The role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By usin...The role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with PIRES2-EGFP- S422D hSGK1 (SD) could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with PIRES2-EGFP (FP) under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with PIRES2-EGFP- K127N hSGK1 (KN) could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.展开更多
Objective: Monocytes/macrophages, proinflammatory cytokines and chemokines are important in the pathogenesis of glomerulonephritis. Interleukin (IL) -13 has been shown to exert potent anti-inflammatory properties. ...Objective: Monocytes/macrophages, proinflammatory cytokines and chemokines are important in the pathogenesis of glomerulonephritis. Interleukin (IL) -13 has been shown to exert potent anti-inflammatory properties. This study was designed to investigate the effect of IL-13 on the expression of proinflammatory cytokines, chemokines and profibrogenic cytokines and the involved molecular mechanism in cultured human mesangial cells (HMCs). Methods: The expressions of proinflammatory cytokines, chemokines and profibrogenic cytokines were determined by ribonuclease protection assay (RPA). Activity of nuclear factor-kappa B (NF-κB) and activa- tor protein-1 (AP-1) was examined by electrophoretic mobility shift assay (EMSA). NF-κB subunit p65 nuclear transportation and c-Jun N-terminal kinase (JNK) activity were assayed by immunoblot. Results: Recombinant IL-13 inhibited tumor necrosis factor-α (TNF-u), IL-1α, IL-1β, monocyte chemoattractant protein-1 (MCP-1), IL-8, and transforming growth factor-β1 (TGF-β1) mRNA expressions in a dose-dependent manner. Lipopoly- sacchorides (LPS) dramatically increased NF-κB DNA binding activity of HMCs, which was inhibited by IL-13 in a dose-dependent manner. LPS-activated NF-κB contained p50 and p65 dimers, but not c-Rel subunit. IL-13 blocked LPS-induced NF-κB subunit p65. LPS stimulated JNK/AP-1 activation, which was inhibited by IL-13 in a dose-dependent manner. Conclusion: IL-13 inhibits proinflammatory cytokines, chemokines, and profibrogenic cytokines synthesis by blocking NF-κB and JNK/AP-1 activation. These observations point to the importance of IL-13 in the modulation of inflammatory processes in the renal glomerulus.展开更多
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...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. It is suggested that cyclosporin A play an important role in antiproliferation mecha-nism of mesangial cells in vitro.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 explore whether there is phenotypic modulation of mesangial cells in streptozotocin (STZ) induced diabetic rats and study the effect of Tujian Mixture (TJM) on it. Methods: SD rats were divided into the ...Objective: To explore whether there is phenotypic modulation of mesangial cells in streptozotocin (STZ) induced diabetic rats and study the effect of Tujian Mixture (TJM) on it. Methods: SD rats were divided into the normal control group , the unilateral nephrectomized control group , the STZ induced diabetes mellitus with unilateral nephrectomy model group , the Valsartan treated group (VT group, n=8) and the TJM treated group , rats in the latter two groups were modeled as in the DM group and treated with Valsartan (20 mg/kg·d) and TJM (20g/kg·d) respectively for 12 weeks. The expression of α-smooth muscle actin (α-SMA) and transforming growth factor-β 1 (TGF-β 1) in rats’ glomeruli were observed by immunohistochemistry assay, and the ratio of α-SMA and TGF-β 1 positive area/total glomerule tuft area (SMA/GT and TGF/GT) were analyzed using computer-assisted image analysis software. Results: In the NC and the QC groups, only trace of α-SMA positive staining was found. But there was prominant α-SMA positive staining in glomeruli of the DM group, with SMA/GT and TGF/GT increased significantly , and marked increase of 24 hrs proteinuria excretion ( P<0 01). As compared with the DM group, the three indexes were all significantly lower in the VT and ZY groups , and the lowering of proteinuria was more significant in the ZY group than that in the VT group (P<0 01). Conclusion: The expression of α-SMA in glomeruli in STZ induced diabetic rats with unilateral nephrectomy is pronounced, indicating that phenotypic modulation of mesangial cells involvement in the pathogenesis of diabetic nephropathy. TJM and Valsartan can reduce 24 hrs proteinuria excretion, inhibit the phenotypic modulation of mesangial cells and the expression of TGF-β 1 in glomeruli of diabetic rats, and the effect of TJM is more potent than that of Valsartan in lowering urinary protein excretion..展开更多
