日粮磷水平和VD_3对Na~+/Pi-Ⅱb mRNA表达和磷吸收的影响
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摘要
磷的大量使用和低利用率造成严重的环境污染和磷资源的浪费,进一步掌握磷吸收的分子机理和调控因素,可为准确掌握磷在日粮中的添加量,提倡低磷耗养殖提供科学依据。
试验一不同磷水平对Na+/Pi-Ⅱb mRNA表达和磷吸收影响的研究
本试验旨在研究影响Na+/Pi-Ⅱb mRNA表达量和磷吸收的适宜低磷水平,并比较不同肠段和肾脏对低磷适应性的差异。选用120只21日龄、平均体重为18-22 g的昆明大白鼠(雄),随机分成4组,每组5个重复,每重复6只大白鼠,分别饲喂含磷(总磷)0.2%P(低磷组)、0.4%P(低磷组)、0.6%P(正常磷组)、0.8%P(高磷组)的饲粮,试验期7 d。测定血钙血磷、骨钙骨磷及不同肠段Na+/Pi-Ⅱb转运载体蛋白mRNA表达量和磷的吸收等指标。结果表明：(1)0.2%低磷组回肠、空肠、十二指肠Na+/Pi-Ⅱb载体转运蛋白mRNA表达量分别是0.6%磷水平组的1.96倍(P<0.01)、2.41倍(P<0.01)、1.21倍(P<0.05)；但肾脏中Na+/Pi-Ⅱb mRNA表达量比其他三个磷水平组分别低9.19倍、12.86倍、23.15倍,差异均极显著(P<0.01)；(2)0.2%低磷组中回肠Na+/Pi-Ⅱb mRNA表达量分别是空肠、十二指肠和肾脏的1.34倍、3.43倍(P<0.05)和6.20倍(P<0.01)；肾脏中Na+/Pi-Ⅱb mRNA的表达量最低,分别比空肠、十二指肠低4.60倍(P<0.01)、1.80倍(P<0.05)；(3)0.2%低磷组回肠中磷摄入量比0.6%和0.8%磷水平组分别提高22.11%(P<0.05)和19.35%(P<0.05),同时提高血清中VD3含量(P<0.05)；不同磷水平对小肠BBMV中磷吸收率影响均不显著(P>0.05),但对肾脏中磷吸收率影响显著,随着磷水平的增高,肾脏BBMV中磷吸收率呈升高趋势。研究表明：(1)低磷提高了回肠中Na+/Pi-Ⅱb载体转运蛋白mRNA表达量和磷的吸收,但显著降低了肾脏中Na+/Pi-Ⅱb载体转运蛋白mRNA表达量和磷的吸收；本研究中,0.2%日粮磷水平可视为从mRNA表达水平影响磷吸收分子机理的日粮磷水平；(2)低磷条件下,大鼠Na+/Pi-Ⅱb载体转运蛋白mRNA表达量以回肠最高,肾脏最低。
试验二VD缺乏时,不同磷水平对Na+/Pi-Ⅱb mRNA表达和磷吸收影响的研究
本试验旨在研究VD3对低磷条件下磷吸收和Na+/Pi-Ⅱb转运的调控作用。选择80只断奶后昆明大白鼠,随机分为0.2%P、0.4%P、0.6%P、0.8%P水平组,以0.6%磷水平为对照组,其它三个磷水平为试验组。每组5个重复,每重复4只大白鼠。日粮中不添加VD,其它参照大白鼠的营养需要标准配制。试验7 d。每晚6点每组大白鼠都以40 mg/kg/d剂量大腿肌注VD3代谢抗干扰药物EHDP(二磷酸盐),使试验动物缺乏VD；第7天早晨6点全部屠宰、取样。
试验显示：(1)在缺乏VD的情况下,两低磷组(0.2%P和0.4%P)骨钙骨磷含量均极显著低于0.6%P和0.8%P组(P<0.01)；不同磷水平对血钙血磷未表现出显著性差异；但随着磷水平的增加,血钙和血磷均表现出上升的趋势(P>0.05)。(2)VD缺乏下,不同磷水平极显著影响血清中VD3、PTH、ALP (P<0.01)。0.2%P处理组与0.8%P处理组比较,血清中VD3、PTH、ALP含量分别提高28.64%(P>0.05)、39.16%(P<0.05)和39.69%。(3)不同磷水平显著影响各肠段中Na+/Pi-Ⅱb mRNA相对表达量(P<0.05)。0.2%P水平组与0.8%P水平组比较,回肠、空肠、十二指肠Na+/Pi-Ⅱb mRNA表达量分别高2.08倍(P<0.01)、1.63倍(P<0.01)、1.75倍(P<0.05),肾脏中却降低1.73倍(P<0.05)。(4)VD缺乏条件下,不同磷水平显著影响磷的摄入量(P<0.05),回肠中两低磷组间磷的摄入量分别为62.24和59.21pmol/mg/60s;正常磷组和高磷组磷摄入量分别为84.25和76.22 pmol/mg/60s; 0.2%磷水平组与0.6%磷水平组比较,磷摄入量降低26.12%(P<0.05)；其它各肠段和肾脏中也均以低磷组中磷吸收为最低。VD缺乏下不同磷水平对BBMV中磷吸收率影响显著(P<0.05)；随着磷水平的降低,磷的吸收率也呈下降趋势,但肾脏中磷吸收率随着磷水平的增高而升高(P<0.05)。
