| Background“Suwen·Yin Yang Big Classification of Natural Phenomena”said,“kidney producing bone marrow,marrow producing liver”.Based on the traditional chinese medicine theory,in the five elements,the kidney is the mother of water and the liver is the son of wood.Therefore,“marrow producing liver”means kidney essence generates liver blood,and embodies parent-child relationship.Besides,Liver stores blood,kidney stores essence,what’s more,both essence and blood can interconvert,so it shows that essence and blood are homologous.Youguiyin is a classic prescription of tonifying kidney and essence,which has the effects of supplementing yin and yang,essence and blood.Our research group hypothesized that“erythropoietin（EPO）may be a vital material basis for kidney essence and can be regarded as a biological marker for it”,specifically,we had verified that Bu Shen Yi Jing chinese medicine improved the constitution of kidney essence deficiency animals while reversing the decrease of EPO in aspects of anti-aging,brain and bone building,improving reproductive function and enhancing immunity.This essay intends to study the improvement effect of You Gui Yin on anemia rat model of kidney essence deficiency and its correlation with EPO,in order to further verify the hypothesis that EPO can be used as a biological marker of kidney essence.The project was funded by the National Natural Science Foundation of China（81473549）.ObjectivesTo observe the ameliorative effect of You Gui Yin on anemia rat model of kidney essence deficiency and its correlation with EPO,and to elucidate the biological mechanism of You Gui Yin’benefiting essence and generating blood.Methods1.Copying method for rat model of renal essence deficiency anemia induced by adenine:male SD rats were treated with 200 mg·kg-1 adenine suspension for 3 weeks,then every other day for 8 weeks to maintain the model.2.Model success criteria:（1）Testing indexes of kidney essence deficiency:Creatinine（CREA）,UREA,BUN,corticotropin releasing hormone（CRH）,adrenocor ticotropic hormore（ACTH）,Corticosterone（CORT）,Three iodine thyroid（T3）,T4,Parathyroid hormone（PTH）,Mg,P,Glu,Fe,IL-1,IL-6,tumor necrosis factor-αand creatinine clearance were measured in serum of rats.（2）Indicators for detecting anemia:the number of red blood cells（RBC）,hematocrit（HCT）and hemoglobin（HGB）were measured.Typically,the result of each rat was compared with the mean value of the normal group,and the rats with these above value had significant difference were selected as the successful model rats for the next step.3.Selecting successful model rats and completing equilibrium test then dividing into groups and administered:the successful rats were randomly divided into five groups:model group,You Gui Yin 10g·kg-1,20g·kg-1,40g·kg-1group and rh EPO group.After grouping and before administration,the model of each group was successfully evaluated by the balance test.Luckily,the statistical results didn’t exhibit any significant difference before administration.Additionally,at the beginning of the 4th week,each You Gui Yin group was given the corresponding dose every morning.As for rh EPO group,500 IU·kg-1 rh EPO was injected subcutaneously once every three days for 8 weeks,meanwhile,every rat except the normal group was treated 200 mg·kg-1adenine every other day in the afternoon in order to maintain this model.4.Observation index and detection method for You Gui Yin ameliorating anemia rats model of kidney essence deficiency:（1）the general morphology of kidney tissue in each group was evaluated,and the kidney was stained with HE and Masson by paraffin section.（2）the levels of CREA,UREA,Mg,P,Glu,TG,ALP and CREA,UREA and TP in urine were tested by automatic blood biochemical analyzer.（3）the contents of CRH,ACTH,CORT,T3,T4,PTH,Fe,Hepcidin,IL-1,IL-2,IL-6 and TNF-αin serum were determined by enzyme-linked immunosorbent assay.