| BackgroundAccording to the theory of traditional Chinese medicine,the deficiency of kidney essence leads to the deficiency of Qi,resulting hypoimmunity.However,the biological material basis and mechanisms of"kidney essence"are still unknown.The preliminary study found that:（1）the connotation of erythropoietin（EPO）was highly similar to"kidney essence",and the hypothesis that"EPO may be an important biological material basis of kidney essence"was put forward,which was preliminarily verified;（2）You-Gui-Yin（YGY）had the effect of improving immune function.However,it has not known whether YGY can improve the phagocytic function of macrophages in rats with kidney deficiency and hypoimmunity（KDH）and its mechanism is related to EPO.This paper aims to observe the effect of YGY on phagocytic function of macrophages in KDH and its correlation with EPO signal pathway.This research was supported by the Natural Science Foundation of China（81473549）.ObjectiveTo explore whether or not YGY can improve the phagocytic function of macrophages and its effect is related to EPO signal pathway.Methods1.Replication,evaluation and detection methods of adenine induced KDH（1）Model replication:male SD rats were administrated with adenine at 150 mg·kg-1 for14 days continuously,and adenine was given for another 30 days every other day.（2）KDH criteria:at 14th days,compared with the control group,CREA and UREA in the serum of the KDH group increased significantly,ACTH,CORT,T,T3,T4,IgG,IgM,C3,C4 decreased significantly.CREA and UREA were detected by BS220 biochemical instrument,ACTH,CORT,T,T3,T4,IgG,IgM,C3 and C4 by ELISA.2.The effect of YGY on macrophage phagocytosis in KDH and its correlation with EPO（1）Group and administration:the rats were randomly divided into model group,rhEPO 500 IU·kg-1 group,YGY 10 g·kg-1,20 g·kg-1,40 g·kg-1 group,10 rats in each group.Another 10 rats were named control group.YGY was given by gavage once a day,rhEPO was given by injection once every 3 days for 30 days continuously,and the rats sacrificed on the 31st day.（2）Macroscopic observation:the changes of animal appearance and kidney tissue morphology.（3）Phagocytosis of macrophages in the spleen:after injecting fluorescent microspheres into the tail vein,the spleen was separated and the fluorescence intensity of the spleen was detected.（4）Phagocytosis test of peritoneal macrophages:non-stimulating method was used to obtain peritoneal macrophages,and colorimetric method was used to detect its phagocytic capacity of neutral red.（5）Phagocytosis test of bone marrow macrophages:obtain bone marrow macrophages by cytokine-induced method,and detect its phagocytic capacity of neutral red by colorimetry;detect the phagocytic situation of macrophages by inverted fluorescence microscope;calculate the rosette formation rate of macrophages.（6）Western blot was used to detect the expression of HIF-1α,EPO,EPOR,p-EPOR,JAK2, p-JAK2,ERK,p-ERK,C/EBPβ,P-C/EBPβ,PPARγ.3.The effect of YGY on the phagocytic capacity of RAW264.7 macrophage and its correlation with EPO（1）MTT method was used to detect the effect of YGY on the activity of RAW264.7 macrophages.（2）RAW264.7 macrophage injury model was made by hypoxic and glucose-deficient injury method（HGI）.（3）The EPO gene of RAW264.7 macrophage was knocked out by lentivirus transduction.（4）Phagocytosis tests:the ability of phagocytosis of neutral red by colorimetry;the ability of phagocytosis of fluorescent microspheres by flow cytometry;the ability of phagocytosis of GFP-E.Coli J96 bacteria and rosette formation by microscopy.