本试验显示：(1)VD缺乏时低磷降低了Na+/Pi-Ⅱb mRNA表达量和磷的吸收；(2)不同的部位中,磷摄入量和吸收率均以回肠中最高,十二指肠最低。
试验三不同磷水平下,补充VD3对Na+/Pi-Ⅱb mRNA表达和磷吸收影响的研究
本研究在试验二的基础上,通过对缺乏VD的试验动物补充VD3后,研究Na+/Pi-Ⅱb转运和磷吸收的变化,以探讨VD3对磷吸收的调控作用。在试验二的基础上,使试验动物先缺乏VD,然后再补充VD3。试验分组和磷水平的设计皆同试验二。试验前6天每晚6点,每组大白鼠仍以40 mg/kg/d剂量大腿肌注VD3代谢抗干扰药物EHDP;屠宰前的12 h,每只老鼠以600 ng/kg·wt(以10ng/mL溶解乙醇中)的剂量腹腔注射1,25-(OH)2D3；第7天早晨屠宰、取样。
试验结果表明：(1)补充VD3后,骨钙骨磷随着磷水平的增加呈增长趋势；低磷降低骨钙骨磷浓度,0.2%磷水平组骨钙、骨磷分别比0.6%磷水平组低3.03倍(P<0.01)、7.06倍(P<0.01)。(2)给VD缺乏动物补充VD3后,0.2%P组较0.6%P组,血清中VD3、PTH、ALP含量分别提高23.25%(P<0.05)、13.97%(P<0.05)、33.55%(P<0.01)。(3)补充VD3后,低磷极显著提高了不同肠段和肾脏中Na+/Pi-Ⅱb mRNA表达量,且以0.2%P组对Na+/Pi-Ⅱb mRNA表达量影响最显著。四个不同部位中,Na+/Pi-Ⅱb mRNA表达量以回肠受低磷影响最大。(4)给缺乏VD的大白鼠补充VD3显著影响磷的摄入量(P<0.05),四个磷水平中,回肠和空肠中皆以0.2%P水平组磷摄入量为最高,但低磷对肾脏Na+/Pi-Ⅱb mRNA表达量的影响趋势与回肠和空肠相反,即0.6%P和0.8%P组中磷摄入量平均值分别比两个低磷组高。不同部位中以回肠中磷摄入量为最高,其次是空肠、肾脏、十二指肠,磷摄入量分别为129.04、90.95、48.75、39.49 pmol/mg/60s。补充VD3后,不同磷水平对BBMV中磷吸收率影响不显著(P>0.05)；但0.2%P与0.6%P组比较,回肠和空肠中磷吸收率高7.57%(P>0.05)、12.57%(P>0.05)；肾脏中磷的吸收率随着磷水平的增高而增高,0.6%P组分别比两个低磷组高13.43%和14.61%,但差异不显著。
综合三个试验结果,在不同日粮磷水平下,无论VD缺乏与否,骨钙骨磷皆随着日粮磷水平增加而升高,低磷增加了PTH和ALP的含量,且在VD缺乏时更为明显；补充VD3后,血清中VD含量增加,PTH和ALP的含量下降；低磷刺激了Na+/Pi-Ⅱb mRNA表达量,但VD缺乏下,磷摄入量随着磷水平下降显著降低,磷吸收率呈相同变化趋势；补充VD3后,Na+/Pi-Ⅱb mRNA和磷摄入量和吸收率增加,且以低磷组提高幅度最大；补充VD3后,不同磷水平对不同部位中磷吸收的影响不一致,低磷刺激回肠和空肠中磷摄入量的提高,但降低了肾脏中磷的摄入量。显示：低磷下,VD3是调控Na+/Pi-Ⅱb和磷吸收的重要因素,不同部位中磷吸收的高低分别为：回肠、空肠、肾脏、十二指肠。
综上所述,本研究得出结论如下：
(1)日粮低磷提高Na+/Pi-Ⅱb载体蛋白mRNA表达和磷的摄入量,本研究中,日粮0.2%P可视为大白鼠从mRNA表达水平影响磷吸收分子机理的日粮磷水平。
(2)大白鼠Na+/Pi-Ⅱb载体蛋白mRNA表达量在回肠最高,肾脏最低；不同肠段或组织中磷吸收高低依次为：回肠、空肠、肾脏、十二指肠。
(3)VD3是调节动物低磷适应性的一个重要因素,且VD3对磷代谢的调控作用符合基因学说观点。
(4)VD缺乏后,补充VD3对磷吸收有提高作用,但这种作用随着磷水平的增加逐步减弱。
(5)不同日粮磷水平和VD3对小肠和肾脏中Na+/Pi-Ⅱb载体转运蛋白mRNA表达量和磷吸收的影响存在差异性。
To reveal the mechanism of phosphate absorption and provide basis for scientific apply Pi or decreased the pollution and waste from phosphate's utilization, and more, define the appropriated factor regulating phosphate uptake, a trial was conducted to determine the effects of gradient dietary Pi on phosphate absorption and a factor regulating low phosphate adaption.
