肾虚免疫低下大鼠EPO的变化及右归饮与外源性EPO的逆转作用
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摘要
观察腺嘌呤致肾虚免疫低下大鼠中促红细胞生成素(EPO)的变化及右归饮与外源性EPO的逆转作用。该研究将SD大鼠随机分为正常组20只与造模组90只,造模组用腺嘌呤150 mg·kg~(-1)连续灌胃14 d后再随机分为8组,模型组20只,右归饮10,20,40 g·kg~(-1)组各10只,rh EPO 500,1 000,1 500 IU·kg~(-1)组各10只,龟鹿二仙膏10 g·kg~(-1)组10只。第15天开始,各组每隔1日给予150 mg·kg~(-1)腺嘌呤灌胃维持模型,同时右归饮各组与龟鹿二仙膏组灌胃给药每日1次,EPO各组颈部皮下注射rh EPO每3日1次,连续30 d。第46天麻醉取血,处死,检测血清中肌酐、尿素、腺体激素、免疫球蛋白、补体、白介素的含量,脾脏中T细胞亚群比例、NK细胞杀伤率及脾细胞的增殖能力。免疫印迹法分别检测肾脏与脾脏中EPO介导的JAK2-STAT5及NF-κB通路中关键蛋白的含量。结果发现,与正常组相比,模型组大鼠血清中肌酐和尿素含量显著上升,ACTH,T,T3含量显著下降,说明大鼠肾功能及肾上腺、性腺、甲状腺功能减退;同时血清中Ig A,Ig G,Ig M,C3,C4,IL-2,IL-6含量均显著下降,脾脏中CD4~+,CD4~+/CD8~+T细胞亚群比例、NK细胞杀伤率及脾淋巴细胞增殖能力显著下降,显示模型组大鼠的免疫功能减退,判定肾虚免疫低下模型构建成功。与模型组相比,右归饮和rh EPO各剂量组均能不同程度地降低大鼠血清中肌酐和尿素含量,增加血清中ACTH,T,T3,T4,Ig A,Ig G,Ig M,C3,C4,IL-2,IL-6的含量,增加脾脏中CD4~+,CD4~+/CD8~+,NK细胞杀伤率及脾淋巴细胞增殖能力。右归饮能显著增加血清中EPO的含量。右归饮和rh EPO各剂量组均能显著调节肾脏与脾脏中EPO介导的JAK2-STAT5及NF-κB通路中关键蛋白EPOR,P-JAK2/JAK2,STAT5,NF-κB p50,NF-κB p65,NF-κB IκB的表达。研究表明,EPO是腺嘌呤致大鼠肾虚免疫低下的重要相关因子;外源性EPO和右归饮对该模型异常指标均有显著的逆转作用;右归饮作用机制可能与上调血清中EPO的含量,并调节肾脏脾脏中EPO介导的JAK2-STAT5及NF-κB通路中的关键蛋白表达有关;外源性EPO的作用机制可能与调节肾脏脾脏中EPO介导的JAK2-STAT5及NF-κB通路中的关键蛋白表达有关。
The aim of this paper was to observe the changes of EPO in rats with chronic renal failure and low immunity induced by adenine and to investigate the reversal effect of Yougui Yin(YGY)and exogenous EPO.SD rats were randomly divided into normal control group(n=20)and adenine-model group(n=90).The adenine-model group rats were given with adenine 150 mg·kg~(-1)for 14days by gavage administration,and then randomly divided into 8 groups as follows:model group(n=20),YGY groups(10,20,40 g·kg~(-1),10 in each group),rh EPO group(500,1 000,1 500 IU·kg~(-1),10 in each group),and Guilu Erxian Gao 10 g·kg~(-1)group(positive control group,n=10).From the 15th day,every group except normal control group received 150 mg·kg~(-1)adenine by gavage administration once every two days to maintain the model.Meanwhile,the rats in each YGY group and Guilu Erxian Gao group received corresponding drugs by gavage administration once a day for 30 days.The rats in rh EPO groups were subcutaneously injected with rh E-PO once every 3 days for 30 days.On day 46,rats were anesthetized to take blood and then sacrificed.The serum levels of creatinine,urea,glandular hormone,immunoglobulin,complement and interleukin,the proportion of T cells in the spleen,the killing rate of NKcells and the proliferative capacity of spleen cells were measured.Western blot was used to detect the key proteins in JAK2-STAT5 and NF-κB pathways mediated by EPO in kidney and spleen.As compared with the normal control group,the serum levels of CREA and UREA were increased significantly and the serum levels of ACTH,T and T3 were decreased significantly in the model group rats,indicating that the functions of kidney,adrenal gland,gonad and thyroid in rats were decreased.At the same time,the serum levels of Ig A,Ig G,Ig M,C3,C4,IL-2 and IL-6 were significantly decreased,the proportion of CD4~+,CD4~+/CD8~+T cell subsets,the killing rate of NK cell and the proliferation ability of spleen lymphocyte in spleen of the model group rats were significantly declined,indicating that the immune function of model group rats was decreased,and the model of kidney deficiency immunodeficiency was successfully constructed.As compared with the model group,both YGY and rh EPO significantly reduced serum levels of CREA and UREA,significantly increased serum levels of ACTH,T,T3,T4,Ig A,Ig G,Ig M,C3,C4,IL-2,and IL-6,increased the proportion of CD4~+,CD4~+/CD8~+T cell subsets,the killing rate of NK cell and the proliferation ability of spleen lymphocyte in spleen.YGY could significantly increase the content of EPO in serum.Both YGY and rh EPO could regulate the expression of EPOR,p-JAK2/JAK2,STAT5,NF-κB p50,NF-κB p65 and NF-κB IκB of EPO-mediated JAK2-STAT5 and NF-κB pathways in kidney and spleen.EPO is an important factor in the chronic renal failure and low immunity induced by adenine in rats.Exogenous EPO and YGY have significant reversal effects for the model rats.The mechanism of YGY may be related to the up-regulation of EPO in serum and regulating the expression of key proteins in EPO-mediated JAK2-STAT5 and NF-κB pathways in kidney and spleen.The mechanism of exogenous EPO may be related to regulating the expression of the key proteins in EPO-mediated JAK2-STAT5 and NF-κB pathways in kidney and spleen.