（5）Western blot was used to detect the expression of HIF-1α,EPO,EPOR,p-EPOR,JAK2,p-JAK2,ERK, p-ERK,C/EBPβ,p-C/EBPβ,PPARγ.Results1.Adenine-induced KDH was successfully replicated and its macrophage phagocytic capacity was significantly reduced.Compared with the control group,the KDH group rats were that（1）thin,hunched back,lost weight,were afraid of cold,had yellowish fur and lacked luster;（2）kidney sections showed more vacuoles,decreased nephrons,dilated tubules,unsaturated glomeruli,and significant infiltration of inflammatory cells in renal tissues;（3）serum CREA and UREA contents increased significantly,while ACTH,CORT,T3,T4 and T contents decreased significantly（all P<0.05）;（4）serum IgG,IgM,C3 and C4 contents were significantly decreased（all P<0.05）;（5）the OD value of neutral red phagocytosed by peritoneal macrophages and bone marrow macrophages decreased significantly;the average fluorescence intensity of phagocytosed fluorescent microspheres and bacteria decreased significantly;the rosette formation rate decreased significantly（all P<0.05）.2.Serum EPO and macrophage EPO pathway were significantly down-regulated in adenine-induced KDH rats.Compared with the control group,the serum EPO concentration in the KDH group was significantly reduced（P<0.05）;the expression levels of EPO,EPOR,p-JAK2,ERK,p-ERK,p-C/EBPβ,PPARγand the ratio of p-JAK2/JAK2,p-ERK/ERK,p-C/EBPβ/C/EBPβin macrophages were significantly decreased（all P<0.05）.3.YGY significantly reversed the decrease of body constitution and macrophage phagocytosis ability in adenine-induced KDH rats.Compared with the KDH group,rats in each dose group of YGY（1）had increased body weight,reduced arched back,improved mental state,more active,less hair loss and better gloss;（2）the number of vacuoles in renal sections decreased,the number of nephrons increased,the dilated renal tubules more narrowed,the glomerular balloon cavity narrowed,and the inflammatory infiltration of renal tissues decreased in YGY 10 g·kg-1 and 20 g·kg-1groups;（3）the contents of serum CREA and UREA in rats in YGY 10 g·kg-1 and 20g·kg-1 groups were significantly decreased,and the contents of serum CORT,ACTH,T,T3 and T4 in YGY 10 g·kg-1,20 g·kg-1 and 40 g·kg-1 groups were all increased in different degrees（all P<0.05）;（4）the serum IgG,IgM,C3 and C4 contents in each dose group of YGY increased to different degrees（all P<0.05）;（5）the OD value of neutral red phagocytosis by peritoneal macrophages was significantly increased in YGY 10g·kg-1 and 20 g·kg-1 groups;the average fluorescence intensity of splenic macrophages phagocytosing fluorescent microspheres in YGY 20 g·kg-1 group was significantly increased;the OD value of neutral red phagocytosis by bone marrow macrophages and the average fluorescence intensity of fluorescent microspheres were significantly increased in YGY all groups;the average fluorescence intensity of phagocytic bacteria was significantly enhanced in YGY 20 g·kg-1,40 g·kg-1 group;the rosette formation rate was significantly increased in all dose groups of YGY（all P<0.05）.4.YGY significantly reversed the decrease of serum EPO and the down-regulation of macrophage EPO pathway in adenine-induced KDH rats.Compared with the KDH group,serum EPO concentration was significantly increased in YGY 10 g·kg-1,20 g·kg-1 and 40 g·kg-1 groups（all P<0.05）;the relative expression of EPO pathway proteins EPO,EPOR,p-JAK2,p-ERK,p-C/EBPβ,PPARγand the ratio of p-JAK2/JAK2,p-ERK/ERK,p-C/EBPβ/C/EBPβin macrophages was significantly increased in YGY all groups（all P<0.05）.5.YGY significantly increased the phagocytic capacity of normal or HGI RAW264.7 macrophages.（1）Normal RAW264.7 macrophage:Compared with the control group,the OD value of neutral red phagocytosis increased significantly in YGY200μg·mL-1,400μg·mL-1,800μg·m L-1 and 1600μg·mL-1 groups;the phagocytic percentage and average fluorescence intensity of fluorescent microspheres and bacteria increased significantly in YGY 400μg·mL-1 and 800μg·mL-1 groups;the rosette formation rate was significantly increased in YGY 400μg·mL-1 group（all P<0.05）.