半胱胺对动物生产性能及其营养生理效应研究
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摘要
本论文以大鼠和肥育猪为实验模型,利用营养代谢小分子(分子量小于1000道尔顿)的组学技术和生物大分子的基因表达技术,研究了半胱胺在营养代谢和生理二个层次上的生物效应和分子基础。研究包括三个实验。
试验一,以Sprague-Dawley大鼠为模型,采用一次性腹腔注射150mg/kgBW半胱胺后,收集0-8,8-24h的尿液以及24h血清,测定1H核磁共振谱和采用PCA或OSC-PLS的实验资料分析法,研究了短期外源半胱胺补充(一次性引入)对大鼠体内物质代谢的影响,结果表明：
①短期外源半胱胺(一次性引入)影响大鼠肠道微生物代谢。明显促进血清乙酸浓度,增加了尿液中二甲胺浓度,降低了0-8h尿液中马尿酸的浓度。
②短期外源半胱胺(一次性引入)影响大鼠三羧酸循环代谢。明显表现为0-8h尿液a-酮戊二酸浓度增加、葡萄糖水平降低。半胱胺也降低尿液琥珀酸和柠檬酸浓度。
③短期外源半胱胺(一次性引入)影响氮代谢。引起尿液中肌酐、二甲基甘氨酸(0-8h)、肌酸(8-24h)浓度以及血清中甘氨酸含量明显增加,肌酸(0～8h)浓度降低。
④短期外源半胱胺(一次性引入)影响脂质代谢。明显降低血清脂蛋白、HDL、VLDL、FA、PUFA、UFA、磷酰胆碱浓度。
结论：短期外源半胱胺(一次性引入)影响机体和内源微生物代谢过程。短期半胱胺补充(一次性引入)降低了能量代谢强度,促进了氮代谢,降低了脂肪代谢。
试验二,以Sprague-Dawley大鼠为模型,采用多次(共8次,一周2次,星期一四各一次)腹腔注射150mg/kg BW半胱胺后收集0～8,8～24h的尿液以及24h血清,测定1H核磁共振谱和采用PCA或者OSC-PLS的实验资料分析法,研究了长期外源半胱胺补充(多次性引入)对大鼠体内物质代谢的影响。结果表明：
①长期外源半胱胺(多次性引入)影响肠道微生物代谢。降低血清乙酸、乙醇、氧化三甲胺、甜菜碱、尿液氧化三甲胺及马尿酸浓度,增加尿液中二甲胺的浓度。
②长期外源半胱胺(多次性引入)影响三羧酸循环代谢。减少尿液琥珀酸浓度,降低尿液柠檬酸(8-24h)及尿液中葡萄糖浓度。
③长期外源半胱胺(多次性引入)影响氮代谢。提高尿液中二甲基甘氨酸、肌酐(0-8h)、肌酸(8-24h)、尿液以及血清甘氨酸浓度,降低尿液肌酐(8～24h)浓度以及三甲基组氨酸浓度。
④长期外源半胱胺(多次性引入)影响脂肪代谢。提高血清HDL水平,降低血清UFA、VLDL和脂蛋白水平。
结论：长期外源半胱胺(多次性引入)影响大鼠生理生化参数和小分子物质代谢。减少了三羧酸循环能量代谢强度,降低了蛋白质的降解。刺激微生物的氨基酸和碳水化合物代谢。
试验三,选用24头PIC肥育猪(初始体重为60.05±1.24公斤、阉公猪和母猪各半),随机分成3组,分别饲喂基础日粮(对照),70和140 mg/kg半胱胺的实验处理饲粮,实验47天,研究了外源半胱胺对肥育猪生产性能和类胰岛素生长因子系统的影响。
结果表明：
①饲料添加70 mg/kg外源半胱胺,显著提高肥育猪平均日增重(19.5%)和日采食量(15.2%),不影响料重比。高剂量(140 mg/kg)半胱胺对肥育猪生产性能没有显著影响。
②饲料添加70 mg/kg外源半胱胺显著(P<0.05)提高血清生长激素(GH)水平(43.1%)和类胰岛素生长因子-I(IGF-I)含量(55.2%)；显著上调上调肝脏、胃、肌肉生长激素受体(GHR)的mRNA的表达量,分别可达到90%、140%、120%；显著上调肝脏、胃、肌肉IGF-I的mRNA的表达量,分别可达到66%、65%、62%；显著上调胃、十二指肠、肌肉中类胰岛素生长因子-I受体(IGF-IR)的mRNA表达量(分别可达到31%、346%、38%)和肝脏中类胰岛素生长因子结合蛋白-3 (IGFBP-3)的mRNA表达量(119%)；下调胃、十二指肠、肌肉中IGFBP-3的mRNA表达量可达到41%、42%、48%。不影响肝脏、胃、十二指肠、肌肉中胰岛素受体(IR)的mRNA表达；也不影响肝脏IGF-IR和十二指肠中GHR、IGF-I的mRNA表达。
③饲料添加140 mg/kg外源半胱胺显著(P<0.05)上调肝脏、胃、十二指肠、肌肉中IGFBP-3的mRNA表达量(114%、212%、196%、194%)；显著下调十二指肠中GHR的水平(22%)以及肝脏、胃、十二指肠、肌肉中IGF-I的mRNA表达量(分别达到46%、51%、42%、54%)和IGF-IR的mRNA表达量(82%、69%、63%、64%)。不影响血清GH、IGF-I水平和肝脏、胃、肌肉GHR的mRNA表达。
结论：外源半胱胺调控动物生长、血清IGF-I浓度以及GHR, IGF-I, IGF-IR, IGFBP-3, IR mRNA水平存在显著剂量效应关系。半胱胺调控GHR, IGF-I, IGF-IR, IGFBP-3 mRNA水平有明显地组织特异性。半胱胺促进肥育猪生长,与GH-IGF轴的生理作用有关。
综上所述：调控微生物代谢,外源半胱胺长期使用优于短期使用。外源半胱胺通过GH-IGF生理轴介导,减少了三羧酸循环能量代谢强度,降低了脂肪代谢和蛋白质的降解,调控了微生物的氨基酸和碳水化合物代谢,促进了动物生长。
Three experiments were conducted to investigate growth performance, nutritional and physiological effects of cysteamine in animals.
