冷应激对鸡肝脏脂肪代谢与炎性因子的影响
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摘要
寒冷是北方地区动物面临的主要应激源,研究表明寒冷地区冷应激可直接影响畜禽的健康及福利。低温可以增加家禽血清中游离脂肪酸、尿酸水平,降低血糖水平、脂质代谢增强。免疫反应和代谢调节高度统一,功能上相互依赖。冷应激后机体大量的能量被用于分解代谢,而保障供给免疫和炎症的活化的能量减少,使机体产生代谢性炎症。本实验探讨了冷应激对鸡肝脏脂质代谢和炎性因子的影响,应用基因克隆技术在原核表达系统中表达了PPARα和AMPKα并制备了多克隆抗体,以雏鸡为研究对象,进行急、慢性冷应激(12±1℃)处理,检测了血清生化指标、肝脏脂联素、PPARα、AMPKα等脂质代谢相关基因表达及含量、肝脏炎症相关基因表达及含量、肝脏热休克蛋白基因表达。结果表明：
1.利用原核表达载体表达PPARα和AMPKα融合蛋白免疫新西兰白兔,成功制备了兔抗鸡PPARα和AMPKα多克隆抗体。
2.急、慢性冷应激可引起鸡血清FFA、TG、GHO、LDL、HDL含量和ALT活性改变,提示冷应激能够使机体脂质代谢增加,为机体提供能量供应;还表明冷应激可引起肝脏的损伤。
3.急、慢性冷应激可引起鸡肝脏脂联素、AdipoR1和AdipoR2含量以及mRNA表达量增加,提示冷应激可能通过脂联素途径增加机体脂质代谢。
4.急、慢性冷应激时,鸡肝脏PPARα、AMPKα蛋白表达增加,肝脏AMPKα1、AMPKα2、 CPT1、ACAO含量增加,肝脏ACC、丙二酰辅酶A含量降低,肝脏PPARα、LXRα、AMPKα1、 AMPKα2、CPT1mRNA表达增加,肝脏ACC mRNA表达降低,提示冷应激可能通过脂联素-PPARα-AMPKα途径增加机体脂质代谢。
5.急、慢性冷应激时,鸡肝脏炎性基因PGE synthase、Cox-2、NF-kB蛋白表达增加,肝脏PGE synthase、Cox-2、NF-kB、TNFα、iNOS、HO-1mRNA表达增加,提示这些基因的表达可能参与了冷应激致鸡肝脏炎症的过程。
6.急、慢性冷应激时,鸡肝脏HSP70、GRP78蛋白及nRNA表达量升高,并首次证实,急、慢性冷应激可引起鸡肝脏HSP60、HSP90mRNA表达增加。提示机体可以通过HSP途径抵抗冷应激致鸡肝脏的损伤。
综上所述,冷应激可通过脂联素-PPARa-AMPKa途径调节鸡肝脏脂质代谢,并引起ALT活性增加,伴有肝脏PGE synthase、Cox-2、NF-kB、TNFα、iNOS、HO-1)和HSP70、GRP78、 HSP60、HSP90表达上调,表明冷应激可致鸡脂质代谢紊乱、肝脏损伤,并伴随着炎症相关基因和热休克蛋白基因的上调,为探讨冷应激致鸡代谢性炎症的机制提供理论基础和实验依据。
Cold stress is the most common stress factor to the animals in the cold region. Studies have demonstrated that cold stress could affect the health and welfare of animals. Low temperature can increase the free fatty acids and trioxypurine levels in serum, decrease the level of blood glucose and enhance the lipid metabolism in poultry. Immune response and metabolic regulation have highly unity and functional interdependence. During cold stress large amounts of body energy is used for anabolic, however the energy to protect the supply of immune and inflammatory activation reduced, which is related to metabolic inflammation. In this study, coding sequences of PPARa and AMPKa were cloned by the methods of molecular biology. As the experimental animals, chickens were treated with acute and chronic cold stress (12±1℃) to investigate the effect of cold stress on lipid metabolism and inflammatory factors in chicken liver. Biochemical indicator in serum, the expression and content of adiponectin, PPARα, AMPKα and lipid metabolism-related genes in liver, and the expression of inflammation-related and HSP genes in liver were detected. The results showed as follows:
1. Fusion proteins of PPARα and AMPKα were expressed in prokaryotic system. The polyclonal antibodies were prepared by using expressed fusion proteins.
