| BackgroundIdiopathic membranous nephropathy(IMN)is one of the most common pathological types of nephrotic syndrome in adults,and it is also the second largest pathological type of primary glomerular disease at present.About 30%of patients can progress to End-stage renal disease(ESRD),within 15 years and 40%of patients can seriously affect their quality of life.In recent years,the clinical incidence of IMN in China is increasing year by year,with an annual growth rate of 13%.Therefore,it is of great social and economic significance to pay attention to the prevention and treatment of IMN.Studies have confirmed that IMN is an autoimmune kidney disease mediated by anti-podocyte antibodies.Membrane attack complex C5b-9 induces podocyte epithelial-mesenchymal transdifferentiation(EMT),resulting in abnormal expression of podocyte-associated proteins and damage of glomerular filtration barrier,resulting in proteinuria.In severe cases,it can lead to podocyte detachment or apoptosis from the glomerular basement membrane,aggravating proteinuria and glomerulosclerosis.At present,Western medicine is mainly treated with immunosuppressants,but because the pathogenesis of IMN is not completely clear,some patients can not be relieved after treatment,and gradually progressed to end-stage kidney disease.In recent years,more and more traditional Chinese medicine scholars have devoted themselves to the research of traditional Chinese medicine in IMN.Based on the results of a large number of domestic studies,traditional Chinese medicine has unique advantages in the treatment of IMN,which can improve patients' clinical symptoms and delay the progress of renal function.Professor Wang Baokui believes that IMN disease belongs to deficiency and excess,and the core pathogenesis is "deficiency of vital qi and evil of wind entering the kidney".Therefore,Fuzhengqufengfang is developed to guide clinical treatment.Previous studies have proved that it can significantly reduce proteinuria in patients with IMN,reduce the level of proteinuria in IMN rats,improve kidney pathology,and regulate the expression of proteins related to some podocytes.The mechanism of its protection of podocytes remains to be further explored.It has been confirmed that there is a close relationship between podocyte EMT and IMN proteinuria.The purpose of this study is to explore the regulation mechanism and target of Fuzhengqufengfang on EMT of IMN podocytes,so as to clarify the effective mechanism of Fuzhengqufengfang in the treatment of IMN,so as to provide scientific basis for popularizing clinical effective prescription and optimizing IMN treatment plan,and provide reliable new ideas for traditional Chinese medicine in the treatment of IMN.PurposeThrough animal experiment,the model of C-BSA glomerulonephritis was established to observe the effect of Fuzhengqufengfang on albuminuria,blood biochemical indexes(BUN,Scr),kidney pathology,podocyte transdifferentiation signal pathway proteins(TGF-?1,Smad2,Smad3,Smad7)and the phenotypic marker Desmin of podocyte transdifferentiation in C-BSA rats,and to explore the regulatory effect of Fuzhengqufengfang on podocyte transdifferentiation in C-BSA rats.Methods1.90 healthy male SD rats of SPF grade,whose growth time was about 6 weeks,were selected and fed adaptively for one week,during which they drank and fed freely.After 24-hour urinary protein content(24 hU-pro)was determined to be less than 5mg,the rats were randomly divided into control group(n=15)and model group(n=75).2.The rat model of C-BSA was established,and the model group referred to Border method.C-BSA was injected into the tail vein,and then the determination of 24 hU-pro>20mg indicated that the model was successful.3.After the establishment of the model,the rats in the model group were randomly divided into model group,high,medium and low dose traditional Chinese medicine group and western medicine group.The high,middle and low dose traditional Chinese medicine groups were given the corresponding dose of Fuzhengqufengfang,the western medicine group was given losartan potassium tablets,and the normal group and model group were given the same amount of deionized water for 4 weeks.4.The general condition of rats in each group was observed and 24 hours after intragastric administration for 2 and 4 weeks,the blood samples of abdominal aorta were taken to detect blood biochemical indexes(BUN and Scr),),and the renal tissues of each group were retained,and the pathological changes of renal tissue were observed under electron microscope and light microscope respectively.The expression of podocyte transdifferentiation signal pathway proteins TGF-?1,Smad2,Smad3,Smad7 and Desmin,a phenotypic marker of podocyte transdifferentiation,were detected by Westernblot method,and the differences among the groups were compared.5.All the data were processed by SPSS22.0 statistical software.The difference was statistically significant(P<0.05).Results1.General situationThe rats in the control group had good mental state,sensitive response,shiny hair and no obvious abnormality in stool and urine.During the modeling period,the model rats gradually appeared lethargy,decreased response sensitivity,yellowed hair,dull hair,even shedding,reduced food intake,increased urine volume,and stool gradually turned loose.Part of the pre-immune site of the back of the neck showed depilation to skin exposure or caudal vein injection site necrosis,given local disinfection treatment,and some rats could see ascites and scrotal edema.After intervention treatment in the drug group,compared with the above model group,the mental state,reaction