Exp.l A total of 14 female Sprague-Dawley rats were randomly assigned to one of two groups, with 7 rats per group. One group was given doses of 150 mg/kg body weight cysteamine intraperitoneally. The other group was given physiological saline as control. Collections of urine were made at 0-8 and 8-24 hours and collections of blood were made at 24 hours after the last injection. The 1H NMR spectra were acquired for each sample. PC A or OSC-PLS was performed on all data. The results indicated that
①Acute CS supplementation also changed microbial metabolites concentration (increasing the concentrations of serum acetate and urine dimethylamine, and reducing urine hippuric acid concentration (0～8 hours)).
②Acute CS also changed TCA metabolites (increasing the concentration of urine a-ketoglutaric acid (0～8 hours), reducing the concentration of urine succinic acid, citric acid), decreased the concentration of urine glucose(0～8 hours)).
③Acute CS also affected the metabolism of nitrogen (increasing the concentration of urine creatinine,2-methylglycine (0-8 hours), creatine (8～24 hours) and serum glycine and reducing the concentration of creatine in urine (0～8 hours)).
④Acute CS also affected the fat metabolism (reducing the concentration of serum lipoprotein, HDL, VLDL, FA, PUFA, UFA, phosphorylcholine) Conclusion:Acute CS supplementation changed animal and microorganisms metabolism, decreased energy metabolism, increased nitrogen metabolism and decreased fat incretion.
Exp.2 A total of 20 female SD rats were randomly assigned to one of two groups, with 10 rats per group for 25 days. One group was given doses of 150 mg/kg body weight cysteamine intraperitoneally (biweekly, Monday and Thursday). The other group was given physiological saline intraperitoneally as control. Collections of urine were made at 0-8 and 8-24 hours and collections of blood were made at 24 hours after the last injection. The1H NMR spectra were acquired for each sample. PCA or OSC-PLS was performed on all data. The results indicated that
①Chronic CS supplementation changed microbial metabolites concentration (decreasing serum concentrations of acetate, ethanol, trimethylamine oxide, betaine hippuric acid and urine concentration of trimethylamine oxide; increasing the concentration of dimethylamine in urine).
②Chronic CS changed TCA metabolites (decreasing the concentration of urine succinic acid and reducing the concentration of urine citric acid (8～24 hours)) and decreased the glucose in urine.
③Chronic CS affected the metabolism of nitrogen (increasing the concentration of urine dimethylglycine, creatinine (0 to 8 hours), creatine (8～24 hours) and glycine in urine or serum; reducing the concentration of urine creatinine (8～24 hours) and 3-methylhistidine).