2. Acute and chronic cold stress could influence the content of FFA, TG, GHO, LDL, HDL and the activity of ALT in serum. It indicated that cold stress could accelerate fat dissolution to provide the energy for the body. It also indicated that cold stress could cause liver damage.
3. Acute and chronic cold stress could increase the adiponectin and its receptors' mRNA expression and concentration in liver. It indicated that cold stress might change the liver lipid metabolism through adiponectin pathway in chickens.
4. Acute and chronic cold stress could increase the expression of PPARα, AMPKα protein in liver, increase the concentration of AMPKα1, AMPKα2, CPT1, ACAO and decrease the concentration of ACC, malonyl CoA in liver, increase the expression of PPARα, LXRα, AMPKα1, AMPKα2, CPT1mRNA and decrease the expression of ACC mRNA in liver. It indicated that cold stress might change the liver lipid metabolism through adiponectin-PPARα-AMPKα pathway in chickens.
5. Acute and chronic cold stress could increase the expression of PGE synthase, Cox-2, NF-κB protein and the expression of PGE synthase, Cox-2, NF-κB, TNFα, iNOS, HO-1mRNA in liver. It indicated that these genes might be involved in the process of liver inflammation of chicken caused by cold stress.
6. Acute and chronic cold stress could increase the HSP70and GRP78' mRNA and protein expression and we first verified that acute and chronic cold stress could increase the HSP60and HSP90' mRNA expression. It indicated that organism could resistance to the damage in chicken's liver through HSP pathway caused by cold stress.
In present study, it indicated that cold stress could change the liver lipid metabolism through adiponectin-PPARα-AMPKα pathway and increase the activity of ALT in chickens. It also clarified that cold stress could increase the expression of PGE synthase, Cox-2, NF-kB, TNFα, iNOS, HO-1gene and HSP gene and the expression of HSP70, GRP78, HSP60, HSP90. It indicated that cold stress could cause lipid metabolism disorders, liver damage and accompanied with increasing in inflammation-related genes and heat shock protein gene. This study provides theoretical basis and experimental evidence for exploring the mechanisms of chicken metabolic inflammation caused by cold stress.
1.利用原核表达载体表达PPARα和AMPKα融合蛋白免疫新西兰白兔,成功制备了兔抗鸡PPARα和AMPKα多克隆抗体。
2.急、慢性冷应激可引起鸡血清FFA、TG、GHO、LDL、HDL含量和ALT活性改变,提示冷应激能够使机体脂质代谢增加,为机体提供能量供应;还表明冷应激可引起肝脏的损伤。
3.急、慢性冷应激可引起鸡肝脏脂联素、AdipoR1和AdipoR2含量以及mRNA表达量增加,提示冷应激可能通过脂联素途径增加机体脂质代谢。
4.急、慢性冷应激时,鸡肝脏PPARα、AMPKα蛋白表达增加,肝脏AMPKα1、AMPKα2、 CPT1、ACAO含量增加,肝脏ACC、丙二酰辅酶A含量降低,肝脏PPARα、LXRα、AMPKα1、 AMPKα2、CPT1mRNA表达增加,肝脏ACC mRNA表达降低,提示冷应激可能通过脂联素-PPARα-AMPKα途径增加机体脂质代谢。
5.急、慢性冷应激时,鸡肝脏炎性基因PGE synthase、Cox-2、NF-kB蛋白表达增加,肝脏PGE synthase、Cox-2、NF-kB、TNFα、iNOS、HO-1mRNA表达增加,提示这些基因的表达可能参与了冷应激致鸡肝脏炎症的过程。
6.急、慢性冷应激时,鸡肝脏HSP70、GRP78蛋白及nRNA表达量升高,并首次证实,急、慢性冷应激可引起鸡肝脏HSP60、HSP90mRNA表达增加。提示机体可以通过HSP途径抵抗冷应激致鸡肝脏的损伤。
综上所述,冷应激可通过脂联素-PPARa-AMPKa途径调节鸡肝脏脂质代谢,并引起ALT活性增加,伴有肝脏PGE synthase、Cox-2、NF-kB、TNFα、iNOS、HO-1)和HSP70、GRP78、 HSP60、HSP90表达上调,表明冷应激可致鸡脂质代谢紊乱、肝脏损伤,并伴随着炎症相关基因和热休克蛋白基因的上调,为探讨冷应激致鸡代谢性炎症的机制提供理论基础和实验依据。