The aim of this paper was to observe the changes of EPO in rats with chronic renal failure and low immunity induced by adenine and to investigate the reversal effect of Yougui Yin(YGY)and exogenous EPO.SD rats were randomly divided into normal control group(n=20)and adenine-model group(n=90).The adenine-model group rats were given with adenine 150 mg·kg~(-1)for 14days by gavage administration,and then randomly divided into 8 groups as follows:model group(n=20),YGY groups(10,20,40 g·kg~(-1),10 in each group),rh EPO group(500,1 000,1 500 IU·kg~(-1),10 in each group),and Guilu Erxian Gao 10 g·kg~(-1)group(positive control group,n=10).From the 15th day,every group except normal control group received 150 mg·kg~(-1)adenine by gavage administration once every two days to maintain the model.Meanwhile,the rats in each YGY group and Guilu Erxian Gao group received corresponding drugs by gavage administration once a day for 30 days.The rats in rh EPO groups were subcutaneously injected with rh E-PO once every 3 days for 30 days.On day 46,rats were anesthetized to take blood and then sacrificed.The serum levels of creatinine,urea,glandular hormone,immunoglobulin,complement and interleukin,the proportion of T cells in the spleen,the killing rate of NKcells and the proliferative capacity of spleen cells were measured.Western blot was used to detect the key proteins in JAK2-STAT5 and NF-κB pathways mediated by EPO in kidney and spleen.As compared with the normal control group,the serum levels of CREA and UREA were increased significantly and the serum levels of ACTH,T and T3 were decreased significantly in the model group rats,indicating that the functions of kidney,adrenal gland,gonad and thyroid in rats were decreased.At the same time,the serum levels of Ig A,Ig G,Ig M,C3,C4,IL-2 and IL-6 were significantly decreased,the proportion of CD4~+,CD4~+/CD8~+T cell subsets,the killing rate of NK cell and the proliferation ability of spleen lymphocyte in spleen of the model group rats were significantly declined,indicating that the immune function of model group rats was decreased,and the model of kidney deficiency immunodeficiency was successfully constructed.As compared with the model group,both YGY and rh EPO significantly reduced serum levels of CREA and UREA,significantly increased serum levels of ACTH,T,T3,T4,Ig A,Ig G,Ig M,C3,C4,IL-2,and IL-6,increased the proportion of CD4~+,CD4~+/CD8~+T cell subsets,the killing rate of NK cell and the proliferation ability of spleen lymphocyte in spleen.YGY could significantly increase the content of EPO in serum.Both YGY and rh EPO could regulate the expression of EPOR,p-JAK2/JAK2,STAT5,NF-κB p50,NF-κB p65 and NF-κB IκB of EPO-mediated JAK2-STAT5 and NF-κB pathways in kidney and spleen.EPO is an important factor in the chronic renal failure and low immunity induced by adenine in rats.Exogenous EPO and YGY have significant reversal effects for the model rats.The mechanism of YGY may be related to the up-regulation of EPO in serum and regulating the expression of key proteins in EPO-mediated JAK2-STAT5 and NF-κB pathways in kidney and spleen.The mechanism of exogenous EPO may be related to regulating the expression of the key proteins in EPO-mediated JAK2-STAT5 and NF-κB pathways in kidney and spleen.
