The Signaling Pathway Of Vitamin D Regulating Phosphate Absorption And NaPi-Ⅱb Expression In The Duodenum Of Broiler Chickens

The regulation of phosphate homeostasis is achieved via complex interactions between the intestine,kidney,and bone in poultry.It is widely accepted that the small intestine play the major role in maintaining blood phosphate concentrations.The nutrient to regulate phosphate absorption in the small intestine is vitamin D3 in poultry feed.Vitamin D3 can be converted into the final active product 1,25-(OH)2-D3.1,25-(OH)2-D3 combines with intestinal nuclear vitamin D receptor(nVDR)and regulates the metabolism of phosphorus in poultry.The signal pathway that 1,25-(OH)2-D3 binds to nVDR to regulate phosphate absorption has not been clarified.Therefore,this experiment aims to explore the main signal pathways of vitamin D affecting phosphate absorption in the small intestine of broiler chickens.Experiment 1 Analysis of differentially expressed genes and signaling pathways of vitamin D regulating phosphorus absorption in duodenum of broiler chickens based on transcriptome sequencing RNA-seqThis study was conducted to investigate the candidate genes and signaling pathways in vitamin D regulating phosphorus absorption in small intestine of Ross 308 broiler chickens from 1 to 16 days of age using RNA-sequencing technology.Three levels(0,5,and 10 μg/kg)of 1,25-dihydroxycholecalciferol(1,25-(OH)2-D3)were added to the basal diet without vitamin D.Broiler chickens were killed and duodenal mucosa was collected at 16 days of age.Results showed that the addition of 5 and 10 μg/kg of 1,25-(OH)2-D3 increased body weight gain and improved tibia and femur mineralization in 1-to 16-day-old broiler chickens compared with the basal diet without vitamin D(P<0.05).The weight,length,and ash weight of the tibia and femur in broilers fed with 10μg/kg 1,25-(OH)2-D3 were lower than those of birds fed with 5μg/kg 1,25-(OH)2-D3(P<0.05).After sequencing,9 samples obtained a total of 61.96 Gb of clean reads,and each sample produced an average of 6.88 Gb of clean reads.The ratio of the obtained sequence by RNA-seq to the reference genome sequence is from 89.98%to 91.08%.The ratio of the gene set by RNA-seq to the reference gene set is from 78.61%to 81.44%.A total of 18.173 genes were detected in the duodenum of broilers,of which 17.557 were known genes and 616 were unknown genes.A total of 1029,939,and 642 candidate genes were screened when comparing the three levels of 1,25-(OH)2-D3(0 vs 5,0 vs 10,and 5 vs 10 μg/kg),respectively.Gene Ontology(GO)analysis identified candidate genes regulating calcium and phosphorus absorption and metabolism,including nuclear vitamin D receptor(VDR),protein disulfide-isomerase A3(PDIA3)(also named membrane vitamin D receptor),type Ⅱb sodium phosphate transporter protein NaPi-Ⅱb(SLC34A2),inorganic phosphorus transporter carrier protein 1 PiT-1(SLC20A1),inorganic phosphorus transporter carrier protein 2 PiT-2(SLC20A2),calbindin D28k(CALB1),ATPase plasma membrane Ca2+transporting 1 PMCA1b(ATP2B1),ATPase plasma membrane Ca2+transporting 2(ATP2B2),solute carrier family 8 member A1 NCX1(SLC8A1),fibroblast growth factor 1(FGF1),and fibroblast growth factor 19(FGF19).Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis indicated pathways associated with the intestinal calcium and phosphorus absorption and metabolism of broiler chickens,including cyclic guanosine monophosphate-protein kinase G(cGMP-PKG)signaling pathway,cyclic adenosine monophosphate-protein kinase A(cAMP-PKA)signaling pathway,mitogen-activated protein kinase(MAPK)signaling pathway,tyrosine kinase-transcription activating factor(Jak-STAT)signaling pathway and phosphatidylinositol-3-kinase-serine/threonine protein Kinase(PI3K-Akt)signaling pathway.The results of qRT-PCR were in accordance with those of RNA-seq.The results showed that MAPK signaling pathway may be involved in the regulation of 1,25-(OH)2-D3 regulation of duodenal phosphorus absorption in broiler chickens.Experiment 2 Signaling pathway of vitamin D regulating NaPi-IIb phosphate transporter expression in the duodenum of broiler chickensThis study was conducted to investigate the effects of vitamin D and MAPK signaling pathway inhibitors on growth performance,bone development,and expressions of phosphate absorption related genes in the duodenum of broiler chickens from 1 to 14 days of age.A total of 90 males of 1-day-old Ross 308 broiler chickens were weighed and randomly allotted to 9 treatments with 10 replicates of 1 bird per replicate.A 3×3 factorial experiment were designed to test 3 diets(basal diet without vitamin D,500 IU/kg vitamin D3,and 500 IU/kg 25-OH-D3)in combination with 3 drugs by intraperitoneal injection(saline,p38MAPK inhibitor SB203580,and ERK1/2 inhibitor PD98059).Broiler chickens were killed and duodenal mucosa was collected at 15 days of age.Real-time quantitative PCR(qRT-PCR)and western blotting were used to detect gene expressions.Results showed that compared with the basal diet(without vitamin D),diets with 500 IU/kg VD3 or 25-OH-D3 significantly increased the weight,length,ash weight,ash content,calcium content,and phosphorus content of femur and tibia in broiler chickens from 1 to 14 days of age(P<0.05).Diets with 500 IU/kg VD3 or 25-OH-D3 did not significantly affect body weight gain,for diameter of femur and tibia(P>0.05).The addition of 500 IU/kg VD3 or 25-OH-D3 significantly increased the mRNA and protein expression levels of NaPi-Ⅱb gene and mRNA expression levels of nVDR,and decreased the protein expression levels of p38MAPK and ERK1/2 in the duodenum of broiler chickens at 14 days of age(P<0.05).By contrast,the addition of 500 IU/kg VD3 or 25-OH-D3 did not affect mRNA expression levels of mVDR,p38MAPK,and ERK1/2.Compared with intraperitoneal injection of saline treatment,intraperitoneal injection of p38MAPK inhibitor and ERK1/2 inhibitor increased mRNA expression level of nVDR and mRNA and protein expression levels of NaPi-Ⅱb in the duodenum of broiler chickens at 14 days of age(P<0.05).Intraperitoneal injection of p38MAPK inhibitor and ERK1/2 inhibitor also decreased the protein levels of p38MAPK and ERK1/2(the phosphorylation level of ERK1/2 at Thr 204 site or the phosphorylation level of p38MAPK at Thr 180 site).By contrast,intraperitoneal injection of p38MAPK inhibitor and ERK1/2 inhibitor did not affect the mRNA expression levels of mVDR,p38MAPK,or ERK1/2.These data indicated that p38MAPK and ERK1/2 participate the process of 1,25-(OH)2-D3 regulating phosphate absorption in the duodenum of broiler chickens.In summary,these data indicate that phosphate absorption-related genes including VDR(nVDR),PDIA3(mVDR),SLC34A2(NaPi-Ⅱb),SLC20A2(PiT-2),FGF1 and FGF19 may participate in the process of vitamin D to regulate phosphorus absorption and metabolism in the small intestine of broiler chickens through MAPK signaling pathway.1,25-(OH)2-D3 combined with nVDR may regulate NaPi-IIb expression and phosphate absorption through p38MAPK and ERK 1/2 signaling pathways.