Cold stress is the most common stress factor to the animals in the cold region. Studies have demonstrated that cold stress could affect the health and welfare of animals. Low temperature can increase the free fatty acids and trioxypurine levels in serum, decrease the level of blood glucose and enhance the lipid metabolism in poultry. Immune response and metabolic regulation have highly unity and functional interdependence. During cold stress large amounts of body energy is used for anabolic, however the energy to protect the supply of immune and inflammatory activation reduced, which is related to metabolic inflammation. In this study, coding sequences of PPARa and AMPKa were cloned by the methods of molecular biology. As the experimental animals, chickens were treated with acute and chronic cold stress (12±1℃) to investigate the effect of cold stress on lipid metabolism and inflammatory factors in chicken liver. Biochemical indicator in serum, the expression and content of adiponectin, PPARα, AMPKα and lipid metabolism-related genes in liver, and the expression of inflammation-related and HSP genes in liver were detected. The results showed as follows:
1. Fusion proteins of PPARα and AMPKα were expressed in prokaryotic system. The polyclonal antibodies were prepared by using expressed fusion proteins.
2. Acute and chronic cold stress could influence the content of FFA, TG, GHO, LDL, HDL and the activity of ALT in serum. It indicated that cold stress could accelerate fat dissolution to provide the energy for the body. It also indicated that cold stress could cause liver damage.
3. Acute and chronic cold stress could increase the adiponectin and its receptors' mRNA expression and concentration in liver. It indicated that cold stress might change the liver lipid metabolism through adiponectin pathway in chickens.
4. Acute and chronic cold stress could increase the expression of PPARα, AMPKα protein in liver, increase the concentration of AMPKα1, AMPKα2, CPT1, ACAO and decrease the concentration of ACC, malonyl CoA in liver, increase the expression of PPARα, LXRα, AMPKα1, AMPKα2, CPT1mRNA and decrease the expression of ACC mRNA in liver. It indicated that cold stress might change the liver lipid metabolism through adiponectin-PPARα-AMPKα pathway in chickens.
5. Acute and chronic cold stress could increase the expression of PGE synthase, Cox-2, NF-κB protein and the expression of PGE synthase, Cox-2, NF-κB, TNFα, iNOS, HO-1mRNA in liver. It indicated that these genes might be involved in the process of liver inflammation of chicken caused by cold stress.
6. Acute and chronic cold stress could increase the HSP70and GRP78' mRNA and protein expression and we first verified that acute and chronic cold stress could increase the HSP60and HSP90' mRNA expression. It indicated that organism could resistance to the damage in chicken's liver through HSP pathway caused by cold stress.
In present study, it indicated that cold stress could change the liver lipid metabolism through adiponectin-PPARα-AMPKα pathway and increase the activity of ALT in chickens. It also clarified that cold stress could increase the expression of PGE synthase, Cox-2, NF-kB, TNFα, iNOS, HO-1gene and HSP gene and the expression of HSP70, GRP78, HSP60, HSP90. It indicated that cold stress could cause lipid metabolism disorders, liver damage and accompanied with increasing in inflammation-related genes and heat shock protein gene. This study provides theoretical basis and experimental evidence for exploring the mechanisms of chicken metabolic inflammation caused by cold stress.
引文
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