引文
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[11]李永勇,张志妮,梁昌盛,等.五味健体口服液对免疫低下小鼠免疫功能的影响[J].中药材,2018(9):1963.
[12]李崭,刘仁慧,康学,等.肾虚衰老模型的建立及其与D-半乳糖衰老模型的比较研究[J].中国中药杂志,2012,37(16):2435.
[13]黄茂林,林伟明,林晓玲,等.辣木叶提取物的制备工艺优化及其对免疫抑制小鼠免疫功能调节的研究[J].中国中药杂志,2018,43(13):2697.
[14]刘永琦,王文.虚证的免疫学本质[J].中国中医基础医学杂志,2003(5):7.
[15]Yokozawa T,Zheng P D,Oura H,et al.Animal model of adenine-induced chronic renal failure in rats[J].Nephron,1986,44(3):230.
[16]Yokozawa T,Chung H Y,Oura H.Urinary constituents and renal function in rats administered with adenine[J].Nihon Jinzo Gakkai Shi,1987,29(9):1129.
[17]童骏峰,徐志伟,杨元宵,等.腺嘌呤与氢化可的松所致大鼠肾阳虚证模型比较研究[J].中华中医药杂志,2015,30(11):3901.
[18]董爱国,霍俊凤,王秀文,等.不同提取工艺生殖营养方对腺嘌呤所致小鼠肾功能损伤的影响[J].中国实验方剂学杂志,2013,19(9):241.
[19]陈俊蓉,陈利国,谢林林.关于腺嘌呤慢性肾衰实验模型的思考[J].实验动物科学,2013,30(2):65.
[20]李许.EPO与“肾精”相似性的体内验证及“补肾益精”中药健脑作用机制研究[D].重庆:西南大学,2016.
[21]李燕君.EPO与肾虚大鼠脑组织神经细胞的关系及右归饮的保护作用研究[D].重庆:西南大学,2015.
[22]杨盛.EPO促脑神经血管单元细胞生长及补肾益精方药促EPO生成作用研究[D].重庆:西南大学,2016.
[23]Jelkmann W.The enigma of the metabolic fate of circulating erythropoietin(Epo)in view of the pharmacokinetics of the recombinant drugs rhEpo and NESP[J].Eur J Haematol,2015,69(5/6):265.
[24]袁颖,马慧,孙璇,等.重组人促红细胞生成素(rhEPO)在老年慢性病贫血(Ac D)中治疗效果的评价及疗效的预测[J].复旦学报:医学版,2013,40(6):724.
[25]Lee Yoon Mi,Choi Jun Ha,Min Wan Kwon,et al.Induction of functional erythropoietin and erythropoietin receptor gene expression by gamma-aminobutyric acid and piperine in kidney epithelial cells[J].Life Sci,2018,215:207.
[2]T9gel F E,Ahlstrom J D,Yang Y,et al.Carbamylated erythropoietin outperforms erythropoietin in the treatment of AKI-onCKD and other AKI models[J].J Am Soc Nephrol,2016,27(11):3394.
