| Renal anemia(RA)is one of the main complications of chronic kidney disease(CKD).It is an important index to aggravate the progression of chronic kidney disease and affect the prognosis of kidney disease.The data show that the anemia rate has reached 19.23%in the CKD 1 stage,and anemia in CKD3-5 period,especially in CKD5 period,up to about 95%,obviously higher than that of CKD patients in European and American.There is a positive correlation between the degree of renal function deterioration and the incidence of anemia.At present,recombinant human erythropoietin has achieved good results in the treatment of renal anemia.Due to a certain degree of iron deficiency in patients with CKD anemia,the rate of reaching the standard is still very low.Studies have shown that fully correcting the abnormal iron metabolism in the body can significantly improve the therapeutic effect of rhu-EPO,and significantly reduce the dosage of it.Hepcidin(hepcidin)is an important regulator of iron homeostasis.BMP/SMAD signaling pathway is the major molecular mechanism regulating the expression of hepcidin.In addition,the treatment of RA with exogenous EPO and iron can produce the side effects of the increase of blood viscosity and hypertension,so the medical treatment of RA just made up the deficiency in this area.The deficiency syndrome of RA TCM pathogenesis is more common in the spleen and kidney yang deficiency,so this study established the RA rat model,and explored the mechanism of the action of the Wenshen Yisui Shengxue Fang on the RA,and the treatment of R for this prescription.A provides experimental basis.Objective:In this study,RA rat model was prepared by unilateral nephrectomy and adenine gavage.The effects of Wenshen Yisui Shengxue Fang on the pharmacodynamics of RA rats and the level of oxidative stress were observed.HE staining and Mallory staining were used to observe the pathological damage of kidney in rats.In situ hybridization,Real time PCR technology,immunological and Western Blot were used.The effects of Wenshen Yisui Shengxue Fang on the expression of BMP6,Smad4,Smadl/5/8,p-Smadl/5/8,hepcidin mRNA and protein in the liver tissue of rats with renal anemia.Method:1 The preparation and treatment of rat model of renal anemia:56 male,SPF and Wister rats,adapted for 1 weeks,divided into 10 rats in the normal group,10 in the sham operation group,10 in the sham operation group and 36 in the model group.The left kidney excision operation was performed in the model rats for second weeks,and the artificial hand group was only stripped of the renal capsule and the kidney was not removed.After 2 weeks of recovery,the rats in the model group were randomly divided into model group,Niao Du Qing group and Wen Shen Sheng Xue group.The model group and the treatment group were given 180mg/Kg D adenine gavage every morning,and the treatment group was given the corresponding medicine in the afternoon treatment group.The normal group,sham operation group and model group were given equal volume of deionized water to the stomach,the dose of gavage was 1ml/100g,and the diet,drinking water and behavior of the animals were closely observed.After 12 weeks of treatment,the rats were harvested for renal tissue and liver tissue.2 related blood test:the rats in each group were taken blood by femoral artery,the serum levels of creatinine(Scr),urea nitrogen(BUN),uric acid(UA),blood Ca2+,P3+,Fe3+ content,serum NO,MDA,SOD content,the other part of the blood test red blood cell(RBC),blood red egg white(HGB)level.3 HE staining and Mallory staining:the pathological changes of the kidneys in rats were observed.4 in situ hybridization and Real time PCR:the expression levels of BMP6,Smad4,Smad1/5/8 and hepcidin mRNA in rat liver tissues were detected.5 immunohistochemistry and Western Blot:the location and expression level of BMP6,Smad4,Smad1/5/8,p-Smad1/5/8 and hepcidin proteins in rat liver tissues were detected.6 data analysis:all experimental data were analyzed by SPSS 22.0.Result:1 Effect of Wenshen Yisui Shengxue Fang on pharmacodynamics of RA ratsCompared with the model group,the normal state of the rats in the warm kidney blood group was obviously improved.Compared with the normal group and the sham operation group,the body mass of the model rats decreased with the increase of the experimental days(P<0.01),the level of serum BUN and Scr increased significantly(P<0.01),and the degree of renal pathological damage was gradually aggravated,and the level of RBC and HGB was significant.The level of serum Ca2+ and Fe3+decreased significantly and the content