ExperimentⅠ. Effects of Dietary Phosphate Content on Na+/Pi-Ⅱb Cotransporter mRNA Expression and Pi absorption
To investigate the effects of low-phosphate diet on Na+/Pi-Ⅱb mRNA and phosphate uptake, and compare the difference between intestinal segments and kidney adaption to low-Pi diet, a study was conducted to determine the effects of phosphate levels on Pi absorption. A total of 120 male Kunming-rats aging 21-days and body weighting 18-22 g was randomly divided into 4 treatments, each treatment consisted of 5 replicates with 6 rats. The four treatments were fed with the basal diet supplemented with 0.2%(low-Pi),0.4%(low-Pi),0.6%(normal-Pi),0.8% P(high-Pi). The experiment lasted for 7 days. On the last day, one of the replicates was slaughtered to determine Ca and Pi concentration of bone and blood or other biochemical indexes.It suggested that (1)Na+/Pi-Ⅱb mRNA expression higher 1.96、2.41、1.21 folds than normal Pi treatment in ileum、jejunum and duodenum compared 0.2%P with 0.6%P. Na+/Pi-Ⅱb mRNA expression in 0.2%P treatment lower 9.19(P<0.01),12.86(P<0.01)、23.15(P<0.01) folds than other three treatments in renal;(2) To 0.2% treatment, Na+/Pi-Ⅱb mRNA expression was higher 1.34(P>0.05)、3.43(P<0.01)、6.20(P<0.01) folds than jejunum、duodenum and renal, and lower 4.60(P<0.01)、1.80(P<0.05) folds in renal compared with jejunum and duodenum. Na+/Pi-Ⅱb mRNA expression is highest in ileum. Pi absorption of 0.2% P treatments higher 22.11% or 19.35% than 0.6%P or 0.8%P treatments(P<0.05), and VD3 concentration of serum is significantly higher than other three treatments. (3) Dietary phosphate no significant effects to Pi absorption rate of BBMV, while affected to it in renal (P<0.05), Pi absorption rate increased with dietary phosphate content(P< 0.05).The results showed that:(1) Low Pi diet significantly increased intestinal Na+/Pi-Ⅱb mRNA expression and Pi absorption,while decreased it in renal. In our study,0.2% dietary phosphate was a optimal low-Pi content that impacted to the mechanism of phosphate absorption from mRNA expression for rat;(2) Expression of Na+/Pi-Ⅱb mRNA is highest in ileum, and lowest in renal.
ExperimentⅡ. Effects of Dietary Phosphate Content on Na+/Pi-Ⅱb mRNA Expression and Phosphate Absorption under Vitamin D Stress
The study was conducted to determine the role of vitamin D3 in regulating phosphate absorption and adaption to a low Pi diet. A total of 80 male Kunming-rats aging 21 days and body weighting 18-22 g was randomly divided into 4 treatments, each treatment consisted of 5 replicates with 4 rats. The treatments were fed with the basal diet supplemented with 0.2%、0.4%、0.6% or 0.8% phosphate. The experiment lasted for 7 days. On the former 6 days at every 6 pm, each of the replicates was treated with EHDP (ethane-1-hydroxy-1,1-diphosphonicacid, tetrasodiumsalt) which is known to decrease the circulating levels of 1,25-(OH)2D3. On the 7th day morning, rats were slaughtered for determination of Ca and Pi concentration and phosphate uptake. It showed that:(1) bone Ca and Pi were significant increased compared two low-Pi treatments with 0.6%P or 0.8%P groups under VD restriction(P<0.01);Dietary phosphate levels didn't affected to serum Ca and Pi; but which tend to rise with dietary Pi content (P>0.05).(2) VD、PTH、ALP concentration of serum were significant affected by diet phosphate contents.Compared 0.2%P with 0.8%P group, VD、PTH and ALP of serum were increased 14.35%(P>0.05)、28.83%(P<0.05) and 36.69%(P <0.05), respectively. (3) Dietary phosphate contents also affected to expression of Na+/Pi-Ⅱb mRNA in small intestine. Compared 0.2%P with 0.8%P group, that of ileum、jejunum、duodenum, respectively, increased 2.08 (P<0.01)、1.63 (P<0.05)、1.75 (P<0.05) folds, while decreased 1.73(P<0.05) folds in renal.(4) Dietary phosphate contents also affected to Pi absorption rate when animal shortage of VD(P< 0.05), Pi absorption of two low-Pi treatments, respectively, were 62.24 and 59.21 pmol/mg/60s; the same of normal-Pi or high-Pi treatments were 84.25 and 76.22 pmol/mg/60s, respectively; Compared 0.2%P with 0.6%P group, Pi absorption decreased to 26.12%(P<0.05), this effects appeared to every intestinal segments and kidney. Dietary phosphate significant affected to Pi absorption rate of BBMV, Pi absorption rate decreased with dietary phosphate in intestine, contrasted to that, Pi absorption rate increased with dietary phosphate in renal.
