神经肽Y对猪小肠上皮细胞NaPi-Ⅱb蛋白表达及无机磷吸收的影响

Effect of Neuropeptide Y on Expression of NaPi-Ⅱ b Protein and Absorption of Inorganic Phosphorus in Porcine Small Intestinal Epithelial Cells

本试验通过猪小肠上皮细胞(Intestinal epithelial cells,IEC)的体外培养,旨在从细胞水平、基因转录和蛋白表达3个层面探讨不同浓度神经肽Y(Neuropeptide Y,NPY)对猪IEC中Ⅱb型钠磷转运蛋白(Sodium dependent phosphate cotransporterⅡb,NaPi-Ⅱb)的表达及无机磷(Inorganic phosphorus,Pi)吸收的影响,阐释NPY调控Pi吸收的机理,为深入研究动物小肠Pi吸收的分子机制和调控通路提供试验依据。试验设有6个处理,对照组未加NPY,其他处理组添加不同浓度的NPY(10-11、10-10、10-9、10-8和10-7 mol·L-1),每个处理设有6~8个重复,通过噻唑蓝(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT)比色法检测小肠上皮细胞的增殖情况;通过磷检测试剂盒,采用磷钼酸法测定细胞上清液Pi含量的变化;利用荧光定量PCR技术检测IEC中NaPi-Ⅱb mRNA的相对表达量,采用蛋白质免疫印迹(Western blotting)技术检测NaPi-Ⅱb蛋白的相对表达水平。结果表明:用不同浓度的NPY刺激IEC 24h后,各处理组细胞增殖无显著性差异(P>0.05)。细胞上清液中Pi含量随时间呈显著线性(P<0.01)和二次曲线(P<0.01)下降;在干预24h后,各处理组细胞上清液中Pi含量组间呈现显著性差异(P<0.01),与对照组相比,10-10、10-9、10-8和10-7 mol·L-1处理组上清液Pi含量呈显著性下降(P<0.01),IEC对Pi的吸收量分别提高了161%、171%、168%和139%。NPY添加浓度与NaPi-Ⅱb mRNA和NaPi-Ⅱb蛋白的相对表达量均呈二次曲线关系(P<0.01,P<0.05)。适宜浓度的NPY可以通过刺激小肠上皮细胞NaPi-Ⅱb mRNA的表达,使NaPi-Ⅱb蛋白表达增多,进而诱导细胞外液中无机磷跨膜转运到细胞内液,促进小肠上皮细胞对无机磷的吸收;培养液中NPY的最佳浓度为10-10~10-8 mol·L-1。

This experiment was conducted to investigate the effect of different concentration of neuropeptide Y on expression of NaPi-Ⅱb protein and absorption of inorganic phosphorus through culturing porcine small intestinal epithelial cells in vitro.To interpret the regulation mechanism of inorganic phosphorus absorption in small intestine affected by neuropeptide Y,and provide experiment basis for further study on molecular mechanism of animal phosphorus absorption and regulation pathways.Six treatments were designed according to different concentrations of neuropeptide Y,and the control group without neuropeptide Y,other treatments of adding different concentrations of neuropeptide Y(10-11,10-10,10-9,10-8 and 10-7 mol·L-1),respectively,with 6or 8repetition for each treatment.Proliferation of porcine small intestinal epithelial cells was determined by MTT assay.Phosphorus content in the cell supernatant fluid was detected by phosphorus detection kits.The relative expression quantity of NaPi-Ⅱb mRNA in porcine small intestinal epithelial cells was detected by RT-PCR.The relative expression levels of NaPi-Ⅱb protein was detected by Western blotting.The results showed that there was no significant difference(P>0.05)in cell proliferation in each treatment group,stimulating intestinal epithelial cells with different concentration of neuropeptide Y after 24h;Phosphorus content in the cell supernatant fluid declined on the form of significant linear(P<0.01)and quadratic curve(P<0.01)over time.Neuropeptide Y and phosphorus content in IEC were very significantly correlated(P<0.01)after stimulated 24 hby NPY.Compared with the control group,the treatment group of10-10,10-9,10-8and 10-7 mol·L-1 very significantly(P<0.01)decreased phosphorus content,and the amount of phosphorus uptake in small intestinal epithelial cells increased by 161%,171%,168%and 139%,respectively than that of the control group.There were significant quadratic correlations between relative expression quantity of NaPi-Ⅱb mRNA and NaPi-Ⅱb protein and neuropeptide Y adding concentration(P<0.01,P<0.05,respectively).We concluded that the appropriate concentration of neuropeptide Y can stimulate NaPi-Ⅱb gene transcription,increase expression levels of NaPi-Ⅱb protein,and then induce inorganic phosphorus in the extracellular fluid to transport across the membrane into the intracellular fluid,so as to promote porcine small intestinal epithelial cells to absorb inorganic phosphorus,and the optimum concentration of neuropeptide Y in the cell culture fluid between 10-10 and 10-8 mol·L-1.