Diabetic kidney disease (DKD)is a microvascular complication of type 2 diabetes.The study of DKD mechanisms is the most important target for the prevention of DKD.Renal senescence is one of the important pathogeneses ...Diabetic kidney disease (DKD)is a microvascular complication of type 2 diabetes.The study of DKD mechanisms is the most important target for the prevention of DKD.Renal senescence is one of the important pathogeneses for DKD,but the mechanism of renal and cellular senescence is unclear.Decreased expression of circulating miR-126 is associated with the development of DKD and may be a promising blood-based biomarker for DKD.This study is to probe the effect and mechanism of miR-126 on the aging of human glomerular mesangial cells (HGMCs)induced by high glucose.HGMCs were cultured with Roswell Park Memorial Institute (RPMI-1640)in vitro.The effect of high glucose on morphology of HGMCs was observed 72h after intervention.The cell cycle was examined by flow cytometry.The telomere length was measured by Southern blotting.The expression levels of p53,p21 and Rb proteins in p53-p21-Rb signaling pathway and p-statl,p-stat3 in JAK/STAT signaling pathway were detected by Western blotting respectively.The expression of miR-126 was examined by qRT-PCR.MiR-126 mimics was transfected into HGMCs.The effects of miR-126 mimics transfection on cell morphology,cell cycle,telomere length,p53,p21,Rb,p-stat1 and p-stat3 were observed. The results showed that high glucose not only arrested the cell cycle in G1phase but also shortened the telomere length.High glucose led to high expression of p53,p21,Rb,p-statl and p-stat3 and premature senescence of HGMCs by activating the telomere-p53-p21-Rb and JAK/STAT signaling pathways.Moreover,the miR-126 was decreased in HGMCs induced by high glucose.It was suggested that the transfection of miR-126 mimics could inhibit the telomere-p53-p21-Rb and JAK/STAT signaling pathway activity in vitro and delay the senescence of HGMCs.The results may serve as a new strategy for the treatment of DKD.展开更多
<Abstrat>The proliferation of mesangial cells on cyclosporin (CsA) test mediumwas studied by MTT assay and TNF-Q in cultured supernatant was examined byusing ELISA. The results showed that cyclosporin A signific...<Abstrat>The proliferation of mesangial cells on cyclosporin (CsA) test mediumwas studied by MTT assay and TNF-Q in cultured supernatant was examined byusing ELISA. The results showed that cyclosporin A significantly inhibited theproliferation of mesangial cells at the concentration between 0. 25 - 15 μg/ml(IC50 1μg/ml). This action appeared to be dose-dependent. Release of TNF-αfrom mesangial cells stimulated by LPS was also dose-dependently suppressed. Itis suggested that cyclosporin A play an important role in antiproliferation mecha-nism of mesangial cells in vitro.展开更多
Background The pathogenesis of diabetic nephropathy (DN) is a complex pathophysiological process.Its precise mechanism is not fully known. In recent years it has been recognized that synthesis of various extracelluar ...Background The pathogenesis of diabetic nephropathy (DN) is a complex pathophysiological process.Its precise mechanism is not fully known. In recent years it has been recognized that synthesis of various extracelluar matrix (ECM) components may increase, and that degradation of ECM may decrease in DN. It was reported heparin could inhibit mesangial cells proliferation in vitro. The main aim of this study is to explore whether heparin inhibits proliferation of mesangial cells grown in high glucose concentration and to measure the effect of heparin on matrix metalloproteinases (MMPs) expression in mesangial cells. Methods The medium contained either low glucose (5 mmol/L) or high glucose (25 mmol/L). The concentrations of heparin in the culture medium were 0, 25, 50,100, 200 or 400 μg/mL. A metabolic (WST-1) assay was used to measure mesangial cell proliferation and Western blot analysis was used to measure MMPs expression of mesangial cells. Results Normal human mesangial cell (NHMC) proliferation was higher in high glucose (HG) medium than in low glucose (LG) medium. They showed a 1.93 fold expansion after 72 h in high glucose in contrast to a 1.63 fold expansion in low glucose. In the presence of heparin, mesangial cells proliferation was inhibited, which was more obvious at high glucose concentrations than at low glucose concentrations. In high glucose, with heparin concentration of 50, 100, 200 and 400 μg/mL, the mesangial cells showed a 0. 61 fold, 0.52 fold, 0.52 fold and 0.41 fold reductions in cell number compared to cells grown without heparin. In low glucose, only concentrations of 200 μg/mL and 400 μg/mL showed reduction in cell number, namely 0.54 fold and 0.45 fold, when compared to cells grown without heparin. In Western blot analysis,MMP1, MMP2, MMP3 and MMP9 was expressed by mesangial cells expressed in both high and low glucose concentrations, which was more prominent in high glucose medium. Incubation of heparin further increased expression of MMP1, MMP2, MMP3 and MMP9. Conclusions This study suggests that glucose can accelerate mesangial cell proliferation while heparin can reduce proliferation, being more obvious at high glucose concentrations. Higher glucose concentrations led to increased MMP expression, which may take part in the regulation of mesangial matrix synthesis and degradation. Addition of heparin resulted in a corresponding increase in MMP expression, most notably at high glucose concentrations, indicating a potentially renoprotective role in DN.展开更多
BACKGROUND:This study aimed to explore the effects of TNF-a on the expression of IP_3R1mRNA and protein in human mesangial cells(HMCs),and to elucidate the mechanism of TNF-a relating to IP_3R1 expression in the occur...BACKGROUND:This study aimed to explore the effects of TNF-a on the expression of IP_3R1mRNA and protein in human mesangial cells(HMCs),and to elucidate the mechanism of TNF-a relating to IP_3R1 expression in the occurrence of hepatorenal syndrome(HRS).METHODS:HMCs were stimulated by tumor(TNF-a) with 100 ng/mL for different hours(2,4,8,and 24 hours).The expression changes of IP_3R1 mRNA and protein were detected by quantitative real-time polymerase chain reaction and immunoblotting.Several inhibitors including D609,U73122,PP1,safingol,rottlerin and non-radioactive protein kinase C(PKC) were used to examine the mechanism of signal transduction ofTNF-a-regulated IP_3R1 in HMCs.RESULTS:The levels of IP_3R1 mRNA at 2 hours after TNF-a exposure were significantly enhanced and peaked at 8 hours in HMCs(P<0.01),then descended at 24 hours(P<0.01).The levels of IP_3R1 protein at 4 hours after TNF-a exposure were obviously increased and peaked at24 hours after TNF-a exposure(P<0.01).Compared to the control group,safingol(PKCa inhibitor)and D609(phosphatidylcholine-specific phospholipase C inhibitor) significantly blocked the TNF-ainduced expression of IP_3R1 mRNA(3.30±0.81 vs.1.95±0.13,P<0.05;2.10±0.49,P<0.01) and IP_3R1protein(3.09±0.13 vs.1.86+0.39,P<0.01;1.98±0.02,P<0.01).TNF-a promoted PKCa activation with maximal PKCa phosphorylation that occurred 8 hours after stimulation measured by non-radioactive PKC assay,and the effect was markedly attenuated by pretreatment with D609 or safingol.CONCLUSION:TNF-a increased the expression of IP_3R1 and this was mediated,at least in part,through the PC-PLC/PKCa signaling pathways in HMCs.展开更多
