Study On The Effect And Mechanism Of Chuankezhi Injection On Rats With Focal Segmental Glomerulosclerosis Model

Focal segmental glomerulosclerosis(FSGS)is one of the important pathological types of steroid-resistant nephrotic syndrome(SRNS)in children.It is easy to develop into endstage renal disease(ESRD)because of its massive proteinuria and rapid progression of renal fibrosis,Causing serious harm to children.A survey in China shows that from 1984 to 2011,children in South China suffered from FSGS,accounting for 21.2%of children with primary nephrotic syndrome(PNS).In the treatment of Western medicine,the effect of conventional hormone therapy on FSGS is poor.Immunosuppressant is the main drug for the treatment of FSGS,but there are also adverse reactions,such as large individual differences in efficacy,destruction of gonads,infertility,increased cardiovascular disease,nephrotoxicity,refractory fungal infection,and so on.For children with poor effects of immunosuppressant treatment,blood purification treatment is even needed.Therefore,it is necessary to explore its mechanism of action and treatment methods.In the course of exploring FSGS,Western medicine has discovered that the central event in the pathogenesis of FSGS is the pathology of the podocyte.FSGS is characterized by damage and loss of podocytes and is clinically characterized by massive proteinuria and fusion loss of ≥80%of podocyte podocytes.Podocytes are an important structural basis for the glomerular capillary filtration membrane and synthesize proteins of the fissure septum and many extracellular matrix components of the glomerular basement membrane.When podocytes are damaged or absent,they cause massive proteinuria as well as progressive glomerulosclerosis.Th17/Treg cell immune disorder is related to podocyte injury.IL-17 secreted by Th17 cells can induce podocyte injury,down-regulate podocyte protein expression,and induce podocyte apoptosis.Overexpression of TGF-β1 secreted by CD4+CD25+Treg cells is closely associated with damage to podocytes.Therefore,by regulating Th17/Treg disorder,podocytes can be protected,proteinuria can be reduced,and glomerulosclerosis can be alleviated.Starting from the pathogenesis theory and combined with the clinical manifestations of FSGS,traditional Chinese Medicine(TCM)found that kidney yang deficiency is one of the important pathogenesis of FSGS.Through the use of single traditional Chinese medicine or prescription to warm and tonify kidney yang,it can alleviate or eliminate hormone resistance,sensitize hormone,protect glomerular podocytes,reduce albuminuria and reduce renal fibrosis.The classic traditional Chinese medicine for warming and Tonifying Kidney Yang,chinese herbs Epimedium sagittatum and Morinda officinalis and their extracts have proved to have good auxiliary efficacy in the study of oral intervention of FSGS.Chuankezhi injection(CKZ)is a TCM injection extracted from Chinese herbs Epimedium sagittatum and Morinda officinalis.It has the effect of warming yang and tonifying kidney.Modern studies have shown that it can improve diabetic nephropathy and regulate Th17/Treg dysfunction.However,CKZ has not been studied in the field of FSGS.Whether it works on FSGS and what is the mechanism of action are worthy of further research and verification.Objective1.This study investigated the efficacy of CKZ in FSGS model rats and its effect on the regulation of Th17/Treg cell immune disorder in the model rats through the animal experiment of adriamycin(ADR).2.This study predicted the possible mechanism of action of CKZ in the treatment of FSGS through network pharmacological analysis and molecular docking,and verified the mechanism of action of CKZ by detecting the mRNA expression and protein expression of the targets in the prediction mechanism of action.MethodsWe prepared the FSGS model in male SD rats by injecting Adriamycin at the tail vein.Based on group intervention and comparison,the primary efficacy parameters of FSGS were observed,including general condition,24-hour urine protein,serum albumin,cholesterol,triglyceride,and renal pathological changes.Differences between groups were determined.Network pharmacological analysis and molecular docking were used to predict the mechanism of action of CKZ.Finally,we used qPCR and WB to detect mRNA expression and protein phosphorylation at specific targets in rat kidney tissue to validate the predicted results in the network pharmacological analysis.1.Efficacy exploration:Modeling:58 male SD rats were randomLy divided into normal control group(N,n=8)and modeling group(n=50).FSGS rats were established by injecting Adriamycin hydrochloride