Overall, Experiment II showed that:(1) Low-Pi diet lowered phosphate absorption under VD stress;(2) The main intestinal segment for phosphate absorption is ileum to rat, and Pi absorption lowest in duodenum.
ExperimentⅢ. Effects of Dietary Phosphate Content on Na+/Pi-Ⅱb mRNA Expression and Absorption after Injected 1,25-(OH)2D3
Based on experimentⅡ, mading rat lack of vitamin D firstly, then administration 1,25-(OH)2D3 to them. The aim of the study was to investigate the tendency of Na+/Pi-Ⅱb mRNA and Pi uptake after administrated 1,25-(OH)2D3 toVD restricted rats. As same as experimentⅡ, the former 6 days, injected EHDP still to rats, before 12h before slaughter, rats were injected with 1,25-(OH)2D3 at 600ng/kg.wt. On the 7th day morning, rats were slaughtered for determination of Ca and Pi concentration and phosphate uptake.
Experiment III showed that:(1) Ca and Pi of bone rised with dietary phosphate after administration 1,25-(OH)2D3 to restricted VD rats. Low-Pi diet declined bone Ca and Pi concentration, compared 0.2%P with 0.6%P group, that decreased 3.03 (P< 0.01) and 7.06 times (P<0.01). (2) Serum VD3、PTH、ALP concentration of 0.2%P treatment increased 23.25%(P<0.05)、13.97%(P<0.05)、33.55%(P<0.01) than 0.2%P treatment after injection 1,25-(OH)2D3.(3) Low-Pi diet significantly elevated Na+/Pi-Ⅱb mRNA expression to every intestinal segments or renal, and more 0.2%P trteatment become the highest, namely mRNA expression of 0.2%P was the impacted treatment mostly after injection 1,25-(OH)2D3. But concerning to different segment, it was highest in ileum.Injection 1,25-(OH)2D3 also significant improved Pi absorption (P <0.05), among four phosphates levels, it is highest in ileum and jejunum to 0.2%P group, while contrasted to that it is higher in duodenum and renal to 0.6%P or 0.8%P treatment, namely Pi absorption in duodenum and renal were higher than in ileum and jejunum when phosphate is 0.6%P or 0.8%P. Ileum was the dominant site to Pi uptake, and secondly, jejunum、rena、duodenum. The value were, respectively,129.04、90.95、48.75、39.49 pmol/mg/60s.Based on the above, it showed Ca and Pi of bone increased with dietary Pi regardless of animal VD varied situation; Low-Pi decreased PTH and ALP concentration of serum, especially nuder situation of restricted VD.Injection 1,25-(OH)2D3 increased serum VD concentration and lowed PTH and ALP concentration.(4)Low-Pi stimulated Na+/Pi-Ⅱb mRNA expression, but Pi uptake downed with diet Pi content when VD is shortaged. Administration 1,25-(OH)2D3 to rats increased expression of Na+/Pi-Ⅱb mRNA, and low-Pi treatment increased most.
Dietary phosphate no significant effects to Pi uptake of BBMV after injection 1,25-(OH)2D3, but compared 0.2%P group to 0.6%P group, Pi absorption higher 7.57% (P>0.05)and 12.57%(P>0.05)in ileum and jejunum.While it increased with dietary phosphate in renal which display contrast effects to small intestine.
ExperimentⅢsuggested vitamin D play an important role for regulation low phosphate adaption. The optimal site for phosphorus absorption was ileum, secondly jejunum, then kidney and duodenum.
In conclusion, it suggested that:(1) Low-Pi diet significantly increased Na+/Pi-Ⅱb mRNA expression and Pi absorption in ileum and jejunum, and decreased in renal.0.2% dietary phosphate was a optimal low-Pi content that impacted to the mechanism of phosphate absorption from mRNA expression for rat. (2) Na+/Pi-Ⅱb cotransporter mRNA expression is highest in ileum, while lowest in kideny for rat under low dietary phosphate. Pi absorption or absorption rate presented a similar effects to small intestine and renal, and the main intestinal segment or organ for Pi absorption is ileum, secondly jejunum and renal, lastly duodenum. (3) VD play an important role for regulating animal adaption to low phosphate, and the mechanism for it was consistent with gene thesis. (4) Injection 1,25-(OH)2D3 to restricted VD rats improve Pi uptake, but this effects decreased with increasing dietary phosphate content. (5) The effects of ditary phosphate content and VD3 on Pi absorption is different between small instine and renal.