Objective:To observe the expression of Long non-coding RNA antisense mitochondrial non-coding RNA-2 (ASncmtRNA-2) in high glucose (HG) treated human renal mesangial cells (HRMCs) and the role of ASncmtRNA-2 in oxidati...Objective:To observe the expression of Long non-coding RNA antisense mitochondrial non-coding RNA-2 (ASncmtRNA-2) in high glucose (HG) treated human renal mesangial cells (HRMCs) and the role of ASncmtRNA-2 in oxidative stress mediated diabetic nephropathy (DN) fibrosis.Methods: The expression levels of ASncmtRNA-2、transforming growth factorβ1 (TGF-β1) and fibronectin (FN) mRNA in cultured HRMCs were measured by qRT-PCR. In addition, relative reactive oxygen species (ROS) levels in HRMCs were detected with the non-fluorescent probe DCFH-DA assays.Results: Compared with 0h, the expression of ASncmtRNA-2 remained unchanged in all groups at 8 h post treatment. However, the level of ASncmtRNA-2 mRNA was increased significantly in HG and HG+NG-nitro-L-Arginine methylester (L-NAME) treated cells compared with low glucose (LG) treated cells from 16h onwards, while the level of ASncmtRNA-2 mRNA in the HG+L-NAME group was decreased compared with the HG group. Moreover, ROS fluorescence was significantly up-regulated in HG-treated cells compared with LG-treated cells, while the ROS fluorescence in HG+L-NAME group was suppressed compared with HG-treated cells. In addition, Levels of ASncmtRNA-2, TGF-β1 and FN mRNA were significantly up-regulated in HG treated cells compared with LG treated cells while Levels of ASncmtRNA-2, TGF-β1 and FN mRNA in HG+L-NAME group were down-regulated compared with HG group. Finally, the expression of ASncmtRNA-2, TGF-β1 and FN mRNA were significantly decreased in HG+ASncmtRNA-2 siRNA group compared with HG group.Conclusion: ASncmtRNA-2 was up-regulated in HG treated cells and may promote glomerular fibrosis in DN via positively regulating the expression of pro-fibrotic factors. These findings may provide novel potential therapeutic treatments for DN.展开更多
Diabetic nephropathy(DN)is a common type of end-stage renal disease and glomerular mesangial cells(GMCs)are widely used as a cell model for DN.This study firstly investigated the inhibitory effects of the Apostichopus...Diabetic nephropathy(DN)is a common type of end-stage renal disease and glomerular mesangial cells(GMCs)are widely used as a cell model for DN.This study firstly investigated the inhibitory effects of the Apostichopus japonicus and Acaudina leucoprocta hydrolysates on cellular growth under high-glucose treatment,better inhibitory effect of A.japonicus hydrolysate was observed compared to that of A.leucoprocta hydrolysate.Subsequently,the global transcription profiles obtained via microarray analysis showed that 6070 and 7015 genes were identified in the A.japonicus and A.leucoprocta groups compared with the model group,respectively.Among them,transcriptions of the slc30a4,slc35dl,tppp3,tp53inpl,bcl-2,apafl,alox12b and adrala genes were restored from the levels of the model group to those of the control group,contributed to cell mitosis and proliferation in both treatment groups.In addition,other apoptosis-related genes,such as bcl-6,clu,foxo3 and akt,showed opposite trends between two groups,which might cause the difference in inhibitory effect.We preliminarily proposed that the regulation effects of A.japonicus and A.leucoprocta on the genes involved in cellular mitosis,proliferation and apoptosis,might contribute to their inhibitory activity on GMCs under high-glucose environment.展开更多
Objective To elucidate the renoprotective effect of resveratrol(RSV)on sphingosine kinase 1(SphK1)signaling pathway and expression of its downstream molecules including activator protein 1(AP-1)and transformation grow...Objective To elucidate the renoprotective effect of resveratrol(RSV)on sphingosine kinase 1(SphK1)signaling pathway and expression of its downstream molecules including activator protein 1(AP-1)and transformation growth factor-β1(TGF-β1)in lipopolysaccharide(LPS)-induced glomerular mesangial cells(GMCs).Methods The rat GMCs line(HBZY-1)were cultured and randomly divided into 5 groups,including control,LPS(100 ng/mL),and 5,10,20µmol/L RSV-treated groups.In addition,SphK1 inhibitor(SK-II)was used as positive control.GMCs were pretreated with RSV for 2 h and treated with LPS for another 24 h.GMCs proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay.The proteins expression of SphK1,p-c-Jun and TGF-β1 in GMCs were detected by Western blot,and DNA-binding activity of AP-1 was performed by electrophoretic mobility shift assay(EMSA).The binding activity between RSV and SphK1 protein was detected by AutoDock Vina and visualized by Discovery Studio 2016.Results LPS could obviously stimulate GMCs proliferation,elevate SphK1,p-c-Jun and TGF-β1 expression levels and increase the DNA-binding activity of AP-1(P<0.05 or P<0.01),whereas these effects were significantly blocked by RSV pretreatment.It was also suggested that the effect of RSV was similar to SK-II(P>0.05).Moreover,RSV exhibited good binding affinity towards SphK1,with docking scores of−8.1 kcal/moL and formed hydrogen bonds with ASP-178 and LEU-268 in SphK1.Conclusion RSV inhibited LPS-induced GMCs proliferation and TGF-β1 expression,which may be independent of its hypoglycemic effect on preventing the development of mesangial cell fibrosis and closely related to the direct inhibition of SphK1 pathway.展开更多
OBJECTIVE:To investigate the effect of mitofusin 2 (Mfn2) and its downstream signaling pathway on glomerular mesangial cells (GMCs) proliferation in IgA nephropathy (IgAN),as well as the mechanism of action of Jixueca...OBJECTIVE:To investigate the effect of mitofusin 2 (Mfn2) and its downstream signaling pathway on glomerular mesangial cells (GMCs) proliferation in IgA nephropathy (IgAN),as well as the mechanism of action of Jixuecao (Herba Centellae Asiaticae,HCA) in the treatment of IgAN.METHODS:Adenovirus-mediated Mfn2 gene transfection and Mfn2 expression were analyzed by real-time polymerase chain reaction (PCR) and Western blotting.IgA1 induced the proliferation of GMCs,which were then treated with HCA.Cell proliferation was detected with cell counting kit-8 (CCK-8),and Mfn2 expression was analyzed by real-time PCR and western blotting.An IgAN animal model was also established and treated with HCA.GMCs proliferation was detected by hematoxylin-eosin staining,mitochondrial structure was analyzed by electron microscopy,mitochondrial function was determined by the Clark oxygen electrode method,and the expression of Mfn2,Phospho-extracellular regulated protein kinases1/2 (P-ERK1/2),Cyclin-dependent kinase 2 (CDK2),Phospho-p27 (p-p27),and cyclin A was analyzed by Western blotting.RESULTS:In vitro,HCA inhibited GMCs in a concentration-dependent manner in association with the upregulation of Mfn2 expression.The overexpression of Mfn2 inhibited IgA1-induced GMCs proliferation and elevated the effect of HCA.In vivo,treatment with HCA could alleviate albuminuria and creatinine and GMCs proliferation.These effects were related to the upregulation of Mfn2,p-p27 and inhibition of p-ERK1/2,CDK2,and cyclinA.Mitochondrial swelling,vacuolar degeneration,and reduction of respiratory control rate were identified in IgAN,but HCA could improve the mitochondrial structure and function.CONCLUSION:HCA inhibited GMCs proliferation via the upregulation Mfn2 and the inhibition of Ras-Raf-ERK/MAPK.We revealed that changes of mitochondrial structure and function are associated with IgAN,but that HCA can improve these mitochondrial features.展开更多
