| Objective:Based on the previous work,this study established a rat model of passive Heymann nephritis(PHN)to further explore the content of Th17/Treg in peripheral blood of the PHN model rats and the fissure membrane of the podocyte.The regulation of α-actinin-4 and CD2 AP provides a basis for the use of traditional Chinese medicine to prevent and treat membranous nephropathy.Method:Male Wistar rats were used to establish the classical PHN model.The successful modeling rats were randomly assigned into model group,cyclosporine group,Yiqi Huoxue Huashi formula group,Yiqi recipe group,Huoxue recipe group and Huashi recipe group,with 10 rats in each group.Another 10 normal rats were used as a blank control group.The treatment groups were given the corresponding drug by gastric gavage,and the rats in the blank control group and the model group were given an equal volume of 0.9% Na Cl solution.The treatment course was 6 weeks.Blood was drawn from the orbital venous plexus of rats at weeks 0,2,4,and 6,and 24-hour urine was collected in metabolic cages.The serum levels of albumin(Alb),creatinine(SCr)and urea nitrogen(BUN),and the 24-hour urine protein quantitation(UPRO)were measured by automatic biochemical analyzer.After intervention,the kidneys were collected.The morphological changes of kidney tissues were observed under light microscope after HE staining.The changes of glomerular Ig G deposition were observed by Ig G immunofluorescence.The morphological changes of glomerular basement membrane,podocytes and podocytic process were observed under a transmission electron microscopy.Changes of Th17/Treg content in peripheral blood of rats by flow cytometry.The m RNA and protein expression levels of α-actinin-4 and CD2 AP in kidney tissues were detected by RT-PCR,immunohistochemistry and Western blot.Result:1.Efficacy index: Compared with the blank control group,the levels of UPRO and SCr increased significantly(P<0.05)and the levels of Alb were significantly decreased(P<0.05)in the model group at each time point;The level of UPRO increased significantly(P<0.05)at weeks 0 and 2,the level of Alb decreased significantly(P<0.05)at each time point,the level of SCr increased significantly(P<0.05)at each time point,and the level of BUN increased significantly(P<0.05)at weeks 2,4 and 6 in the cyclosporine group;The level of UPRO increased significantly(P<0.05)at weeks 0,2,and 4 in Yiqi Huoxue Huashi formula group,the level of Alb decreased significantly(P<0.05),the level of SCr increased significantly(P<0.05)at each time point;The level of UPRO increased significantly(P<0.05),the level of Alb decreased significantly(P<0.05),and the level of SCr increased significantly(P<0.05)at each time point in the decomposed recipes.Compared with the model group,the level of UPRO decreased significantly(P<0.05)at weeks 4and 6,the level of Alb decreased significantly(P<0.05)at weeks 2,4 and 6,the level of SCr and BUN increased significantly(P<0.05)at weeks 2,4 and 6 in the cyclosporine group;The level of UPRO decreased significantly(P<0.05)at week 6 in the Yiqi Huoxue Huashi formula group;there was no significant difference in the decomposed recipes group(P>0.05).Compared with the cyclosporine group,the level of UPRO increased significantly(P<0.05)at weeks 4 and 6 the decomposed recipes group;The level of Alb increased significantly(P<0.05),the levels of SCr and BUN decreased significantly(P<0.05)at weeks 2,4 and 6 in the Chinese medicine treatment group.2.Th17/Treg: Compared with the blank control group,the level of Th17 increased significantly(P<0.05),the level of Treg decreased significantly(P<0.05),and the level of Th17/Treg increased significantly(P<0.05)in the model group at each time point;The level of Th17 increased significantly(P<0.05),the level of Treg decreased significantly(P<0.05),the level of Th17/Treg increased significantly(P<0.05)at week 0 in the treatment group;The level of Treg increased significantly(P<0.05)at weeks 4 and 6 in the cyclosporine group;The level of Treg increased significantly(P<0.05)at week 2 in the Yiqi Huoxue Huashi formula group,Yiqi recipe group and Huashi recipe group.Compared with the model group,the level of Th17 decreased significantly(P<0.05)at weeks 4 and 6,the level of Treg increased significantly(P<0.05)at week 2 and the level of Th17/Treg decreased significantly(P<0.05)at weeks 2,4 and 6 in the cyclosporine group;The level of Th17 and Th17/Treg significantly decreased(P<0.05)in the Chinese medicine treatment group at weeks 2,4 and 6;The level of Treg increased significantly(P<0.05)at weeks 4and 6 in the Yiqi recipe group and Huashi recipe group,and the level of Treg increased significantly(P<0.05)at weeks 2,4,and 6 in the Huoxue group.Compared with the cyclosporine group,the level of Treg increased significantly(P<0.05)at week 6 in the Chinese medicine treatment group.3.Renal pathology:(1)HE staining: Compared with the blank control group,the model group showed obvious glomerular enlargement under the light microscope,diffuse thickening of the glomerular basement membrane,capillary sputum compression,and partial kidney The tubular epithelial cells were swollen,and the damage of each treatment group was lower than that of the model group.