（2）HGI RAW264.7 macrophage:Compared with the control group,the HGI group cells phagocytosed neutral red OD value,the percentage of phagocytosis of fluorescent microspheres,the average fluorescence intensity of bacteria and the rate of rosette formation were significantly reduced（all P<0.05）.Compared with the HGI group,the OD value of phagocytosis neutral red and the percentage of phagocytosis of fluorescent microspheres were significantly increased in the YGY 800μg·mL-1 group;the average fluorescence intensity of phagocytic bacteria was significantly increased in all YGY groups;the rosette formation rate was significantly increased in the YGY 400μg·mL-1and 800μg·mL-1 groups（all P<0.05）.6.YGY regulates the phagocytic capacity of RAW264.7 macrophages through EPO and its signaling pathway.（1）EPO gene knockout（EPO KO）RAW264.7macrophages:Compared with the control group,EPO KO group cells phagocytosed neutral red OD value significantly reduced;phagocytosis percentage of phagocytic fluorescent microspheres significantly decreased;average fluorescence intensity of phagocytic bacteria significantly weakened;rosette formation rate significantly decreased（all P<0.05）.Compared with the EPO KO group,the ability of EPO KO+YGY group to phagocytose neutral red,fluorescent microspheres and bacteria was not significantly different（all P>0.05）,and the rosette formation rate was significantly increased（P<0.05）.Compared with the control+YGY group,the OD value of phagocytic neutral red was significantly reduced in the EPO KO+YGY group;the phagocytic percentage of phagocytic fluorescent microspheres was significantly decreased;the average fluorescence intensity of phagocytic bacteria was significantly weakened;and the rosette formation rate was significantly reduced（all P<0.01）.（2）Normal RAW264.7 macrophage:Compared with the control group,the expression of EPO pathway proteins HIF-1α,EPO,p-EPOR,p-JAK2,p-ERK,p-C/EBPβ,PPARγand the ratio of p-EPOR/EPOR,p-JAK2/JAK2,p-ERK/ERK,p-C/EBPβ/C/EBPβin the YGY group increased significantly（all P<0.05）.（3）HGI RAW264.7 macrophages:Compared with the control group,the expression of EPO pathway proteins HIF-1α,EPO,EPOR,p-JAK2,ERK,p-ERK,p-C/EBPβ,PPARγand the ratio of p-JAK2/JAK2,p-C/EBPβ/C/EBPβin HGI group were significantly decreased（all P<0.05）.Compared with HGI group,the expression of HIF-1α,EPO,EPOR,p-EPOR,JAK2,p-JAK2,p-ERK,C/EBPβ,p-C/EBPβ,PPARγand the ratio of p-EPOR/EPOR,p-JAK2/JAK2,p-ERK/ERK,p-C/EBPβ/C/EBPβin the YGY 800μg·mL-1 group were significantly increased（all P<0.05）.Conclusions1.The rats modeled by adenine showed significant renal function and endocrine disorders（characterization of kidney deficiency）,hypoimmunity with decreased macrophage phagocytosis ability,accompanied by significant reduction in serum EPO content and protein expression of macrophage EPO signaling pathway;2.YGY can significantly improve the body constitution and immune function of KDH rats,and its mechanism is closely related to elevating serum EPO content,up-regulating EPO signaling pathway of macrophages and thus enhancing their phagocytic ability;3.YGY can significantly regulate the phagocytic function of RAW264.7macrophages,and its mechanism is closely related to the regulation of EPO signaling pathway;4.The biological mechanism of KDH treated by YGY is related to EPO,which is of supporting significance to the hypothesis that EPO may be an important biological material basis of kidney essence. |
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