Objective To evaluate activator protein 1(AP 1) mediated mechanisms in thrombin induced qlasmino^gen activator inhibitor 1 (PAI 1) expression in cultured human glomerular mesangial cells (MCs) Methods Electrophoretic ...Objective To evaluate activator protein 1(AP 1) mediated mechanisms in thrombin induced qlasmino^gen activator inhibitor 1 (PAI 1) expression in cultured human glomerular mesangial cells (MCs) Methods Electrophoretic mobility shift assay (EMSA) was employed to assess AP 1 DNA binding activity, and Western blot hybridization was used for quantification of c fos and c jun, two subunits of AP 1 dimers PAI 1 activity and mRNA expression were analysed by the fibrin plate assay and Northern hybridization, respectively Results Thrombin concentration enhanced PAI 1 activity in the supernatant and stimulated PAI 1 mRNA expression in cultured MCs PAI 1 activity was blocked by hirudin, a specific inhibitor of thrombin Further study demonstrated that thrombin promoted AP 1 DNA binding activity but exerted little effect on c fos or c jun Curcumin (AP 1 inhibitor), staurosporine (PKC inhibitor), and genistein (PTK inhibitor) all reduced AP 1 mediated PAI 1 mRNA expression induced by thrombin in cultured MCs Conclusion The present study indicates that in cultured human MCs, thrombin stimulates PAI 1 expression through an AP 1 signal pathway, which may be mediated by PKC and展开更多
Objective To explore the effect of rhein on the regulation of glucose transporter 1 (GLUT1) overexpression and the possible molecular mechanism that rhein antagonize the effect of transforming growth factor β1 (TGF ...Objective To explore the effect of rhein on the regulation of glucose transporter 1 (GLUT1) overexpression and the possible molecular mechanism that rhein antagonize the effect of transforming growth factor β1 (TGF β1) in glomerular mesangial cells Methods Cultured mouse mesangial cells were used The expression of GLUT1 mRNA was detected by Northern blotting; the ability of glucose uptake was determined by 2 deoxy [ 3H] D glucose uptake assay Results Rhein had no effect on glucose uptake in mesangial cells cultured in normal glucose concentration TGF β1 could upregulate the expression of GLUT1 mRNA and glucose uptake in mesangial cells This effect was markedly attenuated by the addition of rhein in a dose dependent manner Conclusions TGF β1 could upregulate the expression of GLUT1 mRNA and glucose uptake in mesangial cells, resulting in excessive glucose consumption and extracellular matrix production in diabetic nephropathy Rhein antagonized the effect of TGF β1 in mesangial cells, so it might be a hopeful remedy for the treatment of patients with diabetic展开更多
Objective To investigate the effect of Astragalin on human renal mesangial cells Methods Cultured human mesangial cells were treated with Astragalin and Astragalin serum in different concentrations in the presence or ...Objective To investigate the effect of Astragalin on human renal mesangial cells Methods Cultured human mesangial cells were treated with Astragalin and Astragalin serum in different concentrations in the presence or absence of PDGF BB, the proliferation and type Ⅳ collagen secretion of mesangial cells were measured by MTT assay and ELISA, and expression of β1 integrin gene was estimated by reverse transcription polymerase chain reaction (RT PCR) method, sespectively Results After 72 hours Astragalin or Astragalin serum treatment, the proliferation of mesangial cells induced by PDGF BB was inhibited significantly in a dose dependent manner compared with untreated controls ( P <0 05 and P <0 01) After 24 hours of Astragalin or Astragalin serum treatment, the secretion of type Ⅳ collagen protein in presence of PDGF BB was significantly decreased and β1 integrin mRNA level decreased significantly compared with untreated control ( P <0 05, P <0 01) Conclusions Astragalin inhibits cell proliferation and matrix over synthesis which might be mediated, at least, partly by decrease of β1 integrin gene over expression The study suggested that Astragalin might play a role in preventing the progression of chronic renal展开更多
The apoptosis of glomerular mesangial cells(GMCs)is considered to be an important contributor to the initiation and development of rat Thy-1 nephritis(Thy-1N)and is accompanied by sublytic C5b-9 deposition.However,the...The apoptosis of glomerular mesangial cells(GMCs)is considered to be an important contributor to the initiation and development of rat Thy-1 nephritis(Thy-1N)and is accompanied by sublytic C5b-9 deposition.However,the mechanism by which sublytic C5b-9 triggers GMC apoptosis has not been elucidated.In this study,functional and histological examinations were performed on GMCs treated with sublytic C5b-9(in vitro)and renal tissues of Thy-1N rats(in vivo).The in vitro studies found that sublytic C5b-9 could trigger GMC apoptosis through upregulating Egr-1,ATF3,and Gadd45 expression.Egr-1-mediated post-transcriptional modulation of ATF3,Egr-1/ATF3-enhanced Gadd45 promoter activity,and p300-mediated ATF3 acetylation were all involved in GMC apoptosis.More importantly,the effective binding elements for Egr-1 and ATF3 to Gadd45β/γpromoters and the ATF3 acetylation site were identified.In vivo,silencing renal p300,Egr-1,ATF3,and Gadd45β/γsignificantly decreased GMC apoptosis,secondary GMC proliferation,and urinary protein secretion in Thy-1N rats.Together,these findings implicate that sublytic C5b-9-induced activation of Egr-1/p300–ATF3/Gadd45 axis plays a critical role in GMC apoptosis in Thy-1N rats.展开更多
OBJECTIVE: To investigate the efficacy of Xiaokeping(XKP)-containing serum on the proliferation of high-glucose-induced mesangial cells(MCs)and the potential underlying mechanism.METHODS: XKP-containing serum was prep...OBJECTIVE: To investigate the efficacy of Xiaokeping(XKP)-containing serum on the proliferation of high-glucose-induced mesangial cells(MCs)and the potential underlying mechanism.METHODS: XKP-containing serum was prepared by the intragastric administration of XKP in rats.HBZY-1 cells were cultured with normal glucose(NC group), high glucose(HG group), and high glucose with different XKP concentrations. Cell proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,and the cell cycle distribution was detected by flow cytometry. The expression of p38 mitogen-activated protein kinase(p38 MAPK) pathway components in MCs was detected by Western blotting and quantitative real-time polymerase chain reaction.RESULTS: The MC proliferation level in the high-glucose group was significantly higher than that in the normal control group, and XKP suppressed the HG-induced proliferation of MCs dose dependently. Moreover, flow cytometry revealed that XKP blocked cell cycle progression by inducing cell cycle arrest in G1 phase and inhibiting S phase entry. XKP down-regulated the protein and m RNA expression of p38 MAPK in MCs(P < 0.05 vs HG).CONCLUSION: The present study demonstrated that XKP-containing serum inhibits high-glucoseinduced proliferation of MCs by causing cell cycle arrest at G1 phase and inhibiting S phase entry. The underlying mechanism involves the down-regulation of the p38 MAPK signaling pathway, providing a theoretical basis for the use of XKP to treat diabetic kidney disease.展开更多