The crude polysaccharide was obtained by means of the decolorization of porphyrized Cordyceps minlitaris stroma with organic solvent, extraction with hot water, precipitation in 80% ethanol, and protein removal with t...The crude polysaccharide was obtained by means of the decolorization of porphyrized Cordyceps minlitaris stroma with organic solvent, extraction with hot water, precipitation in 80% ethanol, and protein removal with the Sevag method. After purification with Sephadex G-75, two of its components, CMP-1 and CMP-2, were obtained. Through the assay of gel chromatography and polarimetry, CMP-1 was identified as pure polysaccharide. The results demonstrated that CMP-1 had favorable oxidation resistance activity, which could scavenge not only oxygen-free radicals in the self-oxidation system of pyrogallic acid, but also the hydroxide-free radicals in the Fenton system. The study focused on the effects of low, medium, and high dosages of CMP-1 in rat blood serum on the proliferation of glomerular mesangial cells in vitro. Through MTT Colorimetric analysis, the activities were compared among the blank control group and the Niaoduqing positive control group CMP-1 and CMP-2. The results shows that CMP-1 was able to inhibit the proliferation of rat glomerular mesangial cells effectively. Therefore, CMP-1, one component of polysaccharides of Cordyceps minlitaris, was certainly a potential remedy for hyperplastic glomerular nephritis, whose antioxidant activity could slow down the process of chronic renal failure(CRF) to some extent.展开更多
The effects of mycophenolic acid (MPA) on high glucose-induced expression of transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) in mesangial cells (MC) were investigated. Rat MC ...The effects of mycophenolic acid (MPA) on high glucose-induced expression of transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) in mesangial cells (MC) were investigated. Rat MC were cultured in the presence of different concentrations of MPA (1.0 and 10.0 μmol/L) or MPA plus high glucose for 72 h. The expression of TGF-β and CTGF was detected by Western blot. The results showed that high glucose could induce the expression of TGF-β and CTGF in MC, but MPA could inhibit this effects. MPA did not influence the expression of TGF-β and CTGF in normal glucose. It was concluded that MPA might prevent the progression of diabetic nephropathy by inhibiting the expression of TGF-β and CTGF in MC.展开更多
Objective:To explore the effect of the Nephritis No. 3 (N-3) recipe on nitric oxide (NO), nitric oxide synthase (NOS) secreted by cultured mesangial cells (MC) and its gene expression of the in-ducible nitric oxide sy...Objective:To explore the effect of the Nephritis No. 3 (N-3) recipe on nitric oxide (NO), nitric oxide synthase (NOS) secreted by cultured mesangial cells (MC) and its gene expression of the in-ducible nitric oxide synthase (iNOS). Methods:The drug (nephritis No. 3)-containing serum was prepared with serum pharmacological technique, and then was applied to react on mesangial cells cultured in fetal calf serum (FCS) and cells cultured in FCS plus lipopolysaccharide. To observe the secretion of NO and NOS and the gene expression of iNOS by means of RT-PCR. Results:Under the two kinds of culture conditions, the content of NO and NOS in the groups with drug-containing serum were higher than those without drug-containing serum (P<0. 05, P<0. 01), and the expression of iNOS mRNA was up-regulated too. Conclusion: The N-3 could significantly promote the secretion of NO and NOS and the mRNA expression of iNOS in rats.展开更多
Objective: To examine whether lipoxin A4 (LXA4) has an inhibitory effect on tumor necrosis factor-α(TNF-α-induced DNA synthesis of glomerular mesangial cells of rat, and explore the molecular mechanisms of LXA4 ...Objective: To examine whether lipoxin A4 (LXA4) has an inhibitory effect on tumor necrosis factor-α(TNF-α-induced DNA synthesis of glomerular mesangial cells of rat, and explore the molecular mechanisms of LXA4 action. Methods: Glomerular mesangial cells of rat were cultured and preincubated with LXA4 at different concentrations, and then treated with TNF-α( 10 ng/ml). DNA synthesis was assessed by the incorporation of [^3H]-thymidine in mesangial cells. Expression of cyclin E protein was determined by Western blotting analysis. Activities of signal transducers and activators of transcription-3 (STAT3) were analyzed by electrophoretic mobility shift assay (EMSA). Results: TNF-α-stimulated DNA synthesis of mesangial cells, upregulafion of cyclin E protein and STAT3 activities were inhibited by LXA4 in a dose-dependent manner. Conclusion: TNF-α-induced DNA synthesis of mesangial cells can be inhibited by TXA4 probably through the mechanism of Jak1/STAT3 pathway-dependent signal transduction.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
To observe the effect of high glucose, angiotensin Ⅱ (AngⅡ) and Losartan on the expression of connective tissue growth factor (CTGF) mRNA in cultured mesangial cells (MCs) Methods MCs of SD rats were isolated and...To observe the effect of high glucose, angiotensin Ⅱ (AngⅡ) and Losartan on the expression of connective tissue growth factor (CTGF) mRNA in cultured mesangial cells (MCs) Methods MCs of SD rats were isolated and cultured High glucose (30 mmol/L) and AngⅡ (10 -9 , 10 - 7 , and 10 -5 mol/L) were added to the medium for 72 hours to observe the influence on CTGF mRNA expression Losartan of 10 -5 mol/L and AngⅡ of 10 -5 mol/L were added to the medium to observe the effects of Losartan on CTGF mRNA expression stimulated by AngⅡ The expressions of CTGF mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR) Results RT-PCR showed that high glucose and AngⅡ up-regulated the expression of CTGF mRNA, and AngⅡ stimulated the expression in a dose-dependent manner Expression of CTGF mRNA induced by AngⅡwas partially suppressed by 10 -5 mol/L Losartan (P<0 05) Conclusions High glucose and AngⅡ can enhance the expression of CTGF mRNA and thus be involved in the process of renal fibrosis Losartan can have a partial fibrogenesis-inhibiting effect, with implications for the treatment of renal fibrosis展开更多