at the tail vein.Specific methods:Adriamycin 4 mg/kg was injected into the tail vein in the first week;Adriamycin 2 mg/kg was injected into the tail vein in the second week;The modeling time was 6 weeks in total.At the end of 6 weeks after modeling,two model rats were randomLy selected to determine that the 24h urinary protein content was more than 100 mg/kg,and focal segmental sclerosis of glomerulus appeared under the optical microscope,then the modeling was successful.Grouping and intervention:After that,the remaining 48 rats were randomLy classified into six groups(n=8):a model group(M),a prednisone group(P),a high-dose group(CKZ1),a medium-dose group(CKZ2),a low-dose group(CKZ3),and a combined therapy group(CKZ1+P).Rats in the N and M groups were intramuscularly injected with normal saline daily(1mL/kg·d)for 6 weeks.P group was orally administered with prednisone aqueous solution(12.6 mg/kg·d).CKZ1,CKZ2,and CKZ3 groups were intramuscularly injected with CKZ,0.72,0.36,and 0.18 mL/kg·d,respectively,daily for 6 weeks.CKZ1+P group was orally administered with prednisone aqueous solution(12.6 mg/kg·d)and intramuscularly injected with CKZ(0.72 mL/kg·d),daily for 6 weeks.Indicators:weigh the body weight once a week,collect 24-hour urine with metabolic cage before the end of the intervention,and measure the quantity of urinary protein;After the intervention,serum albumin,triglyceride,cholesterol,creatinine,and Th17/Treg related immune indexes were measured;After the intervention,the kidney was stained with HE and Masson to make pathological sections of renal tissue,and pathological sections were observed 40x,200x,and 400x under an optical microscope.Imaging software was used to collect images.2.Mechanism of action exploration:determine the metabolic components of CKZ by obtaining the data of mass spectrometry detection,and rat pharmacokinetic experiment;Then,using the method of network pharmacology,construct the "effective component target pathway" network of CKZ,and then construct the protein-protein interaction network(PPI),gene ontology(GO)enrichment and kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis,to obtain the possible core components,disease targets,biological processes,cellular components,molecular functions and signaling pathways of FSGS treated with CKZ.And then the molecular docking verification between the core components of CKZ and the core target of the disease was carried out.Finally,we used real time-polymerase chain reaction(Rt-PCR)and western blot(WB)to detect mRNA expression and protein phosphorylation at specific targets in rat kidney tissue to validate the predicted results in the network pharmacological analysis.Results1.Efficacy exploration results:FSGS rat model was successfully replicated.Intramuscular injection of CKZ had a dose-dependent effect in FSGS model rats,including lowering urine protein,increasing serum albumin,lowering cholesterol and triglyceride,and treating pathological lesions in the kidney.Neither intragastric prednisone nor intramuscular injection of CKZ combined with intragastric prednisone showed the above manifestations.The intervention groups had different degrees of modulating effects on Th17 and IL-17A.The specific results were as follows:(1)General conditions:Compared with group N,group M was depressed,unresponsive,withered hair,reduced food and water intake,loose stools,and decreased body weight.Compared with the M group,the CKZ1 and CKZ2 groups were in good mental condition,responsive,with shiny hair,normal food,and water intake,basically formed stools,and good weight gain,which were close to the normal control group.the performance of the P group,CKZ3 group,and CKZ1+P group was similar to that of the M group.(2)Measurement of 24-hour urine protein:Compared with the N group,the level of 24hour urine proteins in the M group significantly increased(P<0.01).Compared with the M group,the CKZ1 group significantly reduced 24-hour urine protein(P<0.01)and the CKZ2 group significantly reduced 24-hour urine protein(P<0.05).there was no significant difference between the P group,the CKZ3 group,and the CKZ1+P group compared with the M group(P>0.05).(3)Blood biochemistry:Compared with group N,group M had significantly lower ALB(P<0.001),significantly higher CHOL(P<0.001).significantly higher TG(P<0.05).and no significant difference in CR(P>0.05).Compared with the M group,ALB was significantly higher(P<0.01),CHOL was significantly lower(P<0.001),TG was significantly lower(P<0.05),and CR was not significantly different(P>0.05)in the CKZ1 group,and ALB was significantly higher(P<0.05),CHOL was significantly lower(P<0.05),TG was significantly lower(P<0.05),and CR was not significantly different(P>0.05)in the CKZ2 group.There was no significant difference in CR(P>0.05).there was no significant difference in the above blood biochemical indexes in the P group,CKZ3 group,and CKZ1+P group compared with the M group(P>0.05).