The prenent study was designed to test the hypothesis that the inhibitory effect of elevated glucose levels on mesangial cell growth might be mediated by transforming growth factor β (TGF β). Increased glucose level...The prenent study was designed to test the hypothesis that the inhibitory effect of elevated glucose levels on mesangial cell growth might be mediated by transforming growth factor β (TGF β). Increased glucose levels inhibited mesangial cell proliferation in a concentration dependent manner. The addition of a rabbit antipncine TGF β1 neutralizing antibody significantly enchanced the cell proliferation in both normal (5.5mM) and high (50mM) glucose media (12% and 30% respectively), and almost completely blocked the growth inhibition induced by high glucose media. TGF β assay demonstrated mesangial cells produced more active than latent TGF β after growth in high glucose media. We conclude that TGF β functions as an autocrine cytokine in mesangial cells growth and the production of TGF β is modulated by high glucose concentration. The growth inhibition induced by high glucose levels may be largely mediated by endogenous TGF β.展开更多
Objective To investigate the effects of endothelin 1 (ET 1) on mesangial cell proliferation in vitro Methods Antisense oligodeoxynucleotide (As ODN) and its control sequences, sense (Se ODN) and mismatch (Mis ODN) oli...Objective To investigate the effects of endothelin 1 (ET 1) on mesangial cell proliferation in vitro Methods Antisense oligodeoxynucleotide (As ODN) and its control sequences, sense (Se ODN) and mismatch (Mis ODN) oligodeoxynucleotides, targeting preproendothelin 1 (ppET 1) mRNA were delivered into cultured human mesangial cells (HMC) with lipofectin mediated gene transfer method The cytotoxicity of lipofectin was determined with lactate dehydrogenase (LDH) release assay The efficiency of ODNs transfer into HMC was examined with biotinylated As ODN staining The effect of As ODN on expression of ppET 1 mRNA was analyzed with semi quantitative reverse transcription polymerase chain reaction (RT PCR) The action of As ODN on HMC ET 1 secretion was tested by radioimmunoassay (RIA) The influence of As ODN on HMC proliferation was evaluated with MTT method Results As ODN was transferred into HMC with lipofectin without any impairment of cell viability The ppET 1 mRNA expression, the ET 1 secretion and the cell proliferation were inhibited by As ODN transferred into HMC, while Se ODN and Mis ODN transfered into HMC did not show any effects on all of these Conclussion Decrease of ET 1 secretion by HMC, caused by the down regulation of ppET 1 mRNA expression after As ODN transfer, led to inhibition of HMC proliferation These results suggest ET 1 is one of the autocrine growth factors of HMC, and may play an important role in the pathogenesis of proliferative展开更多
Objective To study the effects of high glucose and transforming growth factor β1 (TGF β1) on the expression and function of glucose transporter 1 (GLUT1) in mouse mesangial cells Methods Cultured mouse mesangial cel...Objective To study the effects of high glucose and transforming growth factor β1 (TGF β1) on the expression and function of glucose transporter 1 (GLUT1) in mouse mesangial cells Methods Cultured mouse mesangial cells were used The expression of GLUT1 mRNA was detected by Northern Blot; glucose uptake and its kinetics were determined with a 2 Deoxy [ 3H] D glucose uptake assay Results Mesangial cells exposed to enriched glucose medium (20?mmol/L) for 72 hours demonstrated a decrease in both GLUT1 mRNA and V max for uptake of the glucose analog, 2 deoxy D glucose (2DOG), as compared to mesangial cells cultured in physiologic glucose concentrations(5 5?mmol/L) In contrast, hypertonic mannitol had no effect on GLUT1 mRNA levels TGF β1 treatment for 10 hours stimulated 2DOG uptake, both in 5 5?mmol/L and 20?mmol/L glucose medium, by approximately 4 28 fold in a dose dependent manner (2?ng/ml maximum) Kinetic analysis of 2DOG uptake revealed an increase in V max and a decrease in K m in the presence of TGF β1 TGF β1 also up regulated the expression of GLUT1 mRNA in mesangial cells The addition of anti TGF β neutralizing antibody (30?μg/ml) in mesangial cells cultured in enriched glucose medium (20?mmol/L) led to a 40% decrease in 2DOG uptake Conclusions The expression of GLUT1 can be suppressed by exposure of mesangial cells to high glucose medium, which may serve as a protective mechanism against possible adverse effects of excessive glucose flux into cells TGF β1 stimulates glucose uptake by enhancing the expression and function of GLUT1 in mesangial cells This effect is independent of the glucose milieu in the cultured展开更多
Objective To observe the effects of metformin on expression of Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK),nuclear factor-κB(NF-κB)and transforming growth factorβ1(TGF-β1)in cultured rat glomer...Objective To observe the effects of metformin on expression of Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK),nuclear factor-κB(NF-κB)and transforming growth factorβ1(TGF-β1)in cultured rat glomerular mesangial cells(MCs),and explore its reno-protective mechanisms.Methods MCs were cultured in the medium with normal glucose(group NG,5.6mmol/L),high glucose(group HG,25 mmol/L)and different concentrations of metformin(group M1,M2,M3).After 48 h exposure,the supernatants and展开更多
Objective To investigate the expression of Notch signaling molecules,transforming growth factor-β(TGF-β)and fibronectin(FN)in mesangial cell induced by high glucose,and the underlying mechanism of cordyceps sinensis...Objective To investigate the expression of Notch signaling molecules,transforming growth factor-β(TGF-β)and fibronectin(FN)in mesangial cell induced by high glucose,and the underlying mechanism of cordyceps sinensis.Methods Rat glomerular mesangial cells were divided into following groups:normal展开更多
Objective To explore the clinical significance of complement activation in Ig A nephropathy(IgAN)patients and provide new potential therapy targets.Methods Biopsy-proven IgAN patients admitted in our renal center were...Objective To explore the clinical significance of complement activation in Ig A nephropathy(IgAN)patients and provide new potential therapy targets.Methods Biopsy-proven IgAN patients admitted in our renal center were retrospectively recruited.Demographic,baseline clinical and pathological data were recorded as well as the follow-up results.Patients were divided into three groups,negative,weak positive and strong展开更多
基金This project was supported by grants from the Key Science and Technology Development Program of Nanjing City of the People's Republic of China (No. YKK15057 and No.YKK16097)and the National Natural Science Foundation of China (No.81473684).
