Experimental Study Of Shenshuaiyangzhen Capsule Therapy On Anaemia In Rats With Chronic Renal Failure

It is finally results with various chronic kidney diseases that chronic renal failure (CRF),and intercurrent renal anaemia, malnutrition, osteoporosis etc. At the same time anaemia think as to CRF's symbol. When CRF sufferer's glomerular filtration rate (GFR) low in 30 mls/ min, anemia must be appearing.Anemia is one of the most familiar. Renal anaemia make sufferer the existence rate lower, increasing the cardiovascular affairs' probability, affecting the sufferer's living quantity seriously.The research showed recently that renal anaemia be treated in earlier period, can ease the left ventricles hypertrophy, improve anemia to the damage of important viscera, improve the kidney function probably, raise the sufferer to live the quantity, so rectify t renal anaemia have very important significance. The experiment divides two parts to study Shenshuaiyangzhen(ssyz) capsule therapy on renal anaemia. The first part of examination :Change of haemoglobin (Hb),blood urea nitrogen (BUN), serum creatinine (SCr), transferrin (TF) and reduced glutathione (GSH),erythrocyte brittleness and erythopoietin(EPO) were measured.To investigate the curative effect of ssyz capsule on renal anaemia in rats with chronic renal failure. The first part of examination : The level of the EPO gene expression was messured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).Chapter I Experimental study of ssyz capsule on renal anaemiaObject:To investigate the curative effect of ssyz capsule on anaemia in rats with chronic renal failure.Change of haemoglobin (Hb),blood urea nitrogen (BUN), serum creatinine (SCr), transferrin (TF) and reduced glutathione(GSH),erythrocyte brittleness and EPO were measured Methods:1. Rats were randomly divided into normal group(N group,n=10) and renal anaemic model group. 10% chloral hydrate is injected to abdomen to anaesthesia,Chronic renal anaemia rats were made by remove of right kidney followed by nephrectomy of 70% left kidney ten days later. After Six weeks break the tail to take the blood, serum BUN, Scr and TFs, hemoglobin was examination. Serum was conserved to measure the serum iron, content of EPO. Renal anaemia model rats were randomly divided into five groups: control group,doubling dose group,curative dose group,minimal dose group,yixuesheng group. And rats without any infusion served as normal group. All rats were feeded the standard food in the whole experiment period, water freely. After treatment, blood of celiac artery was collected, serum BUN, Scr and TFs, hemoglobin was examination. Serum was conserved to measure the serum iron, content of EPO.2. Bio-chemical examination of blood:The auto bio-chemical analytical instrument of the OlimpasAU600 measures the BUN, SCr, TF.3. General examination: Hb,GSH,serum iron were measured adopt ultraviolet spectroscope. The serum EPO content examination adopts the ELJSA method4. Erythrocyte brittleness examination: Take blood drop different concentration sodium chloride (2.4g/L, 2.8g/L, 3.2g/L, 3.6g/L, 4.0g/L, 4.4g/L, 4.8g/L, 5.2g/L, 5.6g/L, 6.0g/L), shaked cuvette, put into the refrigerator,conserved at 4°C. After twohours, observed result.5. The rat kidney pathology observation^ the end of examination,each group two rats were choosed, kidney organization to place 10% formalin, fixation,dyeing. Results:1. Variety of hemoglobin: Before treatment,level of hemoglobin did not show the significance of difference between ssyz groups and control group (P>0.05 ) , level of hemoglobin was significantly lower with the normal group( P<0.01). After treatment, hemoglobin level of doubling dose group and curative dose group were higher minimal dose group and yixuesheng group (P<0.05), minimal dose group and yixuesheng group was higher control group (P<0.05 ) .2.Variety of serum iron and TF: Before treatment,level of serum iron and TF did not show the significance of difference between ssyz groups and control group (P>0.05) , level of serum iron and TF was significantly lower with the normal group( P<0.01). After treatment, serum iron and TF level of treated group were higher control group (P<0.05) , serum iron and TF level of doubling dose group, curative dose group and yixuesheng group were higher minimal dose group (P<0.05).3. Erythrocyte brittleness compare: Erythrocyte brittleness of normal group was lower model group (P<0.05) . Erythrocyte brittleness of treated group did not show the significance of difference ( P>0.05 ). Minimal dose