| Chronic kidney disease(CKD)is a chronic progressive renal structural damage and dysfunction caused by a variety of causes.Renal anemia(RA)is one of the major complications in the course of patients with chronic kidney disease,which has a significant impact on the progression of CKD and its prognosis.RA can occur in all stages of CKD,and the rate of anemia increases with the progression of CKD1?5.However,in recent years,more and more clinical investigation data show that the incidence of RA in the early stage of CKD patients is gradually increasing.In CKD,damage to the kidney structure leads to insufficient production of erythropoietin(EPO)and anemia.If the anemia is not corrected in time,it will also aggravate the structural damage of the kidney tissue,worsening the disease and entering a vicious circle.EPO produced by Renal Epo-producing cells(REPC)can stimulate the normal growth,proliferation,differentiation and maturation of erythroid progenitor cells in the bone marrow.It is found that EPO can not only improve the body's anemia,but also It can protect tissues and organs by activating PI3K/AKT signaling pathway,JAK2-STAT5 signaling pathway and NF-?B signaling pathway in tissues against inflammation,anti-fibrosis,anti-apoptosis and affecting oxidative stress.Objective:In this study,a rat model of renal anemia was prepared by a combination of adenine and gavage on the basis of unilateral nephrectomy.The basic state,peripheral blood,renal function and oxidative stress of rats with RA were observed.The effects of the level were observed by HE staining and Mallory staining.Immunohistochemistry and Western Blot were used to detect the expression of EPO,PI3K,AKT,p-AKT and bcl-2 protein in the kidney of RA rats.In situ hybridization and RT-PCR were used to detect EPO,PI3K,AKT and bcl-2 mRNA in the kidney of RA rats.Method:1.Preparation of RA rat animal model and drug treatment:60 male Wister rats of SPF grade were adaptively reared for 1 week,and were randomly divided into 10 groups in the normal group,10 in the sham operation group,and 40 in the model group.In the second week,the left model nephrectomy was performed on the model rats.The sham operation group retained the kidney and only the renal capsule peeling was performed.At the end of the operation,the rats were restoratively reared for 2 weeks,and 40 rats of the model group were randomly divided into body weight groups:model group,EPO group,Shengxuening group and Wenshen Yisuisheng blood group.Rat model rats were given 180mg/Kg-d adenine every morning for intragastric administration.In the afternoon,the corresponding treatment groups were given the corresponding therapeutic drugs.Normal,sham operation and model group were given equal volume of deionized water(1ml/100g),the experiment totaled 12 weeks.2.Rat kidney weight and kidney weight ratioDuring the experiment,the mental state,fur gloss,diet,activity and other conditions of the rats in each group were closely observed.The body weight of the rats was measured weekly and recorded.After the end of the modeling treatment,the kidney tissues of each group were preserved and weighed.3.Blood index testAfter 12 weeks,blood was collected from the femoral artery of the rats,and the levels of RBC,HGB,Scr,BUN,UA,serum NO,MDA and SOD in the blood were measured.4.HE,Mallory stainingThe changes of renal tissue structure in each group were observed under light microscope.5.Immunohistochemistry,Western BlotThe expression of EPO,PI3K,AKT,p-AKT and bcl-2 protein in rat kidney tissues was detected in each group.6.In situ hybridization,RT-PCR experiments:Detection of EPO,PI3K,AKT,p-AKT,and bcl-2 mRNA expression in renal tissues7.Image and data analysis Immunohistochemistry and in situ hybridization were randomly selected from 8 fields.The results were analyzed by Image-Pro Plus 6.0 image analysis software,and the data were statistically analyzed with SPSS 20.0 software.Result:1 Pharmacodynamic study of Wenshen Yisui Shengxue Recipe on RA ratsAfter 12 weeks of drug treatment,compared with the normal and sham-operated groups,the rats in the model group were generally in poor condition,and the body weight was significantly decreased(P<0.01),and the kidney weight-to-weight ratio was significantly increased(P<0.01).Compared with the model group,the general condition of the rats in the Wenshen Yisuishengxue group was significantly improved;the body weight of the rats in the Wenshen Yisuishengxue group was significantly increased(P<0.01),and the weight ratio of kidney to weight was significantly decreased(P<0.01).Compared with the normal and sham-operated groups,the serum levels of BUN,Scr,and UA in the model group were significantly increased(P<0.01),and the levels of RBC and HGB in the whole blood were significantly decreased(P<0.01).Compared with the model group,Wenshen Yisui The levels of serum BUN,Scr and UA in the blood group were significantly decreased(P<0.01),and the levels of RBC and HGB in the whole blood were significantly