文摘Diabetic kidney disease (DKD)is a microvascular complication of type 2 diabetes.The study of DKD mechanisms is the most important target for the prevention of DKD.Renal senescence is one of the important pathogeneses for DKD,but the mechanism of renal and cellular senescence is unclear.Decreased expression of circulating miR-126 is associated with the development of DKD and may be a promising blood-based biomarker for DKD.This study is to probe the effect and mechanism of miR-126 on the aging of human glomerular mesangial cells (HGMCs)induced by high glucose.HGMCs were cultured with Roswell Park Memorial Institute (RPMI-1640)in vitro.The effect of high glucose on morphology of HGMCs was observed 72h after intervention.The cell cycle was examined by flow cytometry.The telomere length was measured by Southern blotting.The expression levels of p53,p21 and Rb proteins in p53-p21-Rb signaling pathway and p-statl,p-stat3 in JAK/STAT signaling pathway were detected by Western blotting respectively.The expression of miR-126 was examined by qRT-PCR.MiR-126 mimics was transfected into HGMCs.The effects of miR-126 mimics transfection on cell morphology,cell cycle,telomere length,p53,p21,Rb,p-stat1 and p-stat3 were observed. The results showed that high glucose not only arrested the cell cycle in G1phase but also shortened the telomere length.High glucose led to high expression of p53,p21,Rb,p-statl and p-stat3 and premature senescence of HGMCs by activating the telomere-p53-p21-Rb and JAK/STAT signaling pathways.Moreover,the miR-126 was decreased in HGMCs induced by high glucose.It was suggested that the transfection of miR-126 mimics could inhibit the telomere-p53-p21-Rb and JAK/STAT signaling pathway activity in vitro and delay the senescence of HGMCs.The results may serve as a new strategy for the treatment of DKD.
文摘<Abstrat>The proliferation of mesangial cells on cyclosporin (CsA) test mediumwas studied by MTT assay and TNF-Q in cultured supernatant was examined byusing ELISA. The results showed that cyclosporin A significantly inhibited theproliferation of mesangial cells at the concentration between 0. 25 - 15 μg/ml(IC50 1μg/ml). This action appeared to be dose-dependent. Release of TNF-αfrom mesangial cells stimulated by LPS was also dose-dependently suppressed. Itis suggested that cyclosporin A play an important role in antiproliferation mecha-nism of mesangial cells in vitro.
文摘Background The pathogenesis of diabetic nephropathy (DN) is a complex pathophysiological process.Its precise mechanism is not fully known. In recent years it has been recognized that synthesis of various extracelluar matrix (ECM) components may increase, and that degradation of ECM may decrease in DN. It was reported heparin could inhibit mesangial cells proliferation in vitro. The main aim of this study is to explore whether heparin inhibits proliferation of mesangial cells grown in high glucose concentration and to measure the effect of heparin on matrix metalloproteinases (MMPs) expression in mesangial cells. Methods The medium contained either low glucose (5 mmol/L) or high glucose (25 mmol/L). The concentrations of heparin in the culture medium were 0, 25, 50,100, 200 or 400 μg/mL. A metabolic (WST-1) assay was used to measure mesangial cell proliferation and Western blot analysis was used to measure MMPs expression of mesangial cells. Results Normal human mesangial cell (NHMC) proliferation was higher in high glucose (HG) medium than in low glucose (LG) medium. They showed a 1.93 fold expansion after 72 h in high glucose in contrast to a 1.63 fold expansion in low glucose. In the presence of heparin, mesangial cells proliferation was inhibited, which was more obvious at high glucose concentrations than at low glucose concentrations. In high glucose, with heparin concentration of 50, 100, 200 and 400 μg/mL, the mesangial cells showed a 0. 61 fold, 0.52 fold, 0.52 fold and 0.41 fold reductions in cell number compared to cells grown without heparin. In low glucose, only concentrations of 200 μg/mL and 400 μg/mL showed reduction in cell number, namely 0.54 fold and 0.45 fold, when compared to cells grown without heparin. In Western blot analysis,MMP1, MMP2, MMP3 and MMP9 was expressed by mesangial cells expressed in both high and low glucose concentrations, which was more prominent in high glucose medium. Incubation of heparin further increased expression of MMP1, MMP2, MMP3 and MMP9. Conclusions This study suggests that glucose can accelerate mesangial cell proliferation while heparin can reduce proliferation, being more obvious at high glucose concentrations. Higher glucose concentrations led to increased MMP expression, which may take part in the regulation of mesangial matrix synthesis and degradation. Addition of heparin resulted in a corresponding increase in MMP expression, most notably at high glucose concentrations, indicating a potentially renoprotective role in DN.