Objective: To investigate the effects of Herbal Compound 861 (Cpd 861) on collagen synthesis and degradation in rat mesangial cells exposed to high glucose. Methods: The third to fifth passage of rat mesangial cel...Objective: To investigate the effects of Herbal Compound 861 (Cpd 861) on collagen synthesis and degradation in rat mesangial cells exposed to high glucose. Methods: The third to fifth passage of rat mesangial cells were exposed to high glucose and Cpd 861 at a concentration of 0.25-4.00 g/L for 24, 48 and 72 h, respectively. Benazepril (107-10s mmol/L) was selected as positive control. The methyl thiazolyl tetrazolium colorimetric assay was used to evaluate the effect of Cpd 861 on cell proliferation. After incubation with Cpd 861 at a concentration of 2.00 g/L for 48 h, the protein secretions of collagen type IV, matrix metallopeptidase 9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor beta 1 (TGF-131), and hepatocyte growth factor (HGF) were detected by enzyme-linked immunosorbent assay method. And rat mesangial cells were harvested to determine MMP-9, TIMP-1, TGF-131 and HGF mRNA expression by revers^transcription polymerase chain reaction. Results: Cpd 861 inhibited cell proliferation induced by high glucose in a dose- and time-dependent manner. Compared with high glucose, collagen type IV production was decreased significantly by Cpd 861 (P〈0.01). Cpd 861 increased the protein secretions and mRNA expressions of MMP-9 and HGF, whereas the protein secretions and mRNA expressions of TIMP-1 and TGF-131 were reduced markedly (P〈0.05). The ratio of MMP-9 to TIMP-1 was enhanced by Cpd 861 significantly. There was no significant difference in all above-mentioned effects between Cpd 861 (2.00 g/L) and benazepril (10-5 mmol/L). Conclusion: The anti-glomerulosclerosis mechanisms of Cpd 861 were partly attributed to its effects of inhibiting mesangial cell proliferation, decreasing collagen synthesis and enhancing collagen degradation.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
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 Electrop...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 PTK展开更多
Background Galectin-3 is the most recently identified advanced glycosylation end products (AGEs) binding protein. This study aimed to investigate the effects of AGEs and rosiglitazone on the expression and secretion...Background Galectin-3 is the most recently identified advanced glycosylation end products (AGEs) binding protein. This study aimed to investigate the effects of AGEs and rosiglitazone on the expression and secretion of galectin-3 in cultured human renal mesangial cells (HRMCs). Methods HRMCs were incubated with different concentrations of AGE-bovine serum albumin (BSA) (0, 50, 100, 200, and 400 mg/L) for different time (0, 24, 36, 48, and 72 hours), and exposed to AGE-BSA in the presence of different concentrations of rosiglitazone (1, 10, and 100 μmol/L). The mRNA and protein expression of galectin-3 in HRMCs were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. The culture medium of HRMCs was collected and concentrated, and the content of galectin-3 in the medium was detected by Western blotting. Results Both RT-PCR and Western blotting revealed that AGE-BSA up-regulated the expression of galectin-3 in HRMCs in a concentration- (P 〈0.05) and time-dependent (P 〈0.05) manner compared with the control. Compared with the control, AGE-BSA elevated the content of galectin-3 in the culture medium of HRMCs time- and concentrationdependently (P 〈0.05, respectively). Both protein and mRNA expression of galectin-3, and its content in the medium of HRMCs exposed to different concentrations of rosiglitazone in the presence of AGE-BSA were increased compared with those of cells exposed to AGE-BSA alone (P 〈0.05). Rosiglitazone increased the expression and secretion of galectin-3 in a dose-dependent manner (P 〈0.05). Conclusions AGEs up-regulates the expression and secretion of galectin-3 in HRMCs. Rosiglitazone further enhances the upregulation of galectin-3 in HRMCs induced by AGEs, which suggests that rosiglitazone may play a role of reno-protection via up-regulation of galectin-3.展开更多