increased(P<0.05).2.Effect of Wenshen Yisui Shengxue Recipe on oxidative stress in RA ratsCompared with the normal and sham-operated groups,the NO of the RA rats in the model group was significantly decreased(P<0.05),and SOD of the RA rats in the model group were significantly decreased(P<0.01),and the MDA was significantly increased(P<0.01).Compared with the model group,the NO of the rats in the Wenshen Yisuisheng group were compared with the model group.The SOD content increased significantly(P<0.01),and the MDA content decreased significantly(P<0.01).3.Effects of Wenshen Yisui Shengxue Recipe on expression of EPO,PI3K,AKT,p-AKT and bcl-2 proteins in kidney of RA ratsImmunohistochemistry showed that the expression of EPO and PI3K protein in the cytoplasm of renal tissue of normal group and sham operation group was significantly higher than that of the model group(P<0.01).Compared with the model group,the renal parenchyma cells in each treatment group were compared.Both EPO and PI3K increased,and the Wenshen Yisuisheng group increased significantly(P<0.01).The expression of AKT was expressed in the cytoplasm and nucleus of the kidney.There was no significant difference in the expression of AKT between the two groups(P>0.05).p-AKT was abundantly expressed in the cytoplasm and nucleus of normal and sham-operated renal parenchyma cells,which was significantly higher than that in the model group(P<0.01).After drug treatment,p-AKT,p-AKT/AKT in each treatment group.The expression was increased compared with the model group,and the Wenshen Yisuisheng blood group was significantly increased(P<0.01).The expression of bcl-2 in renal tissue was significantly higher in the normal group and the sham-operated group,which was significantly higher than that in the model group(P<0.01).The expression of the treatment group was significantly increased after treatment,and the Wenshen Yisuisheng group was significantly increased(P<0.01)..Western Blot results showed that compared with the normal group and the sham operation group,the expression of EPO,PI3K,p-AKT,p-AKT/AKT,and bcl-2 protein in the model group was significantly lower(P<0.01),and EPO in each treatment group.The expressions of PI3K,p-AKT,p-AKT/AKT and bcl-2 protein were significantly higher than those in the model group,and the Wenshen Yisui blood group was significantly increased(P<0.01).There was no significant difference in the expression of AKT protein between the two groups(P>0.05),indicating that there was no significant difference in the expression of AKT between the groups.4.Effect of Wenshen Yisui Shengxue Recipe on expression of EPO,PI3K,AKT and bcl-2mRNA in kidney of RA ratsThe results of in situ hybridization showed that the expression of EPO,PI3K and bcl-2 mRNA in the renal tissue of the model group was significantly lower than that in the normal and sham-operated groups(P<0.01).Compared with the model group,EPO,PI3K and bcl-2 mRNA in each treatment group.The expression was significantly increased,and the Wenshen Yishen blood group was significantly increased(P<0.01).There was no significant difference in AKT expression between the two groups(P>0.05).RT-PCR results showed that a large number of EPO,PI3K,and bcl-2 mRNA were expressed in the renal tissues of the normal group and the sham-operated group,and the expression of the model group was significantly decreased(P<0.01).Compared with the model group,EPO,PI3K,and bcl-2 mRNA were significantly increased in each treatment group,and the blood production of Wenshen Yisui was significantly increased(P<0.01).There was no significant difference in AKT expression between the experimental groups(P>0.05).Conclusion:1.Wenshen Yisui Shengxue Decoction can improve the general state of RA rats,can increase the body weight of RA rats,reduce the weight ratio of kidney weight,and can reduce the pathological damage of renal tissues of RA rats,and protect the kidneys.Play a good role.Wenshen Yisui Shengxue can increase the content of RBC and HGB in peripheral blood of RA rats,improve renal function,reduce serum Scr,BUN,UA,improve anemia and renal function.2.Wenshen Yisui Shengxue Recipe reduced the serum MDA content,increased NO and SOD levels in RA rats,and had a good corrective effect on oxidative stress in rats.3.Wenshen Yisui Shengxue significantly increased the expression of EPO in the kidney,corrected anemia,and activated the downstream apoptosis-related PI3K/AKT pathway through EPO,up-regulated the expression of bcl-2,and played an anti-apoptotic effect on the renal tissue of RA rats.Protect the kidneys.Wenshen Yisui Shengxue Recipe can increase the expression of endogenous EPO in the kidney,thereby activating PI3K/AKT signaling pathway,up-regulating the expression of anti-apoptotic gene bcl-2,and achieving renal protection against renal innate apoptosis,improving anemia On the basis of maintaining normal kidney structure and protecting kidney function. |
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