基金supported by a grant from Health Bureauof Jiangxi Province
文摘BACKGROUND:This study aimed to explore the effects of TNF-a on the expression of IP_3R1mRNA and protein in human mesangial cells(HMCs),and to elucidate the mechanism of TNF-a relating to IP_3R1 expression in the occurrence of hepatorenal syndrome(HRS).METHODS:HMCs were stimulated by tumor(TNF-a) with 100 ng/mL for different hours(2,4,8,and 24 hours).The expression changes of IP_3R1 mRNA and protein were detected by quantitative real-time polymerase chain reaction and immunoblotting.Several inhibitors including D609,U73122,PP1,safingol,rottlerin and non-radioactive protein kinase C(PKC) were used to examine the mechanism of signal transduction ofTNF-a-regulated IP_3R1 in HMCs.RESULTS:The levels of IP_3R1 mRNA at 2 hours after TNF-a exposure were significantly enhanced and peaked at 8 hours in HMCs(P<0.01),then descended at 24 hours(P<0.01).The levels of IP_3R1 protein at 4 hours after TNF-a exposure were obviously increased and peaked at24 hours after TNF-a exposure(P<0.01).Compared to the control group,safingol(PKCa inhibitor)and D609(phosphatidylcholine-specific phospholipase C inhibitor) significantly blocked the TNF-ainduced expression of IP_3R1 mRNA(3.30±0.81 vs.1.95±0.13,P<0.05;2.10±0.49,P<0.01) and IP_3R1protein(3.09±0.13 vs.1.86+0.39,P<0.01;1.98±0.02,P<0.01).TNF-a promoted PKCa activation with maximal PKCa phosphorylation that occurred 8 hours after stimulation measured by non-radioactive PKC assay,and the effect was markedly attenuated by pretreatment with D609 or safingol.CONCLUSION:TNF-a increased the expression of IP_3R1 and this was mediated,at least in part,through the PC-PLC/PKCa signaling pathways in HMCs.
文摘Objective:To observe the expression of Long non-coding RNA antisense mitochondrial non-coding RNA-2 (ASncmtRNA-2) in high glucose (HG) treated human renal mesangial cells (HRMCs) and the role of ASncmtRNA-2 in oxidative stress mediated diabetic nephropathy (DN) fibrosis.Methods: The expression levels of ASncmtRNA-2、transforming growth factorβ1 (TGF-β1) and fibronectin (FN) mRNA in cultured HRMCs were measured by qRT-PCR. In addition, relative reactive oxygen species (ROS) levels in HRMCs were detected with the non-fluorescent probe DCFH-DA assays.Results: Compared with 0h, the expression of ASncmtRNA-2 remained unchanged in all groups at 8 h post treatment. However, the level of ASncmtRNA-2 mRNA was increased significantly in HG and HG+NG-nitro-L-Arginine methylester (L-NAME) treated cells compared with low glucose (LG) treated cells from 16h onwards, while the level of ASncmtRNA-2 mRNA in the HG+L-NAME group was decreased compared with the HG group. Moreover, ROS fluorescence was significantly up-regulated in HG-treated cells compared with LG-treated cells, while the ROS fluorescence in HG+L-NAME group was suppressed compared with HG-treated cells. In addition, Levels of ASncmtRNA-2, TGF-β1 and FN mRNA were significantly up-regulated in HG treated cells compared with LG treated cells while Levels of ASncmtRNA-2, TGF-β1 and FN mRNA in HG+L-NAME group were down-regulated compared with HG group. Finally, the expression of ASncmtRNA-2, TGF-β1 and FN mRNA were significantly decreased in HG+ASncmtRNA-2 siRNA group compared with HG group.Conclusion: ASncmtRNA-2 was up-regulated in HG treated cells and may promote glomerular fibrosis in DN via positively regulating the expression of pro-fibrotic factors. These findings may provide novel potential therapeutic treatments for DN.
基金sponsored by Regional Demonstration Project of Marine Economic Innovation and Development in 2014 and 2016,the National Key Research and Development Program of China(2018YFD0901102)K.C.Wong Magna Fund of Ningbo University.
文摘Diabetic nephropathy(DN)is a common type of end-stage renal disease and glomerular mesangial cells(GMCs)are widely used as a cell model for DN.This study firstly investigated the inhibitory effects of the Apostichopus japonicus and Acaudina leucoprocta hydrolysates on cellular growth under high-glucose treatment,better inhibitory effect of A.japonicus hydrolysate was observed compared to that of A.leucoprocta hydrolysate.Subsequently,the global transcription profiles obtained via microarray analysis showed that 6070 and 7015 genes were identified in the A.japonicus and A.leucoprocta groups compared with the model group,respectively.Among them,transcriptions of the slc30a4,slc35dl,tppp3,tp53inpl,bcl-2,apafl,alox12b and adrala genes were restored from the levels of the model group to those of the control group,contributed to cell mitosis and proliferation in both treatment groups.In addition,other apoptosis-related genes,such as bcl-6,clu,foxo3 and akt,showed opposite trends between two groups,which might cause the difference in inhibitory effect.We preliminarily proposed that the regulation effects of A.japonicus and A.leucoprocta on the genes involved in cellular mitosis,proliferation and apoptosis,might contribute to their inhibitory activity on GMCs under high-glucose environment.
基金Supported by the National Natural Science Foundation of China(No.81603355,81900745)。
文摘Objective To elucidate the renoprotective effect of resveratrol(RSV)on sphingosine kinase 1(SphK1)signaling pathway and expression of its downstream molecules including activator protein 1(AP-1)and transformation growth factor-β1(TGF-β1)in lipopolysaccharide(LPS)-induced glomerular mesangial cells(GMCs).Methods The rat GMCs line(HBZY-1)were cultured and randomly divided into 5 groups,including control,LPS(100 ng/mL),and 5,10,20µmol/L RSV-treated groups.In addition,SphK1 inhibitor(SK-II)was used as positive control.GMCs were pretreated with RSV for 2 h and treated with LPS for another 24 h.GMCs proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay.The proteins expression of SphK1,p-c-Jun and TGF-β1 in GMCs were detected by Western blot,and DNA-binding activity of AP-1 was performed by electrophoretic mobility shift assay(EMSA).The binding activity between RSV and SphK1 protein was detected by AutoDock Vina and visualized by Discovery Studio 2016.Results LPS could obviously stimulate GMCs proliferation,elevate SphK1,p-c-Jun and TGF-β1 expression levels and increase the DNA-binding activity of AP-1(P<0.05 or P<0.01),whereas these effects were significantly blocked by RSV pretreatment.It was also suggested that the effect of RSV was similar to SK-II(P>0.05).Moreover,RSV exhibited good binding affinity towards SphK1,with docking scores of−8.1 kcal/moL and formed hydrogen bonds with ASP-178 and LEU-268 in SphK1.Conclusion RSV inhibited LPS-induced GMCs proliferation and TGF-β1 expression,which may be independent of its hypoglycemic effect on preventing the development of mesangial cell fibrosis and closely related to the direct inhibition of SphK1 pathway.
