| Objective:To explore the protective effect and mechanism of coenzyme Q10(CoQ10)on renal fibrosis induced by unilateral ureteral obstruction(UUO).Methods:In vitro study:1.Human kidney proximal tubule epithelial cells(HK-2 cells)were cultured in a humidified incubator 5%CO2 and 37℃.After 24-h incubation,cells were pretreated with or without different concentrations of CoQ10(5μmoL/L and 10μmoL/L)for 1h,followed by an incubation with or without H2O2(500μmoL/L),Nec-1(30mmoL/L),GSK872(3μmoL/L),and ICG001(10μmoL/L)for 24 h.2.The cell viability of each group was determined by the CCK-8 and MTT assay.3.The levels of intracellular reactive oxygen species(ROS)were measured using 2’,7’-dichlorodihydrofluorescein diacetate(H2DCFDA,Invitrogen),and fluorescence was measured using flow cytometry;Mitochondrial ROS were measured by MitoSOXTM red mitochondrial superoxide and with by a flow cytometer;The cell apoptosis was detected by Annexin V-FITC apoptosis detection.4.Expressions of RIP1-RIP3-MLKL,IL-1β,IL-18,NLRP3,and Wnt/β-catenin/GSK related proteins were detected by Western blot.5.Cellular oxygen consumption rate(OCR)was assessed in real time using an XF24 Extracellular Flux Analyzer.In vivo study:1.A total of 30 7-week-old male SPF Sprague-Dawley rats(weighing between 250 g and 260 g)were randomly divided into 5 groups and treated daily for 7 days.The 5 groups were respectively:sham group(n=6),in which only the rats’ left ureter was exposed and the skin was sutured;UUO group(n=6),in which the rats’left ureter was ligated for 7 days;UUO+CoQ10 group(n=6),in which rats in the UUO group received CoQ10 treatment(20 mg/kg/day,oral gavage);UUO+Nec-1 group(n=6),in which rats in the UUO group received Nec-1 treatment(2 mg/kg/day,oral gavage);UUO+GSK872 group(n=6),in which rats in the UUO group received treatment with GSK872(1 mg/kg/day,intraperitoneal injection).7 days later,urine from rats of each group in 24 h was collected for 24-h UPRO detection with an automatic urine analyzer,and their blood was taken from the right internal jugular vein for Scr and BUN detection.2.Kidney tissues were fixed in periodate-lysine-paraformaldehyde solution and embedded in wax,and sectioned and stained with hematoxylin-eosin(HE)and Masson’s trichrome;Quantitative analysis of fibrosis was performed with a color image auto-analyzer;the cell microstructure was observed by electron microscope,apoptotic cells in renal tissue were observed by TUNEL staining,and expressions of ectodermal dysplasial(ED-1)and 8-OHdG in renal tissue were detected by immunohistochemical staining.3.Detection of protein kinase 1(RIP1),protein kinase 3(RIP3),mixed lineage kinase domain-like protein(MLKL),transforming growth factor-β1(TGF-β1),nterleukin-1β(IL-1β),nterleukin-18(IL-18),NOD-like receptor pyrin domain-containing protein 3 family pyrin domain containing 3(NLRP3),manganese superoxide dismutase(MnSOD),NADPH Oxidase4(NOX-4),optic atrophy 1(OPA1),succinate dehydrogenaseA(SDHA),PINK1/Parkin,CCAAT/enhancer-binding protein-homologuous protein(CHOP),Inositol requiring enzyme1α(IRE-1α),X box binding protein 1(XBP1),B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax).and Wnt/β-catenin/GSK protein expression by the Western blot in renal tissue.4.The urinary 8-OHdG excretion rate was measured by ELISA.Results:In vitro experiments:1.CoQ10 increased the cell viability of HK-2 cells and inhibited apoptotic cells in HK-2 cells in a dose-dependent manner(P<0.05).2.Flow cytometry analysis showed that in HK-2 cells exposed to H2O2,CoQ10 reduced the production of intracellular ROS and MitoSOX in a dose-dependent manner(P<0.05).3.HK-2 cells treated with CoQ10 had higher rates of basal mitochondrial respiration.Compared with cells treated with H2O2 alone,those in the H2O2+CoQ10 group showed higher rates of ATP-associated and total respiration(P<0.05).4.CoQ10 or RIP inhibited the expression of RIP1-RIP3-MLKL induced by H2O2(P<0.05).5.Expression of pyroptosis-related cytokines(pro-IL-1β,pro-IL-18 and NLRP3)in H2O2-treated HK-2 cells was increased,,and this effect was significantly reduced in groups treated with CoQ10 or an RIP inhibitor(Nec-land GSK872)(P<0.05).6.H2O2 activated the expression of Wnt/β-catenin/GSK protein,while their expression was decreased