(4)Pathological observation of the kidney:In this experiment,HE and Masson-stained pathological sections of the kidneys of rats in each group were observed under an optical microscope.The renal tissue structure of group N was normal,and obvious pathological damage occurred in the M group,P group,CKZ3 group,and CKZ1+P group.In the M group,there were glomerular capillary occlusion,mesangial hyperplasia,severe adhesion of balloons,podocyte swelling or vacuolar degeneration,renal tubular expansion,partial abscission of renal tubular epithelial cells,and protein tubular type were observed;we found that the renal tubules were dilated,the epithelial cells of renal tubules were partially exfoliated,and protein casts were observed;Renal interstitial fibrosis increased significantly.Among the other groups,the renal lesions in the CKZ1 group and CKZ2 groups were significantly improved.The CKZ high-dose group showed the slightest pathological changes in the kidney.No apparent lesions were found in the glomerulus and tubules,under an optical microscope,which was consistent with the renal pathological manifestations of the N group.Compared with the N group,the number of FSGS in the M group increased significantly(P<0.001),the number of FSGS in groups CKZ1 and CKZ2 decreased significantly(P<0.001),and there was no significant difference in the number of FSGS in group P,CKZ3 and CKZ1+P compared with group M(P>0.05).The lesion rate of FSGS in each group:compared with group N,the lesion rate of FSGS in group M increased significantly(P<0.001),the lesion rate of FSGS in the CKZ1 group and CKZ2 group decreased significantly(P<0.001),and there was no significant difference in the lesion rate of FSGS in group P,CKZ3 group and CKZ1+P group compared with group M(P>0.05).(5)Cellular immunity:Compared with the N group,the expression levels of Th17 cells were significantly increased(P<0.001),IL-17A expression levels were significantly increased(P<0.001),Treg cell expression levels were not significantly different(P>0.05),and Th17%/Treg%were not significantly different(P>0.05)in the M group.Compared with the M group,the CKZ1 group showed a significant decrease in Th17 cell expression level(P<0.01),IL-17A expression level(P<0.01),no significant difference in Treg cell expression level(P>0.05),and no significant difference in Th17%/Treg%(P>0.05),and the CKZ2 group showed a significant decrease in Th17 cell expression level(P<0.05),no significant difference in IL-17A expression level(P>0.05),no significant difference in Treg cell expression level(P>0.05),no significant difference in Th17%/Treg%(P>0.05),and a significant decrease in Th17 cell expression level(P<0.05),IL-17A expression level(P<0.05),in the CKZ3 group Treg cell expression level was not significantly different(P>0.05),Th17%/Treg%was not significantly different(P>0.05),Th17 cell expression level in CKZ1+P group was significantly decreased(P<0.05),IL-17A expression level was significantly decreased(P<0.05),Treg cell expression level was not significantly different(P>0.05),No significant difference in Th17%/Treg%(P>0.05),and no significant difference in Th17 cell expression level(P<0.05),IL-17A expression level(P>0.05),Treg cell expression level(P>0.05),and Th17%/Treg%(P>0.05)in the P group.2.Mechanism of action exploration results:network pharmacological analysis showed that five active ingredients of Epimedium flavonoids(i.e.Icariin,Icariside II,Epimedin C,Icaritin,and Noricaritin)may be the key ingredients.AKT1 was probably the key target gene.These components may affect protein binding and protein kinase binding in nucleus or cytoplasm,resulting in negative regulation of apoptosis and positive regulation of protein phosphorylation.They play a role in the treatment of FSGS through PI3K-Akt signaling pathway.Results of Rt-PCR and WB showed that intramuscular injection of CKZ significantly increased the relative mRNA expression and protein phosphorylation of PI3K and Akt,respectively.Conclusions1.In this study,CKZ showed significant efficacy in the FSGS model rats,specifically in reducing urine protein,elevating serum albumin,lowering triglycerides and cholesterol,and improving renal pathological damage.2.In this study,the results showed that the mechanism of action of CKZ on FSGS model rats may be associated with the activation of the PI3K-Akt signal pathway to protect podocytes.