The purpose of this study was to examine whether transforming growth factor-β (TGFβ acts as an autocrine cytokine in cultured mesangial cells. Measangial cell conditioned media (CM) were prepared and tested for thei...The purpose of this study was to examine whether transforming growth factor-β (TGFβ acts as an autocrine cytokine in cultured mesangial cells. Measangial cell conditioned media (CM) were prepared and tested for their effect on mesangial cell proliferation. CM showed a concentration dependent inhibition on mesangial cell proliferation and the activity was enhanced by treating conditioned media with acid. Gel filtration analysis showed peak inhibitory activity to reside in fractions with an estimated molecular weight range of 16-30 KD. The activity was partially blocked by anti-TGFβ antibody, but not nonimmune control IgG. The presence of TGFβ was confirmed using the mink lung epithelial cell assay. Furthermore, the addition of anti-TGFβ antibody directly into culture media significantly enhanced mesangial cell proliferation. These results demonstrate that measangial cell produce both active and latent forms of TGFβ, which functions as an autocrine growth inhibitor for mesangial cells.展开更多
Background Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Various treatment regimens and combinations of therapies provide only partial renoprotection. Therefore new approaches are needed...Background Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Various treatment regimens and combinations of therapies provide only partial renoprotection. Therefore new approaches are needed to retard the progression of DN. The aim of the present study was to evaluate the role of a novel spiroalkaloid from Acorus tatarinowii named acortatarin A (AcorA) in inhibiting high glucose-induced extracellular matrix accumulation in mesangial cells (MCs). Methods The cytotoxity of AcorA on MCs was examined by 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay. The expression of fibronectin and collagen IV was examined by real time PCR and western blotting. The expression of p22phox and p47phox was detected by western blot. The interaction between p22phox and p47phox was examined by co-immunoprecipitation. The phosphorylation of p47phox was examined by immunoprecipitation. The phosphorylation of protein kinase C (PKC) α, PKCβ, phospholiase C gamma (PLCγ1), and the p85 subunit of PI3K was determined by Western blotting. Results AcorA significantly inhibited high glucose-induced activation of NADPH oxidase, a ROS-generating enzyme, by increasing phosphorylation of p47phox and enhancing interaction between p22phox and p47phox. Preincubation of AcorA with MCs inhibited high glucose-induced collagen IV and fibronectin production in a dose-dependent manner. Moreover, AcorA attenuated high glucose enhanced phosphorylation of PKCα, PKCβ, PLCγ1, and the p85 subunit of PI3K. Conclusion AcorA inhibits high glucose-induced extracellular matrix production via blockinq NADPH oxidase activation.展开更多
Background Oxidative Stress and p38 mitogen-activated protein kinase (p38MAPK) play a vital role in renal fibrosis. Pioglitazone can protect kidney but the underlying mechanisms are less clear. The purpose of this s...Background Oxidative Stress and p38 mitogen-activated protein kinase (p38MAPK) play a vital role in renal fibrosis. Pioglitazone can protect kidney but the underlying mechanisms are less clear. The purpose of this study was to investigate the effect of pioglitazone on oxidative stress and whether the severity of oxidative stress was associated with the phosphorylation level of p38MAPK.展开更多
Background The plasma concentration of very low density lipoprotein (VLDL) is negatively correlated to renal function in glomerular diseases. Effects of VLDL on renal function have been partially attributed to the p...Background The plasma concentration of very low density lipoprotein (VLDL) is negatively correlated to renal function in glomerular diseases. Effects of VLDL on renal function have been partially attributed to the proliferation of mesangial cells. This study examined the potential role of the p42/44 mitogen activated protein kinase (MAPK) in mesangial cell proliferation induced by VLDL. Methods Mesangial cells were treated with VLDL at different concentrations or for different time. The cell cycle of the mesangial cells was analyzed by Xl-r assay and flow-cytometry; MAPK activity was also assayed. In some experiments, cells were treated with VLDL together with or without 0.1 pmol/L PD 98059. Results Ten to 500 μg/ml VLDL stimulated the proliferation of mesangial cells cultured in vitro in a concentration-dependent manner. The effect was associated with an increase in p42/44 MAPK activity. Increased proliferation of mesangial cells by VLDL was significantly attenuated by PD98059, a specific p42/44 MAPK inhibitor. Conclusion These results indicate that the p42/44 MAPK pathway is an important regulator of mesangial cell proliferation and of renal functions.展开更多
基金a grant from the National Natural Sciences Foundation of China (No. 30600810)
文摘The role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with PIRES2-EGFP- S422D hSGK1 (SD) could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with PIRES2-EGFP (FP) under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with PIRES2-EGFP- K127N hSGK1 (KN) could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.
