| Objective:1.To establish a rat model of IgA nephropathy,and investigate the effect of Gubentongluo Formula on 24-hour urine protein quantification in rats with IgA nephropathy.2.To exploring the mechanism of Gubentongluo Formula on proteinuria by regulating the expression of podocyte-associated proteins podocin,α-actinin-4 and serum B7-13.To explore the correlation between Xuanfu and Slit Dembrane,prove that the kidney Xuanfu is one of the microscopic structures of the podocyte fissure membrane and provide a new basis for TCM multi-target treatment of IgAN.Methods:1.Eighty SPF male SD rats were randomly divided into normal group,model group,Guben Tongluo Formula treatment group and losartan potassium treatment group,with 20 rats in each group.In addition to the normal group,the remaining group was administrated with bovine serum albumin 1ml/100 g every other day,subcutaneously injected with 0.1ml of carbon tetrachloride and 0.5ml of castor oil per week.The stomach lasted for 6 weeks and the injection lasted for 9 weeks.And lipopolysaccharide was injected at a dose of 0.05 mg/tail vein at weeks 6 and 8,respectively.2.We collect 24 h urine in rats immediately after modeling.The Chinese medicine group was given to the “Guben Tongluo Formula” at a dose of 0.5ml/100 g,and Western medicine group was given losartan potassium according to 1ml/100 g,and the treatment lasted for 4W.After treatment for 2W and 4W,10 rats in each group were randomly selected to collect blood,urine and kidney tissue samples for testing.Determination of serum creatinine and urea nitrogen were tested using biochemical methods,renal histopathology was detected by light microscopy,electron microscopy and immunofluorescence.The expression of pococin and α-actinin-4 mRNA in podocyte protein was detected by Real time-PCR,and the expression of B7-1 in serum was detected by ELISA.Immunohistochemical technique was used to detect the expression site and positive expression area of podocyte protein in kidney tissues.Results:1.Blood and urine biochemical results:There was no significant difference in serum creatinine and urea nitrogen between the groups(P >0.05).2.24-hour urine protein quantitative test results:Before treatment,the 24 h urine protein in the Chinese medicine treatment group and losartan group was higher than the normal group(P<0.05).After treatment,the 24 h urinary protein quantitation in the treatment group was lower than that in the model group(P <0.05)and the 24 h urinary protein level of the Chinese medicine group was lower than that of the losartan potassium group(P <0.05).3.The expression of podocyte protein podocin mRNA,α-actinin-4 mRNA:Compared with the model group,the expression of podocin mRNA was increased and the content of α-actinin-4 mRNA was significantly decreased in the Chinese medicine treatment group and losartan group(P<0.05).Compared with 2W treatment,the content of podocin mRNA in the 4W losartan group was significantly increased(P<0.01),and the content of α-actinin-4 mRNA in the 4W Chinese medicine treatment group was significantly lower than that in the losartan potassium group(P <0.05).4.Renal histopathology results:(1)HE staining display:In the model group,mesangial cells and matrix were moderately hyperplasia,inflammatory cells infiltrated,basement membrane thickened,with small blood vessel sclerosis,and lumen narrowing;the Chinese medicine treatment group and losartan group was lighter than the model group.(2)Immunofluorescence staining showed:The fluorescence intensity of IgA in the mesangial area of the model group was extremely high;the fluorescence intensity of the Chinese medicine treatment group and losartan group was significantly lower than that of the model group;the IgA fluorophore was rarely seen in the normal group.(3)The results of electron microscopy show:In the model group,mesangial cells proliferated,the matrix increased,and the mesangial area widened.The foot process is abnormal,some of the foot processes are fused and irregularly arranged,and there are suspicious electron dense deposits.5.Results of renal tissue immunohistochemistry:(1)Results of renal tissue podocyte-associated protein positive rate: after the treatment,compared with the Chinese medicine treatment group and losartan group,the expression of podocin-positive protein in the model group was increased in the kidney tissue,and the expression of α-actinin-4 positive protein in the kidney tissue was decreased.(2)The results of immunohistochemistry test shows: compared with the normal group,there were a large number of podocin and α-actinin-4 protein-positive brown-yellow particles deposited around the glomeruli in the model group,which were less distributed around the renal tubules and collecting ducts(P<0.01);the treatment group was brownish yellow compared with the model group(P >0.05).6.Serum B7-1 expression:Compared with the normal group,the expression of B7-1 was increased in the model group(P<0.05).Compared with the model group,the expression of B7-1 was decreased in the treatment group(P <0.05).Conclusion:1.The proteinuria level of IgAN rats can be reduced by Guben Tongluo Formula,which is more prominent than the losartan potassium group.2.The mechanism of reducing the IgAN proteinuria by Gubentongluo Formula and losartan potassium tablets may be achieved by regulating the expression of podocin and α-actinin-4 in podocytes to reduce the structural and functional damage of podocytes.3.The mechanism of Gubentongluo Recipe to reduce the occurrence and development of proteinuria may be through the increase of α-actinin-4 expression,blocking B7-1activation,reducing the damage of podocyte structure and maintaining the stability of podocyte function. |
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