| Objective: Membranous nephropathy was emphasized as the most common cause of Nephrotic syndrome in adults.In recent years,there was a significant increase in the prevalence of membranous nephropathy,but the etiology of the disease remains to be further clinical research.Membranous nephropathy is diffuse lead to autoimmunity in glomerular epithelial cell immune complex deposition and thickening of the glomerular basement membrane with "spikes" form for the pathological features of a common nephritis type and clinical manifestations for heavy proteinuria,hypoalbuminemia,severe edema and hyperlipidemia.Patients with membranous nephropathy,whose blood is often in a hypercoagulative state,which is an important factor leading to disease progression.Salvia miltiorrhiza can lower the blood viscosity,Improve the blood coagulation state in patients with membranous nephropathy,and improve renal microcirculation to improve renal function and delay the progression of chronic kidney disease.In this study,we established rat models of membranous nephropathy by injecting cationic bovine serum albumin(C-BSA)into tail vein,and with low,medium and high dose of Salvia miltiorrhiza to intervene,and the control group was treated with Benazepril.By observing the rats 24 hours urine protein quantitation(24hUPQ),serum total protein(TP),albumin(ALB),total cholesterol(TC),triglyceride(TG),blood urea nitrogen(BUN),serum creatinine(Scr)and other biochemical indexes and the expression of CD2-associated protein(CD2AP)and Desmin protein in renal tissue.To investigate the therapeutic effect and mechanism of Salvia miltiorrhiza on the membranous nephropathy induced by cationic bovine serum albumin in order to provide theoretical basis for clinical application of Salvia miltiorrhiza polyphenols in the treatment of membranous nephropathy.Methods: The clean grade healthy male SD rats were selected,whose weight were 180±20g.The urine protein was negative after 1 week of adaptive feeding and the measurement of 24 hours urinary protein quantitation was Less than 5mg.The rats were randomly divided into normal group of 10 and build module group of 50,and the normal group rats were numbered.build module group rats were subcutaneously injected with C-BSA emulsified with incomplete adjuvant in Double arm and double groin.Rats in normal group were given equal volumes of normal saline In the same part.They were all premunited 1 week,once every other day.formal immunization was started form the third week.Build module group rats were injected C-BSA in tail vein,and the normal group rats were injected equal amount of normal saline.Immune time: once every other day,continuous for 4 weeks.After the latest immunization,the urine protein of rats in the build module group were all positive tested by urine protein test paper.According to the completely random method,rats of build module group were randomly divided into 5 groups: model group,Benazepril group,Salvia miltiorrhiza of low,medium and high dose groups,and gave them the label.Then taken all the rats 24 hours urine in metabolism cages,and tested the the 24-hour urinary protein quantitative.Benazepril group: Rats were gavaged with Benazepril at a dosage of 10mg/kg/d(mixed with 3ml distilled water before administration),and were received 1.5ml normal saline through intraperitoneal injection every rat simultaneously.Salvia miltiorrhiza of low,medium and high dose groups: Salvia miltiorrhiza were gaven at a dosage of 16.7mg/kg/d,33.3mg/kg/d and 66.7mg/kg/d(mixed into a solution with 1.5ml normal saline before injection)respectively,and 3ml distilled water was orally gaven to each rat simultaneously.Normal group and model group: Every rat was gaven 3ml distilled water orally and 1.5ml normal saline by intraperitoneal injection.Time: 1 times a day,4 weeks.All rats were free to eat and drink.After 4 weeks all rats were taken into metabolic cages for 24 hours urine and tested 24 hour urine protein.Then anesthetized rats by intraperitoneal injection,collected blood from the femoral artery,and measured TP,ALB,TC,TG,BUN,Scr and other indicators of rats in each group.Finally,picked kidney tissue used for light microscopy,electron microscopyand immunofluorescence to observe the glomerular basement membrane,podocyte and immune complex deposition.And were used for Immunohistochemistry and PCR to detect the protein of CD2 AP and Desmin,and their expression in renal tissue.Then,the quantitative analysis was made by computer image analysis system.Results:1 Comparison of 24 hour urinary protein quantityWith intravenous injection of C-BSA 4 weeks,except the normal group,all rats were massive proteinuria,which was significantly larger than that in normal group(P<0.01).While urinary protein of Benazepril group and three Salvia miltiorrhiza groups was declined significantly,there is a obvious difference compared with that in model group(P<0.01);The decrease of urinary protein in three Salvia miltiorrhiza groups was not obvious compared with that in Benazepril group(P>0.05);Compared with the low dose Salvia miltiorrhiza group,the middle and high dose group decreased more significantly,and the high dose group was the most obvious,but there was no statistical significance(P>0.05).2 Comparison of the blood biochemical indexSerum TP,ALB levels were significantly decreased and TC,TG levels were significantly increased in model group,Benazepril group and three Salvia miltiorrhiza groups compared with those in normal group(P<0.05).Levels of TP,ALB were significantly elevated and TC,TG were dramatically decreased in Benazepril group and three Salvia miltiorrhiza groups than those in model group(P<0.05).But there was no obvious change between three Salvia miltiorrhiza groups and Benazepril group(P>0.05).Compared with low dose Salvia miltiorrhiza group,serum TP,ALB levels rised and TC,TG levels decreased more significantly in middle and high dose group,but the changes were not obvious(P>0.05).But there were no obvious changes between three Salvia miltiorrhiza groups and Benazepril group(P>0.05).The differences of BUN and Scr levels were not obvious among each group(P>0.05).3 The pathological changes of renal tissuesLight microscopy