④Chronic CS affected the metabolism of fat (increasing concentration of serum HDL and decreasing the concentration of UFA, VLDL, lipoprotein in urine or serum). Conclusion:Chronic CS changed serum and urine metabolome in rats. Chronic CS decreased energy metabolism (Tricarboxylic acid cycle) and protein breakdown rate. CS can modulate amino acid and carbohydrates metabolism from microbes in animals.
Exp.3 A total of 24 finishing pigs (60.05±1.24 kg; 12 gilts and 12 barrows) were assigned randomly to one of the three dietary groups, with four pens/group (per pen:one gilt, one barrow). The pigs were fed a basal diet containing 0 (control),70 or 140 mg/kg cysteamine feed additive for 47 days. The results indicated that
①the ADG and ADFI (70 mg/kg) were increased (P<0.05) by 19.5%,15.2%, respectively; however, the ADG and ADFI (140 mg/kg) and feed efficiency were not affected.
②CS supplementation (70 mg/kg) increased serum IGF-I level, upregulated mRNA levels of GHR and IGF-I (liver, stomach, muscle), IGF-IR (stomach, duodenum, muscle) and IGFBP-3 (liver), but downregulated IGFBP-3 (stomach, duodenum, muscle). CS supplementation (70 mg/kg) did not affect mRNA levels of GHR and IGF-I (duodenum), IGF-IR (liver), and IR (liver, stomach, duodenum, muscle).
③CS supplementation (140 mg/kg) downregulated GHR (duodenum), IGF-I and IGF-IR mRNA (liver, stomach, duodenum, muscle), but upregulated IGFBP-3 and IR mRNA (liver, stomach, duodenum, muscle) and did not affect ADG, and serum IGF-I concentration.
Conclusion:the results suggest that dietary CS supplementation modulates the growth rate, serum IGF-I concentrations, and the gene expression of GHR, IGF-I, IGF-IR, IGFBP-3, and IR in a dose-dependent manner. CS supplementation has tissue-specific regulation of GHR, IGF-I, IGF-IR, and IGFBP-3 mRNA levels. Moreover, the results also imply the physiologic role of the GH-IGF axis in mediating the dietary CS supplementation-supported growth of finishing pigs.
Collectively, Chronic CS is better than acute CS in modulating microbe metabolism. CS can decrease energy metabolism (Tricarboxylic acid cycle), decrease fat incretion and protein breakdown rate, modulate amino acid and carbohydrates metabolism from microbes and thus mediate the dietary CS supplementation-supported growth of animals through GH-IGF axis.
试验一,以Sprague-Dawley大鼠为模型,采用一次性腹腔注射150mg/kgBW半胱胺后,收集0-8,8-24h的尿液以及24h血清,测定1H核磁共振谱和采用PCA或OSC-PLS的实验资料分析法,研究了短期外源半胱胺补充(一次性引入)对大鼠体内物质代谢的影响,结果表明：
①短期外源半胱胺(一次性引入)影响大鼠肠道微生物代谢。明显促进血清乙酸浓度,增加了尿液中二甲胺浓度,降低了0-8h尿液中马尿酸的浓度。
②短期外源半胱胺(一次性引入)影响大鼠三羧酸循环代谢。明显表现为0-8h尿液a-酮戊二酸浓度增加、葡萄糖水平降低。半胱胺也降低尿液琥珀酸和柠檬酸浓度。
③短期外源半胱胺(一次性引入)影响氮代谢。引起尿液中肌酐、二甲基甘氨酸(0-8h)、肌酸(8-24h)浓度以及血清中甘氨酸含量明显增加,肌酸(0～8h)浓度降低。
④短期外源半胱胺(一次性引入)影响脂质代谢。明显降低血清脂蛋白、HDL、VLDL、FA、PUFA、UFA、磷酰胆碱浓度。