基金Supported by Hangzhou Health Technology Project:Study of the Relationship Between Mitofusin 2 And Cyclosporine A Nephropathy(No.2018A52)National Natural Science Foundation of China:Study on Mechanism of Centella Asiatica Compound in the Treatment of Iga Nephropathy by Regulating Mesangial Cell Proliferation via Mitofusin 2(No.81373631)
文摘OBJECTIVE:To investigate the effect of mitofusin 2 (Mfn2) and its downstream signaling pathway on glomerular mesangial cells (GMCs) proliferation in IgA nephropathy (IgAN),as well as the mechanism of action of Jixuecao (Herba Centellae Asiaticae,HCA) in the treatment of IgAN.METHODS:Adenovirus-mediated Mfn2 gene transfection and Mfn2 expression were analyzed by real-time polymerase chain reaction (PCR) and Western blotting.IgA1 induced the proliferation of GMCs,which were then treated with HCA.Cell proliferation was detected with cell counting kit-8 (CCK-8),and Mfn2 expression was analyzed by real-time PCR and western blotting.An IgAN animal model was also established and treated with HCA.GMCs proliferation was detected by hematoxylin-eosin staining,mitochondrial structure was analyzed by electron microscopy,mitochondrial function was determined by the Clark oxygen electrode method,and the expression of Mfn2,Phospho-extracellular regulated protein kinases1/2 (P-ERK1/2),Cyclin-dependent kinase 2 (CDK2),Phospho-p27 (p-p27),and cyclin A was analyzed by Western blotting.RESULTS:In vitro,HCA inhibited GMCs in a concentration-dependent manner in association with the upregulation of Mfn2 expression.The overexpression of Mfn2 inhibited IgA1-induced GMCs proliferation and elevated the effect of HCA.In vivo,treatment with HCA could alleviate albuminuria and creatinine and GMCs proliferation.These effects were related to the upregulation of Mfn2,p-p27 and inhibition of p-ERK1/2,CDK2,and cyclinA.Mitochondrial swelling,vacuolar degeneration,and reduction of respiratory control rate were identified in IgAN,but HCA could improve the mitochondrial structure and function.CONCLUSION:HCA inhibited GMCs proliferation via the upregulation Mfn2 and the inhibition of Ras-Raf-ERK/MAPK.We revealed that changes of mitochondrial structure and function are associated with IgAN,but that HCA can improve these mitochondrial features.
文摘Objective To evaluate activator protein 1(AP 1) mediated mechanisms in thrombin induced qlasmino^gen activator inhibitor 1 (PAI 1) expression in cultured human glomerular mesangial cells (MCs) Methods Electrophoretic mobility shift assay (EMSA) was employed to assess AP 1 DNA binding activity, and Western blot hybridization was used for quantification of c fos and c jun, two subunits of AP 1 dimers PAI 1 activity and mRNA expression were analysed by the fibrin plate assay and Northern hybridization, respectively Results Thrombin concentration enhanced PAI 1 activity in the supernatant and stimulated PAI 1 mRNA expression in cultured MCs PAI 1 activity was blocked by hirudin, a specific inhibitor of thrombin Further study demonstrated that thrombin promoted AP 1 DNA binding activity but exerted little effect on c fos or c jun Curcumin (AP 1 inhibitor), staurosporine (PKC inhibitor), and genistein (PTK inhibitor) all reduced AP 1 mediated PAI 1 mRNA expression induced by thrombin in cultured MCs Conclusion The present study indicates that in cultured human MCs, thrombin stimulates PAI 1 expression through an AP 1 signal pathway, which may be mediated by PKC and
文摘Objective To explore the effect of rhein on the regulation of glucose transporter 1 (GLUT1) overexpression and the possible molecular mechanism that rhein antagonize the effect of transforming growth factor β1 (TGF β1) in glomerular mesangial cells Methods Cultured mouse mesangial cells were used The expression of GLUT1 mRNA was detected by Northern blotting; the ability of glucose uptake was determined by 2 deoxy [ 3H] D glucose uptake assay Results Rhein had no effect on glucose uptake in mesangial cells cultured in normal glucose concentration TGF β1 could upregulate the expression of GLUT1 mRNA and glucose uptake in mesangial cells This effect was markedly attenuated by the addition of rhein in a dose dependent manner Conclusions TGF β1 could upregulate the expression of GLUT1 mRNA and glucose uptake in mesangial cells, resulting in excessive glucose consumption and extracellular matrix production in diabetic nephropathy Rhein antagonized the effect of TGF β1 in mesangial cells, so it might be a hopeful remedy for the treatment of patients with diabetic
文摘Objective To investigate the effect of Astragalin on human renal mesangial cells Methods Cultured human mesangial cells were treated with Astragalin and Astragalin serum in different concentrations in the presence or absence of PDGF BB, the proliferation and type Ⅳ collagen secretion of mesangial cells were measured by MTT assay and ELISA, and expression of β1 integrin gene was estimated by reverse transcription polymerase chain reaction (RT PCR) method, sespectively Results After 72 hours Astragalin or Astragalin serum treatment, the proliferation of mesangial cells induced by PDGF BB was inhibited significantly in a dose dependent manner compared with untreated controls ( P <0 05 and P <0 01) After 24 hours of Astragalin or Astragalin serum treatment, the secretion of type Ⅳ collagen protein in presence of PDGF BB was significantly decreased and β1 integrin mRNA level decreased significantly compared with untreated control ( P <0 05, P <0 01) Conclusions Astragalin inhibits cell proliferation and matrix over synthesis which might be mediated, at least, partly by decrease of β1 integrin gene over expression The study suggested that Astragalin might play a role in preventing the progression of chronic renal
基金supported by grants from the National Natural Science Foundations of China(81273333,81471626,and 31470853)the Natural Science Foundation of Jiangsu Higher Education Institutions of China(14KJB310006)supported by Priority Academic Program Development(PAPD)of Jiangsu Higher Education Institutions.
文摘The apoptosis of glomerular mesangial cells(GMCs)is considered to be an important contributor to the initiation and development of rat Thy-1 nephritis(Thy-1N)and is accompanied by sublytic C5b-9 deposition.However,the mechanism by which sublytic C5b-9 triggers GMC apoptosis has not been elucidated.In this study,functional and histological examinations were performed on GMCs treated with sublytic C5b-9(in vitro)and renal tissues of Thy-1N rats(in vivo).The in vitro studies found that sublytic C5b-9 could trigger GMC apoptosis through upregulating Egr-1,ATF3,and Gadd45 expression.Egr-1-mediated post-transcriptional modulation of ATF3,Egr-1/ATF3-enhanced Gadd45 promoter activity,and p300-mediated ATF3 acetylation were all involved in GMC apoptosis.More importantly,the effective binding elements for Egr-1 and ATF3 to Gadd45β/γpromoters and the ATF3 acetylation site were identified.In vivo,silencing renal p300,Egr-1,ATF3,and Gadd45β/γsignificantly decreased GMC apoptosis,secondary GMC proliferation,and urinary protein secretion in Thy-1N rats.Together,these findings implicate that sublytic C5b-9-induced activation of Egr-1/p300–ATF3/Gadd45 axis plays a critical role in GMC apoptosis in Thy-1N rats.