group was lower control group(P<0.05)4. GSH compare: GSH content of each group were significantly higher control group (P<0.01) , doubling dose group and curative dose group was higher control group and yixuesheng group (P<0.05) .Content did not show the significance of difference between normal group, doubling dose group and curative dose group(P>0.05) .5. Serum EPO level compare: Before treatment,level of EPO did not show thesignificance of difference in model group(P>0.05), level of EPO was significantly lower with the normal group( P<0.01). After treatment, level of EPO was significantly lower with the normal group all the same( P<0.01). However,EPO level of doubling dose group and curative dose group was significantly increased (P<0.01) , minimal dose group was increased (P<0.05) . yixuesheng group did not show the significance of difference. EPO level of doubling dose group and curative dose group was higher minimal dose group and yixuesheng group (P<0.05) , EPO level of minimal dose group and yixuesheng group were significantly higher control group (P<0.01) .6. The rat kidney pathology compare: Nephron of model group rats were full of albumen, renal arteriolar sclerosis ,fibrosis. In the nephron of doubling dose group rats seldom albumen was found. In the nephron of curative dose group rats albumen wasn't found. In the nephron of yixuesheng group rats albumen, renal arteriolar sclerosis and fibrosis were found. Conclusion:1. EPO level was increased and anemic state was improved by ssyz capsule, ssyz capsule is more effective than yixuesheng capsule. Pathologic picture manifestation that seldom albumen was found. It explained that ssyz capsule can protected nephron and promote synthesize and secrete EPO.2. Ssyz capsule can increase the stability of the erythrocyte membrane, lower erythrocyte brittleness, thus prolong the life of erythrocyte.3. GSH level was increased by ssyz capsule, ssyz capsule is more effective than yixuesheng capsule. It explained that ssyz capsule can raise GSH's level ,increase anti-oxidize system activity, lower oxide toxicity, protect erythrocyte, improve the anemia.4. Level of Serum iron and TF was increased. It explained that ssyz capsule canpromote absorb of iron, raise the exploitation of the iron.Chapter II Experimental study of ssyz capsule on EPO gene expression in ratswith renal anaemiaObjecttTo investigate the effect of ssyz capsule on EPO gene expression anaemia inrats with chronic renal failure.Methods:1. The animal specimen collect:In the end of experimentation, the kidney was washed by prepared 4 °C cold DEPC water again and again. Medulla of kidney was conserved in RNAin, put into the -20 °C refrigerate2. EPO gene expression was messured by semi-quantitative RT-PCR:Total RNA was isolated from rat kidney by homogenization in the presence of Trizol.Using OneStep RT-PCR Kit(QIAGEN), 2jug of total RNA was reverse-transcribed. RT-PCR was performed with EPO-specific primers(5' -AGG CGC GGA GAT GGG GGT GC -3' and 5' -CCC CGG AGG AAG TTG GAG TAG-3' ) to give a 540-bp amplified fragment .As a control for RNA extraction ,integrity,reverse transcription,and amplification,P-actin-specific primers(5' -GTG GGG CGC CCC AGG CAC CA-3' and 5' -CTC CTT AAT GTC ACG CAC GAT TTC-3' ) were used to amplify a 500-bp fragment from the same cDNA preparation.Thermal cycler conditions: Reverse transcription 30min,Initial PCR activation step 15min,Denaturation 30s at 94 °C,Annealing 30s at 56°C,Extension lmin at 72°C,30 cycles,Final extension lOmin at 72°C.An aliquot of the amplified reaction mixture was electrophoresed in a 1.5% agarose gel,dyeing by EB, analyzes by Gel Docl000,content of EPO fragment and P -actin fragment be compared.Results:The each rat RT- PCR amplified fragment was electrophoresed in a 1.5%agarose gel, Marker marking is 540-bp and 500-bp,matching the purpose gene length. EPO gene expression of doubling dose group and curative dose group were significantly higher noemal group's ( P<0.01) EPO gene expression of minimal dose group and control group was higher noemal group's (P<0.05) Conclusion: Ssyz capsule can up-regulated the EPO gene expression. The level of erythropoietin was significantly increased in serum. But EPO content's increment of serum was matched with anemic improvement,it explained that it is not unique reason of improve renal anaemia that EPO content was increased and the gene expression was up-regulated by ssyz capsule. We presume ssyz capsule may enhanced EPO receptor's sensitivity, The accurate mechanism needs the further research.