-siRNA1372 and 1416),respectively.For screening PiT-2-siRNA,the cells were randomly assigned to 1 of 5 treatments with 3 replicates per treatment,and 5 treatments were blank control group(untransfected siRNA),negative control group(transfected negative siRNA)and 3 PiT-2-siRNA treatment groups(transfected PiT-2-siRNA890,996 and 1217),respectively.The results showed that both transfected NaP-IIb-siRNA treatment groups reduced the mRNA expression of NaP-IIb compared with the blank control group and the negative control group,and the decreased level was about 65%,but there was no significant difference(P > 0.10).There was no significant effect in protein expression level of NaP-IIb(P > 0.10),but the protein expression level of transfected NaP-IIb-siRNA1372 was lower than those transfected NaP-IIb-siRNA1416.In the three transfected PiT-2-siRNA treatment groups,PiT-2-siRNA890 significantly reduced the PiT-2 mRNA expression level by about 80%(P < 0.10),and there was no significant difference in protein expression level(P > 0.10).The other PiT-2-siRNA treatment groups had no significant effects on mRNA and protein expression levels(P > 0.10).Therefore,the NaP-IIb-siRNA1372 and PiT-2-siRNA890 were selected for the next experiment 3.Experiment 3 was conducted to study the absorption of phosphorus across normal and NaP-IIb or PiT-2 gene-silenced primary cultured duodenal epithelial cells of broiler embryos in combination with the siRNA interference technique,so as to elucidate whether NaP-IIb and PiT-2 directly participate in the transport and absorption of phosphorus in duodenal epithelial cells of broilers.Both NaP-IIb-siRNA1372 and PiT-2-siRNA890 gene-silencing experiments were completely randomized design involving 2 phosphorus concentrations(0 and 0.25 mmol/L)×2 treaments(transfected siRNA group and blank control group),respectively.There were a total of 4 treaments with 3 replicates per treatment.The results showed that the level of phosphorus incubation had no significant effect on the mRNA and protein expressions of NaP-IIb in the primary cultured duodenal epithelial cells of broiler embryos(P > 0.10).The specific silencing of NaP-IIb by siRNA1372 significantly decreased the NaP-IIb mRNA expression(P < 0.10),but had no significant effect on protein expression level(P > 0.10).The interaction between phosphorus incubation level and transfection of siRNA had a significant effect on the phosphorus absorption percentage of primary cultured duodenal epithelial cells of broiler embryos(P < 0.10).At the 0.25 mmol/L phosphorus incubation level,the siRNA1372 transfection group significantly reduced the phosphorus absorption percentage of duodenal epithelial cells compared with the blank control group(P < 0.10).The results indicated that the decrease of phosphorus absorption could be due to silencing the NaP-Ⅱb mRNA expression in the primary cultured duodenal epithelial cells.The phosphorus incubation level significantly increased the expression of PiT-2 protein and phosphorus absorption in the primary cultured duodenal epithelial cells of broiler embryos(P < 0.10).The specific silencing of PiT-2 gene had no significant effect on protein expression and phosphorus absorption(P > 0.10).The interaction between phosphorus incubation level and transfection of siRNA had a significant effect on PiT-2 mRNA expression(P < 0.10).At the 0.25 mmol/L phosphorus incubation level,the siRNA890 transfection group significantly reduced the expression of PiT-2 mRNA compared with the blank control group(P < 0.10).The results indicated that silencing the PiT-2 gene did not decrease the phosphorus absorption in the primary cultured duodenal epithelial cells.In conclusion,the NaP-IIb might be directly involved in the phosphorus absorption across the primary cultured duodenal epithelial cells of broiler embryos,but PiT-2 might not be involved in that process.