（4）the result of RBC,HCT,HGB,MCV,mean RBC concentration,WBC,monocyte number,monocyte percentage,neutrophil number,lymphocyte percentage,PLT and PCT in blood were measured by automatic blood cell analyzer.（5）immunofluorescence and western blotting were used to detect the relative expression ofα-SMA in kidney.（6）the relative expression of TGFβwas detected by immunohistochemistry and western blotting.（7）the relative expression of PDGFRβin kidney was measured by immunofluorescence.5.Determination of EPO in serum and kidney and EPO signal pathway in kidney tissue:（1）Enzyme-linked immunosorbent assay（ELISA）was used to detect EPO in serum and kidney.（2）the relative expression levels of PHD2,GATA2,HIF2α,PI3K,AKT1,p-AKT,m TOR,p-m TOR,HIF1α,EPOR,JAK2,p-JAK2,STAT5 and p-STAT5 were evaluated by Western blotting.Results1.The constitution and renal function of rats in the model group were significantly reduced by adenine.Compared with the normal group,the rats in the model group had darker hair color,more lethargy,slower weight gain,clearer urine at 24 hours,significantly increased urine output at 24 hours（P<0.01）,and their kidneys were pale and enlarged,presenting as“big white kidney”,Moreover,CRH,ACTH,CORT,PTH,Mg,P,ALP,Hepcidin in serum increased significantly（P<0.05）,while T4,TG,Fe in serum decreased obviously（P<0.05）,showing that the physical constitution of rats was improved.Besides,CREA,UREA and TP in urine,CREA,UREA and BUN in serum respectively were expressed more intensively relative to control（P<0.05）,creatinine clearance rate was significantly decreased（P<0.01）,these data demonstrated that the renal function of the model rats was reduced..2.Adenine-induced anemia,inflammation and renal fibrosis were observed in the model group.The percentage of RBC,HCT,HGB,MCV and lymphocyte in blood of model group was significantly lower than normal one（P<0.05 or P<0.01）,additionally,the mean erythrocyte hemoglobin concentration,WBC,monocyte number,monocyte percentage and neutrophil number were sustantially higher respectively（P<0.05 or P<0.01）,which indicated that the model rats had anemia symptoms.Relative to control,the levels of IL-1,IL-6 and TNF-αin the model group were significantly higher（P<0.01）,however,the concentration of IL-2 were significantly lower（P<0.01）,indicating that inflammation was seen in the model rats.Similarly,the expression of TGFβ,α-SMA and PDGFRβin the kidney tissue of the model rats were significantly increased（P<0.05）respectively compared with the control.3.Lower concentration of EPO and abnormal expression of EPO pathway protein were seen in the kidney essence deficiency anemia rats induced by adenine.Compared with the normal group,the concentration of EPO in serum and kidney tissue of model rats were significantly decreased（P<0.05）,and the expressions of PHD2 and GATA2 in kidney were substantially higher（P<0.05）,typically,HIF2α,p-AKT,m TOR,p-m TOR,HIF1α,JAK2,p-JAK2,STAT5,p-STAT5 reduced significantly（P<0.05 or P<0.01）,respectively.4.You Gui Yin can significantly improve the constitution of kidney essence deficiency anemia rats.Compared with the model group,the hair color and mental state of the rats in You Gui Yin group were improved,and the body weight was increased quickly,what’s more,the histomorphology of the kidney presented better performance,while the renal index was significantly reduced（P<0.05）,CRH,ACTH,CORT,PTH,Mg,P,ALP,,Hepcidin in You Gui Yin group respectively declined significantly（P<0.05 or P<0.01）,Fe increased intensively（P<0.05）,while T4 increased significantly（P<0.05）in You Gui Yin 10 g·kg-1 group and 20g·kg-1 group,the Glu of 20 g·kg-1 group and 40 g·kg-1 group exhibited lower concentration（P<0.05）,these data indicated that You Gui Yin could improve the constitution of renal deficiency anemia rats.5.You Gui Yin can obviously improve the renal function of anemia rats with kidney essence deficiency.Compared with model group,UREA,in You Gui Yin10 