基金supported by grants from the National Natural Science Foundation of China(No.30872803 to Aihua Zhang,No.30772365 to Songming Huang)the Jiangsu Key Medical Talent Foundation(No.RC2007015 to Aihua Zhangthe Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry of China
文摘Objective: Monocytes/macrophages, proinflammatory cytokines and chemokines are important in the pathogenesis of glomerulonephritis. Interleukin (IL) -13 has been shown to exert potent anti-inflammatory properties. This study was designed to investigate the effect of IL-13 on the expression of proinflammatory cytokines, chemokines and profibrogenic cytokines and the involved molecular mechanism in cultured human mesangial cells (HMCs). Methods: The expressions of proinflammatory cytokines, chemokines and profibrogenic cytokines were determined by ribonuclease protection assay (RPA). Activity of nuclear factor-kappa B (NF-κB) and activa- tor protein-1 (AP-1) was examined by electrophoretic mobility shift assay (EMSA). NF-κB subunit p65 nuclear transportation and c-Jun N-terminal kinase (JNK) activity were assayed by immunoblot. Results: Recombinant IL-13 inhibited tumor necrosis factor-α (TNF-u), IL-1α, IL-1β, monocyte chemoattractant protein-1 (MCP-1), IL-8, and transforming growth factor-β1 (TGF-β1) mRNA expressions in a dose-dependent manner. Lipopoly- sacchorides (LPS) dramatically increased NF-κB DNA binding activity of HMCs, which was inhibited by IL-13 in a dose-dependent manner. LPS-activated NF-κB contained p50 and p65 dimers, but not c-Rel subunit. IL-13 blocked LPS-induced NF-κB subunit p65. LPS stimulated JNK/AP-1 activation, which was inhibited by IL-13 in a dose-dependent manner. Conclusion: IL-13 inhibits proinflammatory cytokines, chemokines, and profibrogenic cytokines synthesis by blocking NF-κB and JNK/AP-1 activation. These observations point to the importance of IL-13 in the modulation of inflammatory processes in the renal glomerulus.
文摘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. It is suggested that cyclosporin A play an important role in antiproliferation mecha-nism of mesangial cells in vitro.
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 explore whether there is phenotypic modulation of mesangial cells in streptozotocin (STZ) induced diabetic rats and study the effect of Tujian Mixture (TJM) on it. Methods: SD rats were divided into the normal control group , the unilateral nephrectomized control group , the STZ induced diabetes mellitus with unilateral nephrectomy model group , the Valsartan treated group (VT group, n=8) and the TJM treated group , rats in the latter two groups were modeled as in the DM group and treated with Valsartan (20 mg/kg·d) and TJM (20g/kg·d) respectively for 12 weeks. The expression of α-smooth muscle actin (α-SMA) and transforming growth factor-β 1 (TGF-β 1) in rats’ glomeruli were observed by immunohistochemistry assay, and the ratio of α-SMA and TGF-β 1 positive area/total glomerule tuft area (SMA/GT and TGF/GT) were analyzed using computer-assisted image analysis software. Results: In the NC and the QC groups, only trace of α-SMA positive staining was found. But there was prominant α-SMA positive staining in glomeruli of the DM group, with SMA/GT and TGF/GT increased significantly , and marked increase of 24 hrs proteinuria excretion ( P<0 01). As compared with the DM group, the three indexes were all significantly lower in the VT and ZY groups , and the lowering of proteinuria was more significant in the ZY group than that in the VT group (P<0 01). Conclusion: The expression of α-SMA in glomeruli in STZ induced diabetic rats with unilateral nephrectomy is pronounced, indicating that phenotypic modulation of mesangial cells involvement in the pathogenesis of diabetic nephropathy. TJM and Valsartan can reduce 24 hrs proteinuria excretion, inhibit the phenotypic modulation of mesangial cells and the expression of TGF-β 1 in glomeruli of diabetic rats, and the effect of TJM is more potent than that of Valsartan in lowering urinary protein excretion..