showed that rats glomerular structure had no significant abnormalities in normal group.There were huge addicted fuchsin protein deposition on Rats glomerular basement membrane,the glomerular basement membrane was thickened remarkableiy,and "Spikes" formed outside the glomerular basement membrane in model group.A fewer addicted fuchsin protein deposition on Rats glomerular basement membrane,and glomerular basement membrane thickened lightly and "Spikes" formed lesser in Benazepril group and three Salvia miltiorrhiza groups than that in model group.Electron microscope showed that rats had a clear structure of glomerular podocytes,foot processe arranged in good order in normal group.There were majority and large electron dense deposited on rats glomerular basement membrane,and podocyte foot processes integrated widely or disappeared in model group.A minority and small-sized electron dense deposited on rats glomerular basement membrane,with foot processe fusion was seen partially on rats of Benazepril group and three Salvia miltiorrhiza groups.Immunofluorescence showed that the rats in the normal group had no obvious change in glomerular,there were no fluorescent performance.Immunoglobulin IgG and C3 distributed extensively along the capillary loop,fluorescence intensity was obviously seen in model group.Less immunoglobulin IgG and C3 were visibled along the capillary loop,but the fluorescence intensity was weak in rats glomeruli of Benazepril group and three Salvia miltiorrhiza groups.Immunohistochemistry showed that CD2 AP protein expressed in glomerular subepithelial.There was a large quantity CD2 AP protein expressed in glomerular subepithelial in normal group,which was a linear distribution along the capillary loop,and the staining intensity was strongest.Compared with normal group,CD2 AP protein was significantly reduced in model group(P<0.05),and it could be seen as a granular or patchy distribution along the capillary loop,and the staining intensity was significantly weakened.Compared with model group,CD2 AP protein Increased significantly in Benazepril group and three Salvia miltiorrhiza groups(P<0.05),and the staining intensity was significantly enhanced.There was no obvious difference in staining intensity between Benazepril group and three Salvia miltiorrhiza groups(P > 0.05).Compared with the low dose Salvia miltiorrhiza group,the staining intensity in medium and high dose groups were more stronger,and the highest staining intensity was the high dose group of Salvia miltiorrhiza,but there was no statistical difference(P>0.05).Desmin protein expressed in mesangial cells mainly,weakly expressed in In the normal rat glomeruli.Desmin protein expressed strongerly along the glomerular basement membrane and distributed linearly in model group(P<0.05),compared with normal group.Prompted the expression of Desmin protein increased significantly.Compared with model group,the positive staining was weaker in the Benazepril group and Salvia miltiorrhiza each dose groups,reminded Desmin protein expression decreased,there was a significant difference(P<0.05).There was no obvious difference in staining intensity between Benazepril group and three Salvia miltiorrhiza groups(P>0.05).Compared with the low dose Salvia miltiorrhiza group,the staining intensity in medium and high dose groups were more weaker,and the staining intensity of the high dose Salvia miltiorrhiza group was the weakest,but there was no statistical difference(P>0.05).4 The expression of CD2 AP mRNA and Desmin mRNA in renal tissueStatistical analysis showed that,the expression of CD2 AP mRNA in model group was significantly reduced,and the difference was significant(P <0.05),compared with that in normal group.Compared with the model group,CD2 AP m RNA expression significantly increased in Benazepril group and Salvia miltiorrhiza each dose groups,and the difference is significant(P<0.05).There was no obvious difference in the expression of CD2 AP mRNA between Benazepril group and three Salvia miltiorrhiza groups(P>0.05).Compared with the low dose Salvia miltiorrhiza group,the CD2 AP mRNA in medium and high dose groups increased more obvious,and that was the most obvious in the high dose group of the salvia miltiorrhiza,but there was no statistical difference(P>0.05).Compared with the normal group,the expression of Desmin mRNA in model group was significantly increased,and the difference was significant(P <0.05).Compared with the model group,the expression of Desmin mRNA decreased and the difference is significant(P<0.05).There was no obvious difference in the expression of Desmin mRNA between Benazepril group and three Salvia miltiorrhiza groups(P>0.05).Compared with the low dose Salvia miltiorrhiza group,the Desmin mRNA in medium and high dose groups reduced more obvious,and that was the least in the high dose group of the salvia miltiorrhiza,but there was no statistical difference(P>0.05).Conclusion:1 The expression of CD2 AP reduced and Desmin increased in rats with membranous nephropathy,and which were consistent with the changes of proteinuria and renal pathologic manifestation,suggested that CD2 AP and Desmin are involved in membranous nephropathy podocyte injury and proteinuria.2 Salvia miltiorrhiza can significantly reduce urine protein,total cholesterol and triglycerides levels,and elevated serum albumin levels in membranous nephropathy rats.3 Salvia miltiorrhiza can raise kidney tissue CD2 AP protein expression and reduce Desmin protein expression in membrane nephropathy rats,suggested that the renal protective effects of Salvia miltiorrhiza on membranous nephropathy may be related to the protection of podocytes,maintaining the structure of podocyte and glomerular diaphragm,and the integrity of glomerular filtration function.4 Salvia miltiorrhiza can kidney tissue CD2 AP and desmin protein expression in membrane nephropathy rats,suggested that the renal protective effects of Salvia miltiorrhiza on membranous nephropathy may be related to the protection of podocytes,maintaining the structure of podocyte and glomerular diaphragm,and the integrity of glomerular filtration function. |
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