sensitivity,hair loss,defecation and other general conditions were relatively better,and gradually improved after intragastric administration.There was no significant difference between drug groups.2.24-hour urinary protein quantificationThe results showed that the proteinuria of rats in the model group and drug group was significantly higher than that in the control group and before modeling(P<0.01).The results showed that the proteinuria of rats in the model group and drug group was significantly higher than that in the control group and before modeling(P<0.01).After intragastric administration for 2 weeks and 4 weeks,the level of proteinuria in the drug group was still higher than that in the control group(P<0.01),but lower than that in the model group(P<0.05).Compared with high-dose group and low-dose group,the level of proteinuria in middle-dose traditional Chinese medicine group was significantly lower than that in high-dose group and low-dose traditional Chinese medicine group(P<0.05).Compared with the western medicine group,the decrease of albuminuria was statistically significant(P<0.05).3.Biochemical index changesThe levels of BUN and Scr in the model group were higher than those in the control group.Compared with the model group,there was significant difference in the decrease of BUN in the drug group,and there was no significant difference between the middle dose traditional Chinese medicine group and the high dose traditional Chinese medicine group and the low dose traditional Chinese medicine group,but there was significant difference in the decrease of BUN between the high dose traditional Chinese medicine group,the middle dose traditional Chinese medicine group,the low dose traditional Chinese medicine group and the western medicine group.Compared with the model group,the Scr of the drug group decreased significantly only in the middle dose traditional Chinese medicine group(P<0.05).4.Pathomorphological observation of renal tissue5.Compared with the control group,the foot process of the model group was widely fused,the basement membrane was thickened,a large number of electron densities were deposited under the epithelium,and the glomeruli were enlarged,the basement membrane thickened and vacuolated under light microscope.Edema of renal tubular epithelial cells accompanied by deposition of subepithelial erythrophil.After intragastric administration,all doses of traditional Chinese medicine group and western medicine group were alleviated to a certain extent compared with the model group,the deposition of subepithelial immune complex decreased under electron microscope,the thickening of basement membrane and foot process fusion of podocyte were alleviated,and the thickening of basement membrane and vacuolar degeneration of renal tubular epithelium were alleviated under light microscope,and the deposition of erythrophil complex protein decreased,and the pathological degree of middle dose traditional Chinese medicine group was more significant on the whole.6.Expression of podocyte transdifferentiation signal pathway proteins(TGF-?1,Smad2,Smad3,Smad7)and phenotypic marker Desmin of podocyte transdifferentiationThe results showed that the expression of TGF-?1 protein in glomeruli of model group was significantly higher than that of control group(P<0.01).The expression of),Desmin protein was significantly higher than that of phosphorylated Smad2 and phosphorylated Smad3,while the expression of Smad7 protein was significantly decreased(P<0.05).After 4 weeks of administration,compared with the model group,the expression of TGF-?1 protein in the glomeruli of rats in the drug group decreased significantly in the middle dose traditional Chinese medicine group(P<0.05).The expression of Desmin protein in glomeruli of rats in the drug group showed that the expression of Smad7 protein in the glomeruli of the rats in the high-dose group and the middle-dose group was significantly lower than that in the middle-dose group(P<0.05),and there was no significant difference between the high-dose group and the middle-dose group(P>0.05).The expression of Smad7 protein in the glomerulus in the drug group showed a significant increase(P<0.05),and compared with the middle-dose group,there was no significant difference between the high-dose group and the middle-dose group.The expression of Smad2 and p-Smad2 protein in glomeruli of rats in the drug group showed that the phosphorylated Smad2 in the middle dose group decreased significantly,while the expression of Smad3 and p-Smad3 protein in the glomeruli in the high dose group decreased significantly(P<0.05).ConclusionFuzhengqufengfang can improve the general condition of rats,reduce proteinuria,BUN and Scr,and improve renal pathological damage in C-BSA model rats.At the same time,Fuzhengqufengfang can reduce the expression of TGF-?1 protein in the signal pathway of podocyte transdifferentiation,reduce the ratio of phosphorylated Smad2 to phosphorylated Smad3,increase the expression of Smad7 protein,down-regulate the phenotypic marker Desmin protein of podocyte transdifferentiation in C-BSA model rats,and improve podocyte transdifferentiation.The overall curative effect is more significant in the middle dose of Fuzhengqufengfang.It can also be concluded that the regulation of Fuzhengqufengfang on podocyte transdifferentiation signal pathway proteins(TGF-?1,Smad2,Smad3,Smad7)and Desmin protein,which is a phenotypic marker of podocyte transdifferentiation,may be one of the protective mechanisms of podocyte in rats with membranous nephropathy. |
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