by RIP inhibitor(Nec-1 and GSK872)and CoQ10 or Wnt/β-catenin inhibitor(ICG001)(P<0.05).In vivo experiments1.Induction of UUO resulted in BW loss with or without drug treatment compared with the sham group(P<0.05).2.The results of light microscopy showed that the UUO group was characterized by scattered tubular vacuolization,tubular atrophy,collagen fiber deposition and fibrosis.UUO led to massive inflammatory cell infiltration within the tubulointerstitium,but this effect was abrogated after addition of CoQ10 or RIP inhibitor(Nec-1and GSK872).3.Electron microscopy showed necrotic bodies and cytolysis,tubule epithelium swelling,and abscission of microvilli in the tubular epithelial lumen in obstructed kidneys in the group treated with UUO.Both CoQ10 and RIP inhibitor(Nec-1 or GSK872)attenuated these morphological changes.Electron microscopy showed that UUO disrupted the mitochondrial architecture,manifested by significant reduction and shrinkage of mitochondria,distended mitochondria,cristae fracture,focal vacuolization,mitochondrial deformation(fusion),mitochondria divided into two daughter organelles(fission),and mitophagy formation.CoQ10 treatment prevented this loss of mitochondrial anatomical integrity and preserved the number and size of mitochondria(P<0.05).Induction of UUO was associated with stripping of ribosomes and dilation of vesicle cisternae in the rough ER,however,these alterations were reversed by treatment with CoQ10 and RIP inhibitor.4.Quantitative analysis showed the UUO group had higher fibrosis scores(P<0.01),while the groups given CoQ10 or RIP inhibitor(Nec-1 and GSK872)had lower fibrosis score(P<0.05).Compared with UUO alone,the CoQ10 or RIP inhibitor lowered the expression of TGF-β1(P<0.05).5.Immunoblotting analysis results showed that overexpression of RIP 1-RIP3-MLKL axis proteins was significantly inhibited by treatment with CoQ10 or RIP inhibitor(Nec-1 and GSK872)(P<0.05).6.The results of immunohistochemistry showed that compared with the sham group,the expression of ED-1 in the UUO group was significantly increased(P<0.01),and compared with the UUO group,the expression of ED-1 in the CoQ10 or RIP inhibitor group(Nec-1 and GSK872)was significantly decreased(P<0.05).The expression of pyroptosis-related cytokines(pro-IL-1β,pro-IL 18 and NLRP3)in the UUO group was increased,and this effect was significantly reduced in groups treated with CoQ10 or RIP inhibitor(P<0.05).7.Compared with the UUO group,the expression of 8-OHdG in the CoQ10 or RIP inhibitor group was significantly decreased(P<0.05).CoQ10 significantly reduced the urinary 8-OHdG concentration in the UUO group(P<0.05).CoQ10 scavenged oxidative stress through upregulation of MnSOD and downregulation of NOX4 expression(P<0.05).8.Immunoblotting analysis results showed that the dysregulation of OPA1,SDHA,PINK1,and Parkin expression in the UUO group was counteracted by treatment with CoQ10(P<0.05).9.TUNEL staining results showed that TUNEL-positive cells were mainly distributed in the renal tubule epithelial cells and renal tubule interstitial fibrosis area.Compared with the sham group,the number of TUNEL-positive cells in the UUO group was significantly increased(P<0.01),and compared with the UUO group,the number of TUNEL-positive cells in the CoQ10 or RIP inhibitor group significantly decreased(P<0.05).10.Immunoblotting analysis results showed that CoQ10 regulated the expression of genes controlling ER stress(CHOP,IRE-1α and XBP1S)and apoptosis(Bcl-2 and Bax)(P<0.05).11.Immunoblotting analysis results showed that UUO activated the expression of Wnt/β-catenin/GSK protein(P<0.01),while CoQ10 and RIP inhibitor(Nec-1 and GSK872)decreased their expression(P<0.05).Conclusion:1.Coenzyme Q10 attenuates renal fibrosis by inhibiting RIP1-RIP3-MLKL-mediated necroinflammation.2.Renoprotective effects of Coenzyme Q10 may be related to a reduction in oxidative stress and preservation of mitochondrial fitness.3.Suppression of ER stress and apoptotic cell death may be a mechanism uniderlying the renoprotective properties of Coenzyme Q10.4.Coenzyme Q10 affords renoprotective effects probably by interfering with the the Wnt3α/β-catenin/GSK-3β signaling pathway... |
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