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

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。

Changes of EPO in rats with chronic renal failure and low immunity and reversal effects of Yougui Yin and exogenous EPO

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.