试验一不同磷水平对Na+/Pi-Ⅱb mRNA表达和磷吸收影响的研究
本试验旨在研究影响Na+/Pi-Ⅱb mRNA表达量和磷吸收的适宜低磷水平,并比较不同肠段和肾脏对低磷适应性的差异。选用120只21日龄、平均体重为18-22 g的昆明大白鼠(雄),随机分成4组,每组5个重复,每重复6只大白鼠,分别饲喂含磷(总磷)0.2%P(低磷组)、0.4%P(低磷组)、0.6%P(正常磷组)、0.8%P(高磷组)的饲粮,试验期7 d。测定血钙血磷、骨钙骨磷及不同肠段Na+/Pi-Ⅱb转运载体蛋白mRNA表达量和磷的吸收等指标。结果表明：(1)0.2%低磷组回肠、空肠、十二指肠Na+/Pi-Ⅱb载体转运蛋白mRNA表达量分别是0.6%磷水平组的1.96倍(P<0.01)、2.41倍(P<0.01)、1.21倍(P<0.05)；但肾脏中Na+/Pi-Ⅱb mRNA表达量比其他三个磷水平组分别低9.19倍、12.86倍、23.15倍,差异均极显著(P<0.01)；(2)0.2%低磷组中回肠Na+/Pi-Ⅱb mRNA表达量分别是空肠、十二指肠和肾脏的1.34倍、3.43倍(P<0.05)和6.20倍(P<0.01)；肾脏中Na+/Pi-Ⅱb mRNA的表达量最低,分别比空肠、十二指肠低4.60倍(P<0.01)、1.80倍(P<0.05)；(3)0.2%低磷组回肠中磷摄入量比0.6%和0.8%磷水平组分别提高22.11%(P<0.05)和19.35%(P<0.05),同时提高血清中VD3含量(P<0.05)；不同磷水平对小肠BBMV中磷吸收率影响均不显著(P>0.05),但对肾脏中磷吸收率影响显著,随着磷水平的增高,肾脏BBMV中磷吸收率呈升高趋势。研究表明：(1)低磷提高了回肠中Na+/Pi-Ⅱb载体转运蛋白mRNA表达量和磷的吸收,但显著降低了肾脏中Na+/Pi-Ⅱb载体转运蛋白mRNA表达量和磷的吸收；本研究中,0.2%日粮磷水平可视为从mRNA表达水平影响磷吸收分子机理的日粮磷水平；(2)低磷条件下,大鼠Na+/Pi-Ⅱb载体转运蛋白mRNA表达量以回肠最高,肾脏最低。
试验二VD缺乏时,不同磷水平对Na+/Pi-Ⅱb mRNA表达和磷吸收影响的研究
本试验旨在研究VD3对低磷条件下磷吸收和Na+/Pi-Ⅱb转运的调控作用。选择80只断奶后昆明大白鼠,随机分为0.2%P、0.4%P、0.6%P、0.8%P水平组,以0.6%磷水平为对照组,其它三个磷水平为试验组。每组5个重复,每重复4只大白鼠。日粮中不添加VD,其它参照大白鼠的营养需要标准配制。试验7 d。每晚6点每组大白鼠都以40 mg/kg/d剂量大腿肌注VD3代谢抗干扰药物EHDP(二磷酸盐),使试验动物缺乏VD；第7天早晨6点全部屠宰、取样。
试验显示：(1)在缺乏VD的情况下,两低磷组(0.2%P和0.4%P)骨钙骨磷含量均极显著低于0.6%P和0.8%P组(P<0.01)；不同磷水平对血钙血磷未表现出显著性差异；但随着磷水平的增加,血钙和血磷均表现出上升的趋势(P>0.05)。(2)VD缺乏下,不同磷水平极显著影响血清中VD3、PTH、ALP (P<0.01)。0.2%P处理组与0.8%P处理组比较,血清中VD3、PTH、ALP含量分别提高28.64%(P>0.05)、39.16%(P<0.05)和39.69%。(3)不同磷水平显著影响各肠段中Na+/Pi-Ⅱb mRNA相对表达量(P<0.05)。0.2%P水平组与0.8%P水平组比较,回肠、空肠、十二指肠Na+/Pi-Ⅱb mRNA表达量分别高2.08倍(P<0.01)、1.63倍(P<0.01)、1.75倍(P<0.05),肾脏中却降低1.73倍(P<0.05)。(4)VD缺乏条件下,不同磷水平显著影响磷的摄入量(P<0.05),回肠中两低磷组间磷的摄入量分别为62.24和59.21pmol/mg/60s;正常磷组和高磷组磷摄入量分别为84.25和76.22 pmol/mg/60s; 0.2%磷水平组与0.6%磷水平组比较,磷摄入量降低26.12%(P<0.05)；其它各肠段和肾脏中也均以低磷组中磷吸收为最低。VD缺乏下不同磷水平对BBMV中磷吸收率影响显著(P<0.05)；随着磷水平的降低,磷的吸收率也呈下降趋势,但肾脏中磷吸收率随着磷水平的增高而升高(P<0.05)。
本试验显示：(1)VD缺乏时低磷降低了Na+/Pi-Ⅱb mRNA表达量和磷的吸收；(2)不同的部位中,磷摄入量和吸收率均以回肠中最高,十二指肠最低。
试验三不同磷水平下,补充VD3对Na+/Pi-Ⅱb mRNA表达和磷吸收影响的研究