基金the Science Technology Development Project of Jilin Province, China(No.20020503-2).
文摘The crude polysaccharide was obtained by means of the decolorization of porphyrized Cordyceps minlitaris stroma with organic solvent, extraction with hot water, precipitation in 80% ethanol, and protein removal with the Sevag method. After purification with Sephadex G-75, two of its components, CMP-1 and CMP-2, were obtained. Through the assay of gel chromatography and polarimetry, CMP-1 was identified as pure polysaccharide. The results demonstrated that CMP-1 had favorable oxidation resistance activity, which could scavenge not only oxygen-free radicals in the self-oxidation system of pyrogallic acid, but also the hydroxide-free radicals in the Fenton system. The study focused on the effects of low, medium, and high dosages of CMP-1 in rat blood serum on the proliferation of glomerular mesangial cells in vitro. Through MTT Colorimetric analysis, the activities were compared among the blank control group and the Niaoduqing positive control group CMP-1 and CMP-2. The results shows that CMP-1 was able to inhibit the proliferation of rat glomerular mesangial cells effectively. Therefore, CMP-1, one component of polysaccharides of Cordyceps minlitaris, was certainly a potential remedy for hyperplastic glomerular nephritis, whose antioxidant activity could slow down the process of chronic renal failure(CRF) to some extent.
文摘The effects of mycophenolic acid (MPA) on high glucose-induced expression of transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) in mesangial cells (MC) were investigated. Rat MC were cultured in the presence of different concentrations of MPA (1.0 and 10.0 μmol/L) or MPA plus high glucose for 72 h. The expression of TGF-β and CTGF was detected by Western blot. The results showed that high glucose could induce the expression of TGF-β and CTGF in MC, but MPA could inhibit this effects. MPA did not influence the expression of TGF-β and CTGF in normal glucose. It was concluded that MPA might prevent the progression of diabetic nephropathy by inhibiting the expression of TGF-β and CTGF in MC.
基金The item was supported by Tianjin Scientific Committee (No. 953101311)won the second grade Science and Technology Progress award of Tianjin in 2001
文摘Objective:To explore the effect of the Nephritis No. 3 (N-3) recipe on nitric oxide (NO), nitric oxide synthase (NOS) secreted by cultured mesangial cells (MC) and its gene expression of the in-ducible nitric oxide synthase (iNOS). Methods:The drug (nephritis No. 3)-containing serum was prepared with serum pharmacological technique, and then was applied to react on mesangial cells cultured in fetal calf serum (FCS) and cells cultured in FCS plus lipopolysaccharide. To observe the secretion of NO and NOS and the gene expression of iNOS by means of RT-PCR. Results:Under the two kinds of culture conditions, the content of NO and NOS in the groups with drug-containing serum were higher than those without drug-containing serum (P<0. 05, P<0. 01), and the expression of iNOS mRNA was up-regulated too. Conclusion: The N-3 could significantly promote the secretion of NO and NOS and the mRNA expression of iNOS in rats.
文摘Objective: To examine whether lipoxin A4 (LXA4) has an inhibitory effect on tumor necrosis factor-α(TNF-α-induced DNA synthesis of glomerular mesangial cells of rat, and explore the molecular mechanisms of LXA4 action. Methods: Glomerular mesangial cells of rat were cultured and preincubated with LXA4 at different concentrations, and then treated with TNF-α( 10 ng/ml). DNA synthesis was assessed by the incorporation of [^3H]-thymidine in mesangial cells. Expression of cyclin E protein was determined by Western blotting analysis. Activities of signal transducers and activators of transcription-3 (STAT3) were analyzed by electrophoretic mobility shift assay (EMSA). Results: TNF-α-stimulated DNA synthesis of mesangial cells, upregulafion of cyclin E protein and STAT3 activities were inhibited by LXA4 in a dose-dependent manner. Conclusion: TNF-α-induced DNA synthesis of mesangial cells can be inhibited by TXA4 probably through the mechanism of Jak1/STAT3 pathway-dependent signal transduction.