结论：短期外源半胱胺(一次性引入)影响机体和内源微生物代谢过程。短期半胱胺补充(一次性引入)降低了能量代谢强度,促进了氮代谢,降低了脂肪代谢。
试验二,以Sprague-Dawley大鼠为模型,采用多次(共8次,一周2次,星期一四各一次)腹腔注射150mg/kg BW半胱胺后收集0～8,8～24h的尿液以及24h血清,测定1H核磁共振谱和采用PCA或者OSC-PLS的实验资料分析法,研究了长期外源半胱胺补充(多次性引入)对大鼠体内物质代谢的影响。结果表明：
①长期外源半胱胺(多次性引入)影响肠道微生物代谢。降低血清乙酸、乙醇、氧化三甲胺、甜菜碱、尿液氧化三甲胺及马尿酸浓度,增加尿液中二甲胺的浓度。
②长期外源半胱胺(多次性引入)影响三羧酸循环代谢。减少尿液琥珀酸浓度,降低尿液柠檬酸(8-24h)及尿液中葡萄糖浓度。
③长期外源半胱胺(多次性引入)影响氮代谢。提高尿液中二甲基甘氨酸、肌酐(0-8h)、肌酸(8-24h)、尿液以及血清甘氨酸浓度,降低尿液肌酐(8～24h)浓度以及三甲基组氨酸浓度。
④长期外源半胱胺(多次性引入)影响脂肪代谢。提高血清HDL水平,降低血清UFA、VLDL和脂蛋白水平。
结论：长期外源半胱胺(多次性引入)影响大鼠生理生化参数和小分子物质代谢。减少了三羧酸循环能量代谢强度,降低了蛋白质的降解。刺激微生物的氨基酸和碳水化合物代谢。
试验三,选用24头PIC肥育猪(初始体重为60.05±1.24公斤、阉公猪和母猪各半),随机分成3组,分别饲喂基础日粮(对照),70和140 mg/kg半胱胺的实验处理饲粮,实验47天,研究了外源半胱胺对肥育猪生产性能和类胰岛素生长因子系统的影响。
结果表明：
①饲料添加70 mg/kg外源半胱胺,显著提高肥育猪平均日增重(19.5%)和日采食量(15.2%),不影响料重比。高剂量(140 mg/kg)半胱胺对肥育猪生产性能没有显著影响。
②饲料添加70 mg/kg外源半胱胺显著(P<0.05)提高血清生长激素(GH)水平(43.1%)和类胰岛素生长因子-I(IGF-I)含量(55.2%)；显著上调上调肝脏、胃、肌肉生长激素受体(GHR)的mRNA的表达量,分别可达到90%、140%、120%；显著上调肝脏、胃、肌肉IGF-I的mRNA的表达量,分别可达到66%、65%、62%；显著上调胃、十二指肠、肌肉中类胰岛素生长因子-I受体(IGF-IR)的mRNA表达量(分别可达到31%、346%、38%)和肝脏中类胰岛素生长因子结合蛋白-3 (IGFBP-3)的mRNA表达量(119%)；下调胃、十二指肠、肌肉中IGFBP-3的mRNA表达量可达到41%、42%、48%。不影响肝脏、胃、十二指肠、肌肉中胰岛素受体(IR)的mRNA表达；也不影响肝脏IGF-IR和十二指肠中GHR、IGF-I的mRNA表达。
③饲料添加140 mg/kg外源半胱胺显著(P<0.05)上调肝脏、胃、十二指肠、肌肉中IGFBP-3的mRNA表达量(114%、212%、196%、194%)；显著下调十二指肠中GHR的水平(22%)以及肝脏、胃、十二指肠、肌肉中IGF-I的mRNA表达量(分别达到46%、51%、42%、54%)和IGF-IR的mRNA表达量(82%、69%、63%、64%)。不影响血清GH、IGF-I水平和肝脏、胃、肌肉GHR的mRNA表达。
结论：外源半胱胺调控动物生长、血清IGF-I浓度以及GHR, IGF-I, IGF-IR, IGFBP-3, IR mRNA水平存在显著剂量效应关系。半胱胺调控GHR, IGF-I, IGF-IR, IGFBP-3 mRNA水平有明显地组织特异性。半胱胺促进肥育猪生长,与GH-IGF轴的生理作用有关。
综上所述：调控微生物代谢,外源半胱胺长期使用优于短期使用。外源半胱胺通过GH-IGF生理轴介导,减少了三羧酸循环能量代谢强度,降低了脂肪代谢和蛋白质的降解,调控了微生物的氨基酸和碳水化合物代谢,促进了动物生长。
Three experiments were conducted to investigate growth performance, nutritional and physiological effects of cysteamine in animals.
Exp.l A total of 14 female Sprague-Dawley rats were randomly assigned to one of two groups, with 7 rats per group. One group was given doses of 150 mg/kg body weight cysteamine intraperitoneally. The other group was given physiological saline as control. Collections of urine were made at 0-8 and 8-24 hours and collections of blood were made at 24 hours after the last injection. The 1H NMR spectra were acquired for each sample. PC A or OSC-PLS was performed on all data. The results indicated that
①Acute CS supplementation also changed microbial metabolites concentration (increasing the concentrations of serum acetate and urine dimethylamine, and reducing urine hippuric acid concentration (0～8 hours)).
②Acute CS also changed TCA metabolites (increasing the concentration of urine a-ketoglutaric acid (0～8 hours), reducing the concentration of urine succinic acid, citric acid), decreased the concentration of urine glucose(0～8 hours)).