[3]Stoyanoff Tania Romina,Rodríguez Juan Pablo,Todaro Juan Santiago,et al.Erythropoietin attenuates LPS-induced microvascular damage in a murine model of septic acute kidney injury[J].Biomed Pharmacother,2018,107:1046.
[4]Hertzberg-Bigelman E,Barashi R,Levy R,et al.Down-regulation of cardiac erythropoietin receptor and its downstream activated signal transducer phospho-STAT-5 in a rat model of chronic kidney disease[J].Isr Med Assoc J,2016,18(6):326.
[5]Cravedi P,Manrique J,Hanlon K E,et al.Immunosuppressive effects of erythropoietin on human alloreactive T cells[J].J Am Soc Nephrol,2014,25(9):2003.
[6]Cadman E T,Thysse K A,Bearder S,et al.Eosinophils are important for protection,immunoregulation and pathology during infection with nematode microfilariae[J].PLoS Pathogens,2014,10(3):e1003988.
[7]Icardi A,Paoletti E,De N L,et al.Renal anaemia and EPO hyporesponsiveness associated with vitamin D deficiency:the potential role of inflammation[J].Nephrol Dial Transplant,2013,28(7):1672.
[8]段富津.方剂学[M].上海:上海科学技术出版社,2010.
[9]刘逢琴,张峰,李贵海,等.右归饮对小鼠免疫功能的影响[J].山东中医学院学报,1996(1):43.
[10]孙筱婷,刘宣,李琦.肾虚证动物模型研究概况[J].中华中医药杂志,2017,32(2):659.
[11]李永勇,张志妮,梁昌盛,等.五味健体口服液对免疫低下小鼠免疫功能的影响[J].中药材,2018(9):1963.
[12]李崭,刘仁慧,康学,等.肾虚衰老模型的建立及其与D-半乳糖衰老模型的比较研究[J].中国中药杂志,2012,37(16):2435.
[13]黄茂林,林伟明,林晓玲,等.辣木叶提取物的制备工艺优化及其对免疫抑制小鼠免疫功能调节的研究[J].中国中药杂志,2018,43(13):2697.
[14]刘永琦,王文.虚证的免疫学本质[J].中国中医基础医学杂志,2003(5):7.
[15]Yokozawa T,Zheng P D,Oura H,et al.Animal model of adenine-induced chronic renal failure in rats[J].Nephron,1986,44(3):230.
[16]Yokozawa T,Chung H Y,Oura H.Urinary constituents and renal function in rats administered with adenine[J].Nihon Jinzo Gakkai Shi,1987,29(9):1129.
[17]童骏峰,徐志伟,杨元宵,等.腺嘌呤与氢化可的松所致大鼠肾阳虚证模型比较研究[J].中华中医药杂志,2015,30(11):3901.
[18]董爱国,霍俊凤,王秀文,等.不同提取工艺生殖营养方对腺嘌呤所致小鼠肾功能损伤的影响[J].中国实验方剂学杂志,2013,19(9):241.
[19]陈俊蓉,陈利国,谢林林.关于腺嘌呤慢性肾衰实验模型的思考[J].实验动物科学,2013,30(2):65.
[20]李许.EPO与“肾精”相似性的体内验证及“补肾益精”中药健脑作用机制研究[D].重庆:西南大学,2016.
[21]李燕君.EPO与肾虚大鼠脑组织神经细胞的关系及右归饮的保护作用研究[D].重庆:西南大学,2015.
[22]杨盛.EPO促脑神经血管单元细胞生长及补肾益精方药促EPO生成作用研究[D].重庆:西南大学,2016.
[23]Jelkmann W.The enigma of the metabolic fate of circulating erythropoietin(Epo)in view of the pharmacokinetics of the recombinant drugs rhEpo and NESP[J].Eur J Haematol,2015,69(5/6):265.
[24]袁颖,马慧,孙璇,等.重组人促红细胞生成素(rhEPO)在老年慢性病贫血(Ac D)中治疗效果的评价及疗效的预测[J].复旦学报:医学版,2013,40(6):724.
[25]Lee Yoon Mi,Choi Jun Ha,Min Wan Kwon,et al.Induction of functional erythropoietin and erythropoietin receptor gene expression by gamma-aminobutyric acid and piperine in kidney epithelial cells[J].Life Sci,2018,215:207.