基金Supported by the National Natural Science Foundation of China: To investigate the molecular mechanism of Xiaokeping mixture on early diabetic nephropathy through the miR-192/TGF-β/Smad signal pathway (No. 81774270)the New Medical Talent Training Plan of Zhejiang Province in 2017111 Talent Training Plan in Tongde Hospital of Zhejiang Province。
文摘OBJECTIVE: To investigate the efficacy of Xiaokeping(XKP)-containing serum on the proliferation of high-glucose-induced mesangial cells(MCs)and the potential underlying mechanism.METHODS: XKP-containing serum was prepared by the intragastric administration of XKP in rats.HBZY-1 cells were cultured with normal glucose(NC group), high glucose(HG group), and high glucose with different XKP concentrations. Cell proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,and the cell cycle distribution was detected by flow cytometry. The expression of p38 mitogen-activated protein kinase(p38 MAPK) pathway components in MCs was detected by Western blotting and quantitative real-time polymerase chain reaction.RESULTS: The MC proliferation level in the high-glucose group was significantly higher than that in the normal control group, and XKP suppressed the HG-induced proliferation of MCs dose dependently. Moreover, flow cytometry revealed that XKP blocked cell cycle progression by inducing cell cycle arrest in G1 phase and inhibiting S phase entry. XKP down-regulated the protein and m RNA expression of p38 MAPK in MCs(P < 0.05 vs HG).CONCLUSION: The present study demonstrated that XKP-containing serum inhibits high-glucoseinduced proliferation of MCs by causing cell cycle arrest at G1 phase and inhibiting S phase entry. The underlying mechanism involves the down-regulation of the p38 MAPK signaling pathway, providing a theoretical basis for the use of XKP to treat diabetic kidney disease.
文摘The prenent study was designed to test the hypothesis that the inhibitory effect of elevated glucose levels on mesangial cell growth might be mediated by transforming growth factor β (TGF β). Increased glucose levels inhibited mesangial cell proliferation in a concentration dependent manner. The addition of a rabbit antipncine TGF β1 neutralizing antibody significantly enchanced the cell proliferation in both normal (5.5mM) and high (50mM) glucose media (12% and 30% respectively), and almost completely blocked the growth inhibition induced by high glucose media. TGF β assay demonstrated mesangial cells produced more active than latent TGF β after growth in high glucose media. We conclude that TGF β functions as an autocrine cytokine in mesangial cells growth and the production of TGF β is modulated by high glucose concentration. The growth inhibition induced by high glucose levels may be largely mediated by endogenous TGF β.
文摘Objective To investigate the effects of endothelin 1 (ET 1) on mesangial cell proliferation in vitro Methods Antisense oligodeoxynucleotide (As ODN) and its control sequences, sense (Se ODN) and mismatch (Mis ODN) oligodeoxynucleotides, targeting preproendothelin 1 (ppET 1) mRNA were delivered into cultured human mesangial cells (HMC) with lipofectin mediated gene transfer method The cytotoxicity of lipofectin was determined with lactate dehydrogenase (LDH) release assay The efficiency of ODNs transfer into HMC was examined with biotinylated As ODN staining The effect of As ODN on expression of ppET 1 mRNA was analyzed with semi quantitative reverse transcription polymerase chain reaction (RT PCR) The action of As ODN on HMC ET 1 secretion was tested by radioimmunoassay (RIA) The influence of As ODN on HMC proliferation was evaluated with MTT method Results As ODN was transferred into HMC with lipofectin without any impairment of cell viability The ppET 1 mRNA expression, the ET 1 secretion and the cell proliferation were inhibited by As ODN transferred into HMC, while Se ODN and Mis ODN transfered into HMC did not show any effects on all of these Conclussion Decrease of ET 1 secretion by HMC, caused by the down regulation of ppET 1 mRNA expression after As ODN transfer, led to inhibition of HMC proliferation These results suggest ET 1 is one of the autocrine growth factors of HMC, and may play an important role in the pathogenesis of proliferative
文摘Objective To study the effects of high glucose and transforming growth factor β1 (TGF β1) on the expression and function of glucose transporter 1 (GLUT1) in mouse mesangial cells Methods Cultured mouse mesangial cells were used The expression of GLUT1 mRNA was detected by Northern Blot; glucose uptake and its kinetics were determined with a 2 Deoxy [ 3H] D glucose uptake assay Results Mesangial cells exposed to enriched glucose medium (20?mmol/L) for 72 hours demonstrated a decrease in both GLUT1 mRNA and V max for uptake of the glucose analog, 2 deoxy D glucose (2DOG), as compared to mesangial cells cultured in physiologic glucose concentrations(5 5?mmol/L) In contrast, hypertonic mannitol had no effect on GLUT1 mRNA levels TGF β1 treatment for 10 hours stimulated 2DOG uptake, both in 5 5?mmol/L and 20?mmol/L glucose medium, by approximately 4 28 fold in a dose dependent manner (2?ng/ml maximum) Kinetic analysis of 2DOG uptake revealed an increase in V max and a decrease in K m in the presence of TGF β1 TGF β1 also up regulated the expression of GLUT1 mRNA in mesangial cells The addition of anti TGF β neutralizing antibody (30?μg/ml) in mesangial cells cultured in enriched glucose medium (20?mmol/L) led to a 40% decrease in 2DOG uptake Conclusions The expression of GLUT1 can be suppressed by exposure of mesangial cells to high glucose medium, which may serve as a protective mechanism against possible adverse effects of excessive glucose flux into cells TGF β1 stimulates glucose uptake by enhancing the expression and function of GLUT1 in mesangial cells This effect is independent of the glucose milieu in the cultured
文摘Objective To observe the effects of metformin on expression of Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK),nuclear factor-κB(NF-κB)and transforming growth factorβ1(TGF-β1)in cultured rat glomerular mesangial cells(MCs),and explore its reno-protective mechanisms.Methods MCs were cultured in the medium with normal glucose(group NG,5.6mmol/L),high glucose(group HG,25 mmol/L)and different concentrations of metformin(group M1,M2,M3).After 48 h exposure,the supernatants and
文摘Objective To investigate the expression of Notch signaling molecules,transforming growth factor-β(TGF-β)and fibronectin(FN)in mesangial cell induced by high glucose,and the underlying mechanism of cordyceps sinensis.Methods Rat glomerular mesangial cells were divided into following groups:normal
文摘Objective To explore the clinical significance of complement activation in Ig A nephropathy(IgAN)patients and provide new potential therapy targets.Methods Biopsy-proven IgAN patients admitted in our renal center were retrospectively recruited.Demographic,baseline clinical and pathological data were recorded as well as the follow-up results.Patients were divided into three groups,negative,weak positive and strong
