| Objective: To explore the mechanism of action of Huangkui capsule in the treatment of chronic glomerulonephritis(CGN)by combining proteomics and network pharmacology(NP),to provide a reference for the rational clinical use of drugs and a more adequate basis for the clinical promotion and application of Huang Kui capsule.Methods:(1)Cationic bovine serum albumin(C-BSA)was used to induce 40 SPF-grade SD rats to establish CGN rat model.The experiment was divided into 6 groups: Normal group(NOR,n=8),Model group(MOD,n=8),Losartan potassium group(LST,n=8),Huangkui capsule low-dose(HK-L,n=8),Huangkui capsule medium-dose(HK-M,n=8)and Huangkui capsule high-dose(HK-H,n=8)groups.After 4 weeks of administration,the levels of 24 h urine protein content,protein-to-creatinine ratio(PCR),Serum creatinne(Scr),blood urea nitrogen(BUN),serum tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),malondialdehyde(MDA)content and superoxide dismutase(SOD)activity were measured.Histopathological changes in the kidney were observed by hematoxylineosin staining(HE),and Ig G deposition in rat kidney was observed by immunofluorescence.(2)Kidney tissues from NOR,MOD and HK-M groups(denoted as HK group in subsequent experiments)were analyzed by proteomic assays to screen for differential proteins.Gene ontology(GO),Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis and protein-protein interactions(PPI)network analysis were performed on the differential proteins to explore the targets of CGN treatment with Huangkui capsule.(3)Based on network pharmacology technology,the chemical constituents of Huangkui capsule were collected through literature review.The 2D-SDF structures of each component were obtained using the Pub Chem database and then imported into the Swiss Target Prediction database to retrieve the action targets of Huangkui capsule using "human" as the study species.The CGN-related genes were retrieved from the Gene Cards database using the keywords of "Chronic Nephritic" and "Chronic glomerulonephritis".Map the action target of Huangkui capsule and CGN-related targets and draw a Venny diagram.The intersection part of the two is the potential action target of Huangkui capsule in treating CGN.Cytoscape 3.8.0 software was used to construct the componenttarget-CGN network of Huangkui capsule.GO and KEGG enrichment analysis and PPI network analysis were performed for the potential targets by Metascape and STRING databases.(4)The combined proteomics and network pharmacology analysis was performed to explore the action targets of Huangkui capsule for the treatment of CGN,and relevant experimental validation of key targets and pathways was performed through relevant physicochemical experiments.Results:(1)The pharmacological effect study of Huangkui capsule indicated that compared with NOR,the 24 h urine protein content,PCR,BUN,Scr,serum TNF-α,IL-6 and MDA content were significantly higher(P<0.05)and SOD activity was significantly lower(P<0.05)in rats of MOD group;after the intervention of Huangkui capsule,compared with MOD,the 24 h urine protein content,PCR,BUN,Scr,serum TNF-α,IL-6and MDA content were significantly lower(P<0.05)and SOD activity were significantly higher(P<0.05)in each group after intervention with Huangkui capsule compared with MOD.Through pathological examination,it was revealed that the glomerular volume of the rats in the MOD group showed signs of significant enlargement and the thickness of the basement membrane also showed different degrees of increase,and the diffuse deposition of Ig G fluorescence along the glomerular thylakoid or basement membrane was clearly visible in the kidney tissues.After treatment with Huangkui capsule,the increase in glomerular volume and basement membrane thickening of the rats were significantly reduced,and the phenomenon of Ig G deposition was significantly improved.