g·kg-1 group was significantly increased（P<0.05）,and TP in 40 g·kg-1 group was significantly increased（P<0.05）,and CREA,UREA and BUN in youguiyin group were significantly reduced（P<0.05）,respectively,the creatinine clearance rate of 20 g·kg-1 group was significantly increased（P<0.05）,which indicated that You Gui Yin could improve the renal function of renal deficiency anemia rats.6.You Gui Yin can obviously improve the blood routine indexes of anemia rats with kidney essence deficiency.Compared with model group,RBC,HCT and HGB in blood of rats in 10 g·kg-1and 20 g·kg-1group were significantly increased（P<0.05 or P<0.01）,and MCV in You Gui Yin 20 g·kg-1group were significantly increased（P<0.05）,the mean hemoglobin concentration of 20 g·kg-1 group was significantly reduced（P<0.05）,and the value of WBC in10 g·kg-1 group was significantly decreased（P<0.01）,besides,the number of monocytes,the percentage of monocytes and the number of neutrophil in youguiyin 20 g·kg-1group presented significantly lower concentration（P<0.05）,and the percentage of lymphocyte in 20 g·kg-1group was significantly higher（P<0.05）.7.You Gui Yin can obviously improve the inflammation index of anemia rats with kidney essence deficiency.Compared with model group,the concentration of IL-1,IL-6 and TNF-αin youguiyin group were significantly decreased（P<0.05）,while IL-2 was significantly increased（P<0.01）.8.You Gui Yin can obviously improve the renal fibrosis of anemia rats with kidney essence deficiency.Compared with the model group,the collagen volume fraction in You Gui Yin group was significantly decreased（P<0.05）,and the relative expression of TGFβ,α-SMA,PDGFRβin kidney were significantly decreased（P<0.05）,respectively,these results showed that You Gui Yin could reduce the degree of renal fibrosis in model rats.9.You Gui Yin can significantly reverse the content of EPO and the abnormality of signal pathway in renal deficiency anemia rats.Compared with the model group,the levels of EPO in the serum of rats in youguiyin 20 g·kg-1 group and 40 g·kg-1group were significantly increased（P<0.01）,while in the kidney tissue,You Gui Yin 10 g·kg-1group,20 g·kg-1 group and40 g·kg-1group were significantly increased（P<0.05）,the relative expression of PHD2 and GATA2 protein in 20 g·kg-1 group decreased significantly（P<0.05）.Additionally,the relative expression of HIF2α,p-AKT and p-JAK2 in youguiyin group increased significantly（P<0.05）,and the relative expression levels of PI3K and HIF1αprotein in youguiyin 20 g·kg-1 group and40 g·kg-1 group were significantly increased（P<0.05）,while m TOR in 40 g·kg-1 group was significantly increased（P<0.05）,the expression of p-m TOR was significantly increased in youguiyin 10 g·kg-1group（P<0.05）,and the relative expression of STAT5 protein was significantly increased in youguiyin 20 g·kg-1 group（P<0.05）.Conclusions1.Renal deficiency anemia rat model was successfully induced by intragastric administration of 200 mg·kg-1 adenine for 3 weeks and at intervals for 8 weeks.2.The content of EPO in serum and the expression of EPO in kidney tissue of the animal model were significantly decreased,and the expression level of PHD2/HIF2α,PI3K/AKT/m TOR/HIF1αand JAK2/STAT5 pathway were significantly changed.3.Kidney function,inflammation,renal fibrosis and blood routine indexes of anemia rat model of kidney deficiency can be significantly improved by You Gui Yin,which may be related to the increase of EPO in serum and the kidney tissue,as well as the regulation of PHD2/HIF2α,PI3K/AKT/m TOR/HIF1αand JAK2/STAT5 pathway protein content.4.These results prove that You Gui Yin has the function of enriching essence and promoting blood production,which provide experimental in favor of the theory of“Essence and blood are homologious”,supporting the hypothesis that EPO can be regarded as a biological marker of kidney essence. |
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