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ThestudywassupportedbythegrantsfromtheNationalNatureScienceFoundationofChina (No 39870 2 97)
文摘To observe the effect of high glucose, angiotensin Ⅱ (AngⅡ) and Losartan on the expression of connective tissue growth factor (CTGF) mRNA in cultured mesangial cells (MCs) Methods MCs of SD rats were isolated and cultured High glucose (30 mmol/L) and AngⅡ (10 -9 , 10 - 7 , and 10 -5 mol/L) were added to the medium for 72 hours to observe the influence on CTGF mRNA expression Losartan of 10 -5 mol/L and AngⅡ of 10 -5 mol/L were added to the medium to observe the effects of Losartan on CTGF mRNA expression stimulated by AngⅡ The expressions of CTGF mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR) Results RT-PCR showed that high glucose and AngⅡ up-regulated the expression of CTGF mRNA, and AngⅡ stimulated the expression in a dose-dependent manner Expression of CTGF mRNA induced by AngⅡwas partially suppressed by 10 -5 mol/L Losartan (P<0 05) Conclusions High glucose and AngⅡ can enhance the expression of CTGF mRNA and thus be involved in the process of renal fibrosis Losartan can have a partial fibrogenesis-inhibiting effect, with implications for the treatment of renal fibrosis
文摘Objective: To investigate the effects of Herbal Compound 861 (Cpd 861) on collagen synthesis and degradation in rat mesangial cells exposed to high glucose. Methods: The third to fifth passage of rat mesangial cells were exposed to high glucose and Cpd 861 at a concentration of 0.25-4.00 g/L for 24, 48 and 72 h, respectively. Benazepril (107-10s mmol/L) was selected as positive control. The methyl thiazolyl tetrazolium colorimetric assay was used to evaluate the effect of Cpd 861 on cell proliferation. After incubation with Cpd 861 at a concentration of 2.00 g/L for 48 h, the protein secretions of collagen type IV, matrix metallopeptidase 9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor beta 1 (TGF-131), and hepatocyte growth factor (HGF) were detected by enzyme-linked immunosorbent assay method. And rat mesangial cells were harvested to determine MMP-9, TIMP-1, TGF-131 and HGF mRNA expression by revers^transcription polymerase chain reaction. Results: Cpd 861 inhibited cell proliferation induced by high glucose in a dose- and time-dependent manner. Compared with high glucose, collagen type IV production was decreased significantly by Cpd 861 (P〈0.01). Cpd 861 increased the protein secretions and mRNA expressions of MMP-9 and HGF, whereas the protein secretions and mRNA expressions of TIMP-1 and TGF-131 were reduced markedly (P〈0.05). The ratio of MMP-9 to TIMP-1 was enhanced by Cpd 861 significantly. There was no significant difference in all above-mentioned effects between Cpd 861 (2.00 g/L) and benazepril (10-5 mmol/L). Conclusion: The anti-glomerulosclerosis mechanisms of Cpd 861 were partly attributed to its effects of inhibiting mesangial cell proliferation, decreasing collagen synthesis and enhancing collagen degradation.
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 PTK
文摘Background Galectin-3 is the most recently identified advanced glycosylation end products (AGEs) binding protein. This study aimed to investigate the effects of AGEs and rosiglitazone on the expression and secretion of galectin-3 in cultured human renal mesangial cells (HRMCs). Methods HRMCs were incubated with different concentrations of AGE-bovine serum albumin (BSA) (0, 50, 100, 200, and 400 mg/L) for different time (0, 24, 36, 48, and 72 hours), and exposed to AGE-BSA in the presence of different concentrations of rosiglitazone (1, 10, and 100 μmol/L). The mRNA and protein expression of galectin-3 in HRMCs were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. The culture medium of HRMCs was collected and concentrated, and the content of galectin-3 in the medium was detected by Western blotting. Results Both RT-PCR and Western blotting revealed that AGE-BSA up-regulated the expression of galectin-3 in HRMCs in a concentration- (P 〈0.05) and time-dependent (P 〈0.05) manner compared with the control. Compared with the control, AGE-BSA elevated the content of galectin-3 in the culture medium of HRMCs time- and concentrationdependently (P 〈0.05, respectively). Both protein and mRNA expression of galectin-3, and its content in the medium of HRMCs exposed to different concentrations of rosiglitazone in the presence of AGE-BSA were increased compared with those of cells exposed to AGE-BSA alone (P 〈0.05). Rosiglitazone increased the expression and secretion of galectin-3 in a dose-dependent manner (P 〈0.05). Conclusions AGEs up-regulates the expression and secretion of galectin-3 in HRMCs. Rosiglitazone further enhances the upregulation of galectin-3 in HRMCs induced by AGEs, which suggests that rosiglitazone may play a role of reno-protection via up-regulation of galectin-3.
