| Objective:To reveal the effects and underlying mechanism of Fufang Zhenzhu Tiaozhi Capsule(FTZ)on renal fibrosis based on network pharmacology and animal experiments.Methods:1.Network pharmacologyInformation about active compounds of herbs in FTZ was screened from Traditional Chinese medicine systems pharmacology(TCMSP)public database and literature mining.Then,the potential targets of those active compounds were predicted through a search of TCMSP,Pub Chem Search,ALOGPS 2.1 and Swiss Target Prediction databases.In addition,the therapeutic targets of renal fibrosis were obtained from Gene Cards,Online Mendelian Inheritance in Man(OMIM)and Dis Ge NET databases.The renal fibrosis related targets’ proteinprotein interaction(PPI)network and the FTZ related targets’ PPI network were constructed by Biso Genet plugin to obtain core targets.The FTZ-renal fibrosis core target PPI network was established by using String database.The abovementioned data were visualized and constructed using Cytoscape 3.7.2.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses was performed by using Clue GO and Clue Pedia plugin,which were carried out to further explore the renal fibrosis mechanism and therapeutic effect of FTZ.2.In vivo animal experiments2.1 Pharmacodynamic evaluation of renal protective effect of FTZ40 male C57BL/6 mice were randomly divided into five groups: sham operation group(Sham),UUO model group and 3 UUO groups treated with low,medium or high-dose of FTZ formula(0.6,1.2,2.4 g/kg/d).The mice were intragastrically administrated with corresponding FTZ once a day for 7 days before UUO surgery as well as 7 days after UUO surgery.The 24-h urine,blood and kidney tissues were used to detect the 24-h excretion amount of urine protein(24 h-Upr),urine creatinine(Ucr)excretion and the levels of serum creatinine(Scr)and blood urea nitrogen(BUN).HE and Sirius red staining were used to evaluate renal morphological changes and renal collagen accumulation.The protein levels of fibronectin(FN1),α-SMA and Collagen I were determined by Western blotting.The protein and m RNA levels of TGF-β1,β-arrestin1 and β-arrestin2 were evaluated by Western blotting and q RT-PCR.2.2 The effect and mechanism of FTZ on renal fibrosis180 male C57BL/6 mice were randomly divided into six groups: sham operation group(Sham),UUO model group and 4 UUO groups treated with low,medium or high-dose of FTZ formula(0.6,1.2,2.4 g/kg/d)and Bena(1.67mg/kg/d).The mice were intragastrically administrated with corresponding FTZ or Bena once a day for 3,7 and 14 days after UUO surgery.The blood and kidney tissues were used to detect the levels of serum creatinine(Scr)and blood urea nitrogen(BUN).HE staining was used to evaluate renal morphological changes.The protein levels of fibronectin(FN1),α-SMA,TGF-β1 and β-arrestin1 were determined by Western blotting.The m RNA levels of TGF-β1,β-arrestin1 andβ-arrestin2 were evaluated by q RT-PCR.Result:1.Network pharmacologyA total of 237 active compounds were obtained from FTZ,involving 235 main active compounds and 591 potential targets.quercetin,kaempfero,luteolin and other important active components were analyzed.And there were 1544 gene targets related to renal fibrosis,of which 325 overlapped with the targets of FTZ.There were 68 core targets screened,among which the targets such as TNF,CASP3,VEGFA,CXCL8,IL6,MAPK3,AKT1,ALB,TP53,FN1,JUN,STAT3,EGF,EGFR and TGFB1 may be the core targets of FTZ in the treatment of renal fibrosis.GO function enrichment analysis showed that it mainly involved biological process such as regulation of nitric-oxide synthase biosynthetic process,regulation of transcription from RNA polymerase II promoter,regulation of vascular smooth muscle cell proliferation and positive regulation of gene erpression,and molecular function such as protein phosphatase binding,cytokine activity,protein binding,transcription factor binding and protein kinase binding.KEGG pathway enrichment analysis of 68 core targets,FTZ can regulate 31 important signal pathway including Fluid shear stress and atherosclerosis,AGE-RAGE signaling pathway in diabetic complications,HIF-1 signaling pathway,VEGF signaling pathway,Bladder cancer,and other pathways.It is inferred that FTZ plays an anti-fibrosis role mainly act on the core targets such as TGFB1,FN1,TNF and so on,participating in diabetic complications AGE-RAGE signal pathway and other related signal pathways such as TGF-β1 signal pathway,MAPK signal pathway,PI3K-AKT signal pathway based on the visualization of KEGG pathway map.2.In vivo animal experiments2.1 Compared with the UUO model group,FTZ obviously reduced the ratio of kidney weight to body weight and the levels of 24 h-Upr,Ucr excretion,Scr and BUN(P < 0.01)induced by UUO.FTZ significantly alleviated renal injury,including reducing the accumulation of collagen as well as improving the pathological levels of glomeruli and renal tubules(P < 0.01).Correspondingly,the UUO-induced protein levels of FN1,α-SMA,Collagen I were significantly blunt by FTZ(P < 0.05,P < 0.01).Mechanically,the UUO-induced protein and m RNA levels of TGF-β1,β-arrestin1 andβ-arrestin2 were markedly attenuated by FTZ treatment(P<0.05,P<0.01).2.2 In the progression of renal fibrosis,compared with the UUO model group,FTZ and Bena obviously reduced the ratio of kidney weight to body weight,the levels of Scr and BUN,the pathological injury degree of glomeruli and renal tubules induced by UUO(P < 0.05,P < 0.01).Correspondingly,the UUO-induced protein levels of FN1 and α-SMA were significantly blunt by FTZ and Bena(P < 0.05,P < 0.01).Mechanically,the UUO-induced expression levels of TGF-β1,β-arrestin1 and β-arrestin2 were markedly attenuated by FTZ and Bena treatment(P < 0.05,P < 0.01).Conclusion:1.This study can reflects multi-components,multi-targets and multipathway characteristics of FTZ in the treatment of renal fibrosis,which provides a novel approach for further research of its mechanism.2.Our results suggested that FTZ effectively ameliorates renal fibrosis in UUO-induced mice and its effects may be conducted by inhibiting the expression of TGF-β1/β-arrestin1/β-arrestin2 pathway and downregulating the expression of fibrosis associated proteins such asα-SMA,FN1 and Collagen-I. |