本研究在试验二的基础上,通过对缺乏VD的试验动物补充VD3后,研究Na+/Pi-Ⅱb转运和磷吸收的变化,以探讨VD3对磷吸收的调控作用。在试验二的基础上,使试验动物先缺乏VD,然后再补充VD3。试验分组和磷水平的设计皆同试验二。试验前6天每晚6点,每组大白鼠仍以40 mg/kg/d剂量大腿肌注VD3代谢抗干扰药物EHDP;屠宰前的12 h,每只老鼠以600 ng/kg·wt(以10ng/mL溶解乙醇中)的剂量腹腔注射1,25-(OH)2D3；第7天早晨屠宰、取样。
试验结果表明：(1)补充VD3后,骨钙骨磷随着磷水平的增加呈增长趋势；低磷降低骨钙骨磷浓度,0.2%磷水平组骨钙、骨磷分别比0.6%磷水平组低3.03倍(P<0.01)、7.06倍(P<0.01)。(2)给VD缺乏动物补充VD3后,0.2%P组较0.6%P组,血清中VD3、PTH、ALP含量分别提高23.25%(P<0.05)、13.97%(P<0.05)、33.55%(P<0.01)。(3)补充VD3后,低磷极显著提高了不同肠段和肾脏中Na+/Pi-Ⅱb mRNA表达量,且以0.2%P组对Na+/Pi-Ⅱb mRNA表达量影响最显著。四个不同部位中,Na+/Pi-Ⅱb mRNA表达量以回肠受低磷影响最大。(4)给缺乏VD的大白鼠补充VD3显著影响磷的摄入量(P<0.05),四个磷水平中,回肠和空肠中皆以0.2%P水平组磷摄入量为最高,但低磷对肾脏Na+/Pi-Ⅱb mRNA表达量的影响趋势与回肠和空肠相反,即0.6%P和0.8%P组中磷摄入量平均值分别比两个低磷组高。不同部位中以回肠中磷摄入量为最高,其次是空肠、肾脏、十二指肠,磷摄入量分别为129.04、90.95、48.75、39.49 pmol/mg/60s。补充VD3后,不同磷水平对BBMV中磷吸收率影响不显著(P>0.05)；但0.2%P与0.6%P组比较,回肠和空肠中磷吸收率高7.57%(P>0.05)、12.57%(P>0.05)；肾脏中磷的吸收率随着磷水平的增高而增高,0.6%P组分别比两个低磷组高13.43%和14.61%,但差异不显著。
综合三个试验结果,在不同日粮磷水平下,无论VD缺乏与否,骨钙骨磷皆随着日粮磷水平增加而升高,低磷增加了PTH和ALP的含量,且在VD缺乏时更为明显；补充VD3后,血清中VD含量增加,PTH和ALP的含量下降；低磷刺激了Na+/Pi-Ⅱb mRNA表达量,但VD缺乏下,磷摄入量随着磷水平下降显著降低,磷吸收率呈相同变化趋势；补充VD3后,Na+/Pi-Ⅱb mRNA和磷摄入量和吸收率增加,且以低磷组提高幅度最大；补充VD3后,不同磷水平对不同部位中磷吸收的影响不一致,低磷刺激回肠和空肠中磷摄入量的提高,但降低了肾脏中磷的摄入量。显示：低磷下,VD3是调控Na+/Pi-Ⅱb和磷吸收的重要因素,不同部位中磷吸收的高低分别为：回肠、空肠、肾脏、十二指肠。
综上所述,本研究得出结论如下：
(1)日粮低磷提高Na+/Pi-Ⅱb载体蛋白mRNA表达和磷的摄入量,本研究中,日粮0.2%P可视为大白鼠从mRNA表达水平影响磷吸收分子机理的日粮磷水平。
(2)大白鼠Na+/Pi-Ⅱb载体蛋白mRNA表达量在回肠最高,肾脏最低；不同肠段或组织中磷吸收高低依次为：回肠、空肠、肾脏、十二指肠。
(3)VD3是调节动物低磷适应性的一个重要因素,且VD3对磷代谢的调控作用符合基因学说观点。
(4)VD缺乏后,补充VD3对磷吸收有提高作用,但这种作用随着磷水平的增加逐步减弱。
(5)不同日粮磷水平和VD3对小肠和肾脏中Na+/Pi-Ⅱb载体转运蛋白mRNA表达量和磷吸收的影响存在差异性。
To reveal the mechanism of phosphate absorption and provide basis for scientific apply Pi or decreased the pollution and waste from phosphate's utilization, and more, define the appropriated factor regulating phosphate uptake, a trial was conducted to determine the effects of gradient dietary Pi on phosphate absorption and a factor regulating low phosphate adaption.