of P3+increased significantly(P<0.01).Compared with the model group,the serum level of BUN and Scr decreased significantly(P<0.01)and the level of RBC and HGB increased(P<0.01),and the levels of serum Ca2+ and Fe3+ increased significantly,and the content of P3+decreased significantly.2 Effect of Wenshen Yisui Shengxue Fang on serum oxidative stress in RA ratsCompared with the normal group and the sham operation group,the content of NO and SOD in the serum of the model rats decreased significantly(P<0.01),and the content of MDA increased significantly(P<0.01).Compared with the model group,the content of NO and SOD in the serum of the warm kidney blood group was significantly higher(P<0.01),and the content of MDA decreased significantly(P<0.01).3 Effect of Wenshen Yisui Shengxue Fang on expression of BMP6,Smad4,Smad1/5/8 and hepcidin mRNA in liver tissue of RA ratsIn situ hybridization:the liver tissues of the normal group and the sham operation group expressed a small amount of BMP6,Smad4,Smad1/5/8 and hepcidin mRNA,which were significantly lower than that of the model group(P<0.01).Compared with the model group,the expression of BMP6,Smad4,Smad1/5/8 and hepcidin mRNA in the group of warm kidney and kidney blood decreased significantly,with significant difference(P<0.01).Real time-PCR:compared with the normal group and the sham operation group,the rat liver tissue in the model group expressed a large number of BMP6,Smad4,Smad1/5/8 and hepcidin mRNA,which had significant difference(P<0.01).Compared with the model group,the expression of BMP6,Smad4,Smadl/5/8 and hepcidin,in the rats of the warm kidney blood group was significantly reduced,and had a significant difference.Difference(P<0.01).4 Effect of Wenshen Yisui Shengxue Fang on expression of BMP6,Smad4,Smad1/5/8.p-Smad1/5/8 and hepcidin proteins in liver tissue of RA ratsImmunohistochemistry:the expression of BMP6 and hepcidin protein in the liver cytoplasm of the normal group and the sham operation group was lower than that of the model group(P<0.01).Compared with the model group,the expression of BMP6 and hepcidin protein in the warm kidney blood group decreased(P<0.01);the normal group and the sham operation group expressed a small amount of Smad4,Smadl/5/8 and p-Smadl/5/8 in the cytoplasm and nucleus of the sham operation group.The protein expression was significantly lower than that of the model group(P<0.01).Compared with the model group,the expression of Smad4,Smadl/5/8 and p-Smadl/5/8 in the warm kidney blood group decreased significantly(P<0.01),and there was no significant difference between the groups of Smadl/5/8 and p-Smadl/5/8(P>0.05).Western Blot:compared with the normal group and the sham operation group,the expression of BMP6,Smad4,Smadl/5/8,p-Smadl/5/8 and hepcidin in the liver tissue of the model group increased significantly(P<0.01).Compared with the model group,the expression of BMP6,Smad4,Smadl/5/8,p-Smadl/5/8 and egg white in the liver tissue of the warm kidney blood group decreased significantly.There was no significant difference in the ratio of Smadl/5/8 to p-Smadl/5/8 between the groups(P>0.05).Conclusion:1 RA model rats,with the increase of model time,the body mass decreased,the renal function decreased,the electrolyte metabolism was disordered,the renal pathological changes were obvious.The blood routine indexes suggested anemia,and the model was successfully established.2 Wenshen Yisui Shengxue Fang can improve the general state of RA rats,increase the body mass of renal anemia rats,increase the level of RBC,HGB and serum Fe3+ in the blood of RA rats,and improve the anemia effectively:3 Wenshen Yisui Shengxue Fang can increase blood Ca2+,reduce serum P3+ level,reduce serum Scr,BUN and UA levels,improve serum electrolyte metabolism and renal function of RA rats,and reduce the content of MDA in serum of renal anemia rats,increase the level of NO and SOD,and effectively improve the oxygen of rats.Stress the state of stress.4 The expression of BMP6,Smad4,Smadl/5/8,p-Smadl/5/8,hepcidin protein and BMP6,Smad4,Smadl/5/8 and hepcidin mRNA in the liver tissue of RA rats can be reduced by warming kidney and kidney nourishing blood to reduce the expression of BMP6,Smad4,Smadl/5/8,hepcidin mRNA,effectively correcting iron deficiency and improving anemia.In conclusion,Wen Shen Yi Sheng Xue Fang has a positive effect on the anemia of RA rats.It may affect the expression of hepcidin in the liver of rats with renal anemia through the BMP/SMAD signaling pathway and correct the iron deficiency. |
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