Study On The Effect And Mechanism Of Zishen Pill On Renal Tubular Epithelial Cell Pyroptosis In Diabetic Nephropathy

[Aim]To investigate the effects and mechanisms of ZiShenWan(ZSW)on renal tubular epithelial cell pyroptosis in diabetic nephropathy(DN),and to investigate the targets and pathways of ZSW in DN therapy by network pharmacology analysis on the purpose of providing evidence for ZSW in DN treatment.[Methods]1.Experiment in vivo:Male 6-week-old db/db mice were randomly divided into five groups including model group,positive control group,high dose ZSW group,medium dose ZSW group and low dose ZSW group(n=10).Non diabetic db/m mice were served as blank control group(n=10).After 2-week long adaptive feeding,ZSW groups were given ethanol extract by gavage(high dose:6.0 g/kg;medium dose:3.0 g/kg;low dose:1.5 g/kg).Mice in positive control group were given dapagliflozin by gavage(1.0 mg/kg);Mice in model group and blank control group were given equal volume of deionized water by gavage at the same time.Administration was performed once a day and lasted for 12 weeks.Body weight and FBG of tail vein were detected every 2 weeks.Urine ACR,NAGase,NGAL,and CysC were detected every 4 weeks.After 12 weeks of administration,eyeballs were taken for blood collection,right kidney weight was measured,and kidney tissues were fixed and frozed.Serum HbAlc,BUN,and CRE were detected.Pathological changes of renal tissues stained with HE,PAS,and Masson were observed by light microscope.The transmission electron microscope was used for observations of the ultrastructural changes.The expression of epithelial-mesenchymal transition(EMT)markers including ?-SMA and E-cadherin in renal tubular epithelium were analyzed by immunohistochemistry staining.TUNEL staining was used to analyze nuclear damage of renal tubular epithelial cells.Western blot and RT-qPCR were used to detect the protein and mRNA expressions of NLRP3 inflammasome including NLRP3,ASC,and Caspase-1,as well as pyroptosis-related inflammatory cytokines including IL-1?,cleaved IL-1?,and IL-18 in renal tissues.2.Experiment in vitro:Human proximal tubular epithelial(HK-2)cells were resuscitated subcultured.The culture conditions and drug concentration for administration were selected via MTT assay.After 48 h stimulation by hypertonic glucose(HG)(45 ?mol/L)for induction of pyroptosis and EMT,cells were divided into five groups,including HG group,low dose ZSW group,medium dose ZWS group,high dose ZSW group and positive control group.All dose of ZSW groups were treated with alcohol extract(high dose:15 ?g/ml;medium dose:7.5?g/ml;low dose:3.75 ?g/ml);the positive control group was treated with dapagliflozin(0.1?mol/L).In addition,the blank group and mannitol group were set as normal control and hypertonic control.The inverted microscope was used for cell morphological observation.Transwell assay was used for evaluation of migration ability.The nucleus and cell membrane injuries were evaluated by TUNEL staining and LDH test separately.Pyrototic level was detected by Annexin V-PI staining.The concentration levels IL-1? and IL-18 in cell supernatant were detected by ELISA;The protein and mRNA expression levels of NLRP3,ASC,Caspase-1,IL-1?,cleaved IL-1?,and IL-18 were respectively detected by WB and RT-qPCR.3.Network pharmacology analysis:The key targets of ZSW in DN therapy were obtained by searching databases for drug active components of ZSW and disease targets of DN.The"drug-component-target" network of ZSW in DN therapy was established by Cytoscape software.The protein-protein interaction(PPI)network of ZSW in DN therapy was established by STRING database to screen the core targets of ZSW in DN therapy;The Cytoscape software and DAVID database were applied for enrichment analysis of gene function and signaling pathways.[Results]1.Experiment in vivo:(1)Curative effect evaluation:Compared with the normal control group,the levels of FBG,HbAlc,BUN,serum CRE,as well as urinary ACR,NAGase,NGAL,and CysC in the model group were significantly increased(P<0.01),and the above indexes were significantly decreased by ZSW treatment(P<0.01).(2)Pathological observation:The enlargement of glomerular volume,glomerular basement membrane(GBM)thickening,mesangial matrix proliferation,tubular vacuolar degeneration,and inflammatory cell infiltration were observed in the model group,and the collagen fiber staining area,GBM thickness and tubular injury index were significantly increased in the model group(P<0.01).ZSW alleviated the pathological changes and significantly reduced the quantitative indexes mentioned above(P<0.01).