ExperimentⅠ. Effects of Dietary Phosphate Content on Na+/Pi-Ⅱb Cotransporter mRNA Expression and Pi absorption
To investigate the effects of low-phosphate diet on Na+/Pi-Ⅱb mRNA and phosphate uptake, and compare the difference between intestinal segments and kidney adaption to low-Pi diet, a study was conducted to determine the effects of phosphate levels on Pi absorption. A total of 120 male Kunming-rats aging 21-days and body weighting 18-22 g was randomly divided into 4 treatments, each treatment consisted of 5 replicates with 6 rats. The four treatments were fed with the basal diet supplemented with 0.2%(low-Pi),0.4%(low-Pi),0.6%(normal-Pi),0.8% P(high-Pi). The experiment lasted for 7 days. On the last day, one of the replicates was slaughtered to determine Ca and Pi concentration of bone and blood or other biochemical indexes.It suggested that (1)Na+/Pi-Ⅱb mRNA expression higher 1.96、2.41、1.21 folds than normal Pi treatment in ileum、jejunum and duodenum compared 0.2%P with 0.6%P. Na+/Pi-Ⅱb mRNA expression in 0.2%P treatment lower 9.19(P<0.01),12.86(P<0.01)、23.15(P<0.01) folds than other three treatments in renal;(2) To 0.2% treatment, Na+/Pi-Ⅱb mRNA expression was higher 1.34(P>0.05)、3.43(P<0.01)、6.20(P<0.01) folds than jejunum、duodenum and renal, and lower 4.60(P<0.01)、1.80(P<0.05) folds in renal compared with jejunum and duodenum. Na+/Pi-Ⅱb mRNA expression is highest in ileum. Pi absorption of 0.2% P treatments higher 22.11% or 19.35% than 0.6%P or 0.8%P treatments(P<0.05), and VD3 concentration of serum is significantly higher than other three treatments. (3) Dietary phosphate no significant effects to Pi absorption rate of BBMV, while affected to it in renal (P<0.05), Pi absorption rate increased with dietary phosphate content(P< 0.05).The results showed that:(1) Low Pi diet significantly increased intestinal Na+/Pi-Ⅱb mRNA expression and Pi absorption,while decreased it in renal. In our study,0.2% dietary phosphate was a optimal low-Pi content that impacted to the mechanism of phosphate absorption from mRNA expression for rat;(2) Expression of Na+/Pi-Ⅱb mRNA is highest in ileum, and lowest in renal.
ExperimentⅡ. Effects of Dietary Phosphate Content on Na+/Pi-Ⅱb mRNA Expression and Phosphate Absorption under Vitamin D Stress
The study was conducted to determine the role of vitamin D3 in regulating phosphate absorption and adaption to a low Pi diet. A total of 80 male Kunming-rats aging 21 days and body weighting 18-22 g was randomly divided into 4 treatments, each treatment consisted of 5 replicates with 4 rats. The treatments were fed with the basal diet supplemented with 0.2%、0.4%、0.6% or 0.8% phosphate. The experiment lasted for 7 days. On the former 6 days at every 6 pm, each of the replicates was treated with EHDP (ethane-1-hydroxy-1,1-diphosphonicacid, tetrasodiumsalt) which is known to decrease the circulating levels of 1,25-(OH)2D3. On the 7th day morning, rats were slaughtered for determination of Ca and Pi concentration and phosphate uptake. It showed that:(1) bone Ca and Pi were significant increased compared two low-Pi treatments with 0.6%P or 0.8%P groups under VD restriction(P<0.01);Dietary phosphate levels didn't affected to serum Ca and Pi; but which tend to rise with dietary Pi content (P>0.05).(2) VD、PTH、ALP concentration of serum were significant affected by diet phosphate contents.Compared 0.2%P with 0.8%P group, VD、PTH and ALP of serum were increased 14.35%(P>0.05)、28.83%(P<0.05) and 36.69%(P <0.05), respectively. (3) Dietary phosphate contents also affected to expression of Na+/Pi-Ⅱb mRNA in small intestine. Compared 0.2%P with 0.8%P group, that of ileum、jejunum、duodenum, respectively, increased 2.08 (P<0.01)、1.63 (P<0.05)、1.75 (P<0.05) folds, while decreased 1.73(P<0.05) folds in renal.(4) Dietary phosphate contents also affected to Pi absorption rate when animal shortage of VD(P< 0.05), Pi absorption of two low-Pi treatments, respectively, were 62.24 and 59.21 pmol/mg/60s; the same of normal-Pi or high-Pi treatments were 84.25 and 76.22 pmol/mg/60s, respectively; Compared 0.2%P with 0.6%P group, Pi absorption decreased to 26.12%(P<0.05), this effects appeared to every intestinal segments and kidney. Dietary phosphate significant affected to Pi absorption rate of BBMV, Pi absorption rate decreased with dietary phosphate in intestine, contrasted to that, Pi absorption rate increased with dietary phosphate in renal.