(2)Proteomic results showed that a total of 8055 proteins were quantified in kidney tissues.2079 differential proteins(1238 up-regulated proteins and 841 down-regulated proteins)were identified from the kidney tissues of NOR and MOD groups,1374 differential proteins(502 upregulated proteins and 872 down-regulated proteins)were identified from MOD and HK groups.and a total of 962 differential proteins(576 upregulated proteins and 386 down-regulated proteins)were identified from NOR,MOD and HK groups.Bioinformatics analysis showed that the cellular localization of the differential proteins among the three groups was mainly in mitochondria,involving precursor metabolites,energy production,organic acid catabolism,cell respiration and other biological processes;the main molecular functions involved oxidoreductase activity,cell adhesion molecule binding,and electron transfer activity,etc.The KEGG pathway enrichment analysis indicated that the differential proteins among the three groups were enriched in the regulation of actin cytoskeleton,phosphatidylinositol 3 kinase-protein kinase B(PI3K-Akt),Peroxisome proliferator-activated receptor(PPAR)signaling pathways and other signaling pathways.Protein interactions revealed Actin cytoplasmic1(ACTB),protein-Serine-threoninekinase(AKT1),ATP synthase subunit alpha(ATP5A1),Fibronectin1(FN1),Succinate dehydrogenase(SDHA),Aconitase2(ACO2),ATP synthase subunit beta(ATP5B),Malatedehydrogenase2(MDH2),catalase(CAT),and Cytochrome c(CYCS)are important target proteins.(3)The network pharmacology study revealed that there were 462 targets corresponding to 50 main components of Huangkui capsule and1835 CGN-related targets,and 147 potential targets of Huangkui capsule for the treatment of CGN were obtained after taking the intersection.GO enrichment analysis showed 1053 biological processes(BP),55 cellular components(CC),and 88 molecular functions(MF).149 related pathways were enriched by KEGG,which mainly concentrated in PI3K-AKT,Tumor necrosis factor(TNF),Ras-related protein 1(Rap1),Mitogen activated protein kinase(MAPK),Nuclear factor-κB(NF-κB)and other signaling pathways.Protein interactions identified TNF,Albumin(ALB),AKT1,Caspase 3(CASP3),Signal transducer and activator of transcription 3(STAT3),Epidermal growth factor receptor(EGFR),Matrix metalloproteinase 9(MMP9),Steroid receptorcoactivator(SRC),Prostaglandin G/H synthase 2(PTGS2)and Toll-like receptor 4(TLR4)were the more critical proteins.(4)The combined proteomics and network pharmacology analysis revealed 13 targets,including Phosphoinositide-3-kinase regulatory subunit1(PIK3R1),AKT1,STAT3,F2,Poly ADP ribose polymerase(PARP1),Intercellular adhesion molecule-1(ICAM1),Dipeptidyl peptidase 4(DPP4),Plasma Kallikrein B1(KLKB1),Coagulation factor X(F10),Non-receptor protein tyrosine phosphatase 6(PTPN6),Fatty Acid Binding Protein 3(FABP3),Glucose-6-phosphate exchanger SLC37A4(SLC37A4)and Caspase 1(CASP1).A total of 72 pathways were coenriched,including Hypoxia inducible factor-1(HIF-1),Vascular endothelial growth factor(VEGF),PI3K-Akt,Mechanistic target of rapamycin kinase(m TOR),Rap1,PPAR,FCγR mediated phagocytosis,regulation of the actin cytoskeleton and other signaling pathways related to nephropathy.Among them,STAT3,PIK3R1 and AKT1 were common targets of proteomics and network pharmacology and participated in the regulation of several signaling pathways related to nephropathy,which could be the core targets of Huangkui capsule in the treatment of CGN and played an important role in the treatment of CGN with Huangkui capsule.The target validation revealed that the expression of STAT3,PIK3R1 and AKT1 proteins were increased in the kidney tissues of MOD rats compared to NOR(P<0.05).the expression of STAT3,PIK3R1 and AKT1 proteins were decreased in the kidney tissues of HK rats compared to MOD(P<0.05),and this result was consistent with the results of the previous proteomics experiments.In addition,the levels of related factors in the HIF-1/VEGF signaling pathway,in which STAT3,PIK3R1 and AKT1 are all involved,were verified,and the results showed that the expression of HIF-1α and VEGF proteins were increased in the kidney tissues of rats in the MOD group compared with NOR(P<0.05);the expression of HIF-1α and VEGF proteins were decreased in the kidney tissues of rats in the HK group compared with MOD(P<0.05).It was suggested that the HIF-1/VEGF signaling pathway could be regulated by STAT3,PIK3R1 and AKT1 proteins to treat CGN.Conclusions:(1)Huangkui capsule significantly improved renal function index of CGN rats,reduced inflammation and oxidative response,improved the pathological damage and protected rat kidney tissues in CGN rats.(2)Combined network pharmacology and proteomics analysis and experimental validation revealed that proteins such as PIK3R1,AKT1,STAT3,F2,PARP1,ICAM1,DPP4,KLKB1,F10,PTPN6,FABP3,SLC37A4 and CASP1 were the targets of the action of Huangkui capsule in the treatment of CGN,which exerted their effects on the treatment of CGN through the regulation of signaling pathways such as HIF-1,VEGF,PI3K-Akt,MAPK,NF-κB,Rap1 and PPAR. |