(2)Ig G immunofluorescence: compared with the blank control group,Ig G fluorescence deposition in the glomerular capillary vasospasm and mesangial area of the model group and each treatment group was Diffuse scattered granularity,the fluorescence intensity of the model group was +++~++++,and the fluorescence intensity of each treatment group was ++~+++;(3)Electron microscopy: compared with the blank control group,the model group was visible.A large number of electron dense deposits are deposited under the skin,and the foot processes are widely fused or disappeared.Compared with the model group,the degree of glomerular lesions in each drug intervention group was alleviated.Comparison of the width of the foot process:Compared with the blank control group,the width of the foot process of the model group widened significantly(P<0.05),and the foot process fused.Compared with the model group,the foot process width of each treatment group reduced significantly(P<0.05),the foot process fusion alleviated.Compared with the cyclosporine group,there was no significant difference in the width of the foot process between the Chinese medicine treatment groups(P>0.05).4.Immunohistochemistry: Compared with the blank control group,the expression of α-actinin-4 protein in the kidney of the model group up-regulated significantly(P<0.05),and the expression of CD2 AP protein down-regulated significantly(P<0.05).The expression of α-actinin-4 protein up-regulated significantly(P<0.05)in Yiqi Huoxue Huashi formula group,Huoxue recipe group and Huashi recipe group.The expression of CD2 AP protein down-regulated significantly(P<0.05)in Yiqi Huoxue Huashi formula group and Yiqi recipe group.Compared with the model group,the expression of α-actinin-4 protein down-regulated significantly(P<0.05)and the expression of CD2 AP protein up-regulated significantly(P<0.05)in each treatment group.Compared with the cyclosporine group,the expression levels of α-actinin-4 protein up-regulated significantly(P<0.05)in the Yiqi Huoxue Huashi formula group,Huoxue recipe group and Huashi recipe group.5.RT-PCR: Compared with the blank control group,the expression level of α-actinin-4 m RNA up-regulated significantly(P<0.05),and the expression level of CD2 AP m RNA down-regulated significantly(P<0.05),there was no significant difference in each treatment group(P>0.05).Compared with the model group,the expression level of α-actinin-4 m RNA down-regulated significantly(P<0.05),and the expression level of CD2 AP m RNA up-regulated significantly(P<0.05)in the cyclosporine group.The expression of α-actinin-4 m RNA down-regulated Significantly(P<0.05)in Yiqi recipe group,the expression of CD2 AP m RNA up-regulated significantly(P<0.05)in Huoxue recipe group.Compared with the cyclosporine group,there was no significant difference in the expression levels of α-actinin-4 and CD2 AP m RNA in each Chinese medicine treatment group(P>0.05).6.WB: Compared with the blank control group,the expression level of α-actinin-4 protein up-regulated significantly(P<0.05)in the model group,and the expression level of CD2 AP protein down-regulated significantly(P<0.05).The expression level α-actinin-4 protein up-regulated significantly(P<0.05)in each treatment group;the expression level of CD2 AP protein significantly down-regulated(P<0.05)in Yiqi recipe group.Compared with the model group,the expression level of α-actinin-4 protein down-regulated significantly(P<0.05)and the expression level of CD2 AP protein up-regulated significantly(P<0.05)in the cyclosporine group.The expression level of α-actinin-4protein down-regulated significantly(P<0.05)in the Yiqi Huoxue Huashi formula group,Yiqi recipe group and Huoxue recipe;the expression of CD2 AP protein increased significantly(P<0.05)in Huoxue recipe group.Compared with the cyclosporine group,there was no significant difference in the expression levels of α-actinin-4 and CD2 AP between the Chinese medicine treatment groups(P>0.05).Conclusion:1.Yiqi Huoxue Huashi formulae and each decomposed group can regulate the balance of Th17/Treg by down-regulating Th17 cells and up-regulating Treg cells,thereby achieving the effect of preventing and treating passive Heymann nephritis rats.2.Yiqi Huoxue Huashi formula significantly reduces the urinary protein level and alleviates glomerular injury in PHN model rats,and the mechanism of action may be related to down-regulation of α-actinin-4 expression and up-regulation of CD2 AP expression.Moreover,among its decomposed recipes,Yiqi recipe can inhibit α-actinin-4expression and Huoxue recipe can promote CD2 AP expression. |
PDF Full Text Request