Overall, Experiment II showed that:(1) Low-Pi diet lowered phosphate absorption under VD stress;(2) The main intestinal segment for phosphate absorption is ileum to rat, and Pi absorption lowest in duodenum.
ExperimentⅢ. Effects of Dietary Phosphate Content on Na+/Pi-Ⅱb mRNA Expression and Absorption after Injected 1,25-(OH)2D3
Based on experimentⅡ, mading rat lack of vitamin D firstly, then administration 1,25-(OH)2D3 to them. The aim of the study was to investigate the tendency of Na+/Pi-Ⅱb mRNA and Pi uptake after administrated 1,25-(OH)2D3 toVD restricted rats. As same as experimentⅡ, the former 6 days, injected EHDP still to rats, before 12h before slaughter, rats were injected with 1,25-(OH)2D3 at 600ng/kg.wt. On the 7th day morning, rats were slaughtered for determination of Ca and Pi concentration and phosphate uptake.
Experiment III showed that:(1) Ca and Pi of bone rised with dietary phosphate after administration 1,25-(OH)2D3 to restricted VD rats. Low-Pi diet declined bone Ca and Pi concentration, compared 0.2%P with 0.6%P group, that decreased 3.03 (P< 0.01) and 7.06 times (P<0.01). (2) Serum VD3、PTH、ALP concentration of 0.2%P treatment increased 23.25%(P<0.05)、13.97%(P<0.05)、33.55%(P<0.01) than 0.2%P treatment after injection 1,25-(OH)2D3.(3) Low-Pi diet significantly elevated Na+/Pi-Ⅱb mRNA expression to every intestinal segments or renal, and more 0.2%P trteatment become the highest, namely mRNA expression of 0.2%P was the impacted treatment mostly after injection 1,25-(OH)2D3. But concerning to different segment, it was highest in ileum.Injection 1,25-(OH)2D3 also significant improved Pi absorption (P <0.05), among four phosphates levels, it is highest in ileum and jejunum to 0.2%P group, while contrasted to that it is higher in duodenum and renal to 0.6%P or 0.8%P treatment, namely Pi absorption in duodenum and renal were higher than in ileum and jejunum when phosphate is 0.6%P or 0.8%P. Ileum was the dominant site to Pi uptake, and secondly, jejunum、rena、duodenum. The value were, respectively,129.04、90.95、48.75、39.49 pmol/mg/60s.Based on the above, it showed Ca and Pi of bone increased with dietary Pi regardless of animal VD varied situation; Low-Pi decreased PTH and ALP concentration of serum, especially nuder situation of restricted VD.Injection 1,25-(OH)2D3 increased serum VD concentration and lowed PTH and ALP concentration.(4)Low-Pi stimulated Na+/Pi-Ⅱb mRNA expression, but Pi uptake downed with diet Pi content when VD is shortaged. Administration 1,25-(OH)2D3 to rats increased expression of Na+/Pi-Ⅱb mRNA, and low-Pi treatment increased most.
Dietary phosphate no significant effects to Pi uptake of BBMV after injection 1,25-(OH)2D3, but compared 0.2%P group to 0.6%P group, Pi absorption higher 7.57% (P>0.05)and 12.57%(P>0.05)in ileum and jejunum.While it increased with dietary phosphate in renal which display contrast effects to small intestine.
ExperimentⅢsuggested vitamin D play an important role for regulation low phosphate adaption. The optimal site for phosphorus absorption was ileum, secondly jejunum, then kidney and duodenum.
In conclusion, it suggested that:(1) Low-Pi diet significantly increased Na+/Pi-Ⅱb mRNA expression and Pi absorption in ileum and jejunum, and decreased in renal.0.2% dietary phosphate was a optimal low-Pi content that impacted to the mechanism of phosphate absorption from mRNA expression for rat. (2) Na+/Pi-Ⅱb cotransporter mRNA expression is highest in ileum, while lowest in kideny for rat under low dietary phosphate. Pi absorption or absorption rate presented a similar effects to small intestine and renal, and the main intestinal segment or organ for Pi absorption is ileum, secondly jejunum and renal, lastly duodenum. (3) VD play an important role for regulating animal adaption to low phosphate, and the mechanism for it was consistent with gene thesis. (4) Injection 1,25-(OH)2D3 to restricted VD rats improve Pi uptake, but this effects decreased with increasing dietary phosphate content. (5) The effects of ditary phosphate content and VD3 on Pi absorption is different between small instine and renal.
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