(3)EMT in renal tubular epithelium:Compared with the normal control group,the expression intensity of ?-SMA in renal tubular,as well as the protein and mRNA expression levels in renal tissue were significantly increased,while the expression intensity of E-cadherin in renal tubular as well as the expression of protein and mRNA in renal tissues were significantly decreased in the model group(P<0.01).ZSW treatment significantly changed the expression intensities of ?-SMA and E-cadherin.The protein and mRNA expression levels of?-SMA and E-cadherin were changed by ZSW at varying degrees(P<0.05 or P<0.01).(3)Pyroptosis of renal tubular epithelium cells:Compared with the normal control group,the cell nuclear fragmentation increased significantly in the model group showed by TUNEL staining(P<0.01).Meanwhile,the protein and mRNA expressions of IL-1?,cleaved IL-1?,and IL-18 in renal tissues of model group were significantly increased(P<0.01).ZSW alleviated cell nuclear fragmentation,significantly decreased the protein and mRNA expression levels of IL-1?,cleaved IL-1?,and IL-18(P<0.01).(4)NLRP3 inflammasome activation in renal tissues:Compared with the normal control group,the protein and mRNA expressions of NLRP3,ASC and,Caspase-1 in renal tissues of model group were significantly increased(P<0.01).ZSW decreased the protein expression levels of NLRP3,ASC,and Caspase-1 at varying degrees(P<0.05 or P<0.01)and significantly decreased the mRNA expression levels of NLRP3 and ASC(P<0.01).High dose ZSW significantly decreased the mRNA expression levels of Caspase-1(P<0.01).2.Experiment in vitro:(1)EMT:Stimulation by HG for 48 h induced HK-2 cells to interstitial-like change and increased the migration ability.The protein and mRNA expression levels of ?-SMA were significantly increased,while the protein and mRNA expression levels of E-cadherin were significantly decreased(P<0.01).ZSW significantly inhibited the interstitial-like change and migration ability of HG-induced HK-2 cells,and regulated the protein and mRNA expression levels of ?-SMA and E-cadherin at varying degrees(P<0.05 or P<0.01).(3)Pyroptosis:HG strengthened the damages of cell membrane and nuclear as well as pyroptosis index of HK-2 cells.The release of IL-1? and IL-18 in HK-2 cells significantly increased by HG.The protein and mRNA expressions of IL-1?,cleaved IL-1?,and IL-18 in HG-induced HK-2 cells were significantly increased(P<0.01).ZSW alleviated the damages of cell membrane and nuclear,decreased the pyroptosis level of HG-induced HK-2 cells.The concentration of IL-1? and IL-18 in the HG-induced HK-2 cell culture supernatants as well as the protein and mRNA expressions were significantly decreased by ZSW treatment(P<0.01).(4)NLRP3 inflammasome activation:The protein and mRNA expressions of NLRP3,ASC,and Caspase-1 in HG-induced HK-2 cells were significantly increased(P<0.01).The protein and mRNA expressions of NLRP3,ASC,and Caspase-1 were decreased by ZSW treatment at varying degrees(P<0.05 or P<0.01).3.Network pharmacology analysis:A total of 56 active ingredients of ZSW were screened,51 of whose as well as 166 DN-related targets were selected from databases for "drug-component-target" network construction of ZSW in DN therapy.A total of 154 key core targets involving considerable inflammation-related factors were obtained from PPI network.The core targets were involved in cell components such as calcium channel complex,membrane region,synapse and cytoplasm;molecular function terms such as G-protein coupled receptor activity and neurotransmitter binding;biological processes such as lipopolysaccharide-mediated signaling pathway,oxidation-reduction process,and synaptic transmission.The core targets were enriched in signaling pathways such as advanced glycation end products,cell proliferation and differentiation,endocrine resistance,nerve tissue conduction,and inflammatory response including NOD-like receptor(NLR)signaling pathway.[Conclusions]1.ZSW could attenuate hyperglycaemia and proteinuria,improve renal tubular early injury indexes and renal function,alleviate pathological damage of renal tissue,and inhibit renal tubular EMT and pyroptosis in db/db mice,which might be related to suppressing the NLRP3 inflammasome activation.2.ZSW could inhibit HK-2 cell pyroptosis and EMT induced by HG,whose mechanism might be mediated by suppressing NLRP3 inflammasome activation.3.A considerable number of components of ZSW might perform therapeutic effects on DN by regulating multiple targets and signaling pathways.Anti-inflammation might be the key mechanism of DN therpy.