③Acute CS also affected the metabolism of nitrogen (increasing the concentration of urine creatinine,2-methylglycine (0-8 hours), creatine (8～24 hours) and serum glycine and reducing the concentration of creatine in urine (0～8 hours)).
④Acute CS also affected the fat metabolism (reducing the concentration of serum lipoprotein, HDL, VLDL, FA, PUFA, UFA, phosphorylcholine) Conclusion:Acute CS supplementation changed animal and microorganisms metabolism, decreased energy metabolism, increased nitrogen metabolism and decreased fat incretion.
Exp.2 A total of 20 female SD rats were randomly assigned to one of two groups, with 10 rats per group for 25 days. One group was given doses of 150 mg/kg body weight cysteamine intraperitoneally (biweekly, Monday and Thursday). The other group was given physiological saline intraperitoneally as control. Collections of urine were made at 0-8 and 8-24 hours and collections of blood were made at 24 hours after the last injection. The1H NMR spectra were acquired for each sample. PCA or OSC-PLS was performed on all data. The results indicated that
①Chronic CS supplementation changed microbial metabolites concentration (decreasing serum concentrations of acetate, ethanol, trimethylamine oxide, betaine hippuric acid and urine concentration of trimethylamine oxide; increasing the concentration of dimethylamine in urine).
②Chronic CS changed TCA metabolites (decreasing the concentration of urine succinic acid and reducing the concentration of urine citric acid (8～24 hours)) and decreased the glucose in urine.
③Chronic CS affected the metabolism of nitrogen (increasing the concentration of urine dimethylglycine, creatinine (0 to 8 hours), creatine (8～24 hours) and glycine in urine or serum; reducing the concentration of urine creatinine (8～24 hours) and 3-methylhistidine).
④Chronic CS affected the metabolism of fat (increasing concentration of serum HDL and decreasing the concentration of UFA, VLDL, lipoprotein in urine or serum). Conclusion:Chronic CS changed serum and urine metabolome in rats. Chronic CS decreased energy metabolism (Tricarboxylic acid cycle) and protein breakdown rate. CS can modulate amino acid and carbohydrates metabolism from microbes in animals.
Exp.3 A total of 24 finishing pigs (60.05±1.24 kg; 12 gilts and 12 barrows) were assigned randomly to one of the three dietary groups, with four pens/group (per pen:one gilt, one barrow). The pigs were fed a basal diet containing 0 (control),70 or 140 mg/kg cysteamine feed additive for 47 days. The results indicated that
①the ADG and ADFI (70 mg/kg) were increased (P<0.05) by 19.5%,15.2%, respectively; however, the ADG and ADFI (140 mg/kg) and feed efficiency were not affected.
②CS supplementation (70 mg/kg) increased serum IGF-I level, upregulated mRNA levels of GHR and IGF-I (liver, stomach, muscle), IGF-IR (stomach, duodenum, muscle) and IGFBP-3 (liver), but downregulated IGFBP-3 (stomach, duodenum, muscle). CS supplementation (70 mg/kg) did not affect mRNA levels of GHR and IGF-I (duodenum), IGF-IR (liver), and IR (liver, stomach, duodenum, muscle).
③CS supplementation (140 mg/kg) downregulated GHR (duodenum), IGF-I and IGF-IR mRNA (liver, stomach, duodenum, muscle), but upregulated IGFBP-3 and IR mRNA (liver, stomach, duodenum, muscle) and did not affect ADG, and serum IGF-I concentration.
Conclusion:the results suggest that dietary CS supplementation modulates the growth rate, serum IGF-I concentrations, and the gene expression of GHR, IGF-I, IGF-IR, IGFBP-3, and IR in a dose-dependent manner. CS supplementation has tissue-specific regulation of GHR, IGF-I, IGF-IR, and IGFBP-3 mRNA levels. Moreover, the results also imply the physiologic role of the GH-IGF axis in mediating the dietary CS supplementation-supported growth of finishing pigs.
Collectively, Chronic CS is better than acute CS in modulating microbe metabolism. CS can decrease energy metabolism (Tricarboxylic acid cycle), decrease fat incretion and protein breakdown rate, modulate amino acid and carbohydrates metabolism from microbes and thus mediate the dietary CS supplementation-supported growth of animals through GH-IGF axis.
引文
[1]Millard WJ, Sagar SM, Martin JB. Cysteamine induced depletion of somatostatin and prolactin. Federation Proceedings,1988,44:2546-2550.
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