文摘The purpose of this study was to examine whether transforming growth factor-β (TGFβ acts as an autocrine cytokine in cultured mesangial cells. Measangial cell conditioned media (CM) were prepared and tested for their effect on mesangial cell proliferation. CM showed a concentration dependent inhibition on mesangial cell proliferation and the activity was enhanced by treating conditioned media with acid. Gel filtration analysis showed peak inhibitory activity to reside in fractions with an estimated molecular weight range of 16-30 KD. The activity was partially blocked by anti-TGFβ antibody, but not nonimmune control IgG. The presence of TGFβ was confirmed using the mink lung epithelial cell assay. Furthermore, the addition of anti-TGFβ antibody directly into culture media significantly enhanced mesangial cell proliferation. These results demonstrate that measangial cell produce both active and latent forms of TGFβ, which functions as an autocrine growth inhibitor for mesangial cells.
基金This stucty was supported by the National "973" Program (No. 2012CB517700) and the National Natural Science Foundation of China (No. U0932002) to Dr. HOU Fan-fan the Knowledge Innovation Program of Chinese Academy of Sciences (No. KSCX2-YW-R-211) and the Project of Natural Compound Library Construction from Chinese Academy of Sciences (No. KSCX2-EW-R-15) to Dr. CHENG Yong-xian.
文摘Background Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Various treatment regimens and combinations of therapies provide only partial renoprotection. Therefore new approaches are needed to retard the progression of DN. The aim of the present study was to evaluate the role of a novel spiroalkaloid from Acorus tatarinowii named acortatarin A (AcorA) in inhibiting high glucose-induced extracellular matrix accumulation in mesangial cells (MCs). Methods The cytotoxity of AcorA on MCs was examined by 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay. The expression of fibronectin and collagen IV was examined by real time PCR and western blotting. The expression of p22phox and p47phox was detected by western blot. The interaction between p22phox and p47phox was examined by co-immunoprecipitation. The phosphorylation of p47phox was examined by immunoprecipitation. The phosphorylation of protein kinase C (PKC) α, PKCβ, phospholiase C gamma (PLCγ1), and the p85 subunit of PI3K was determined by Western blotting. Results AcorA significantly inhibited high glucose-induced activation of NADPH oxidase, a ROS-generating enzyme, by increasing phosphorylation of p47phox and enhancing interaction between p22phox and p47phox. Preincubation of AcorA with MCs inhibited high glucose-induced collagen IV and fibronectin production in a dose-dependent manner. Moreover, AcorA attenuated high glucose enhanced phosphorylation of PKCα, PKCβ, PLCγ1, and the p85 subunit of PI3K. Conclusion AcorA inhibits high glucose-induced extracellular matrix production via blockinq NADPH oxidase activation.
文摘Background Oxidative Stress and p38 mitogen-activated protein kinase (p38MAPK) play a vital role in renal fibrosis. Pioglitazone can protect kidney but the underlying mechanisms are less clear. The purpose of this study was to investigate the effect of pioglitazone on oxidative stress and whether the severity of oxidative stress was associated with the phosphorylation level of p38MAPK.
文摘Background The plasma concentration of very low density lipoprotein (VLDL) is negatively correlated to renal function in glomerular diseases. Effects of VLDL on renal function have been partially attributed to the proliferation of mesangial cells. This study examined the potential role of the p42/44 mitogen activated protein kinase (MAPK) in mesangial cell proliferation induced by VLDL. Methods Mesangial cells were treated with VLDL at different concentrations or for different time. The cell cycle of the mesangial cells was analyzed by Xl-r assay and flow-cytometry; MAPK activity was also assayed. In some experiments, cells were treated with VLDL together with or without 0.1 pmol/L PD 98059. Results Ten to 500 μg/ml VLDL stimulated the proliferation of mesangial cells cultured in vitro in a concentration-dependent manner. The effect was associated with an increase in p42/44 MAPK activity. Increased proliferation of mesangial cells by VLDL was significantly attenuated by PD98059, a specific p42/44 MAPK inhibitor. Conclusion These results indicate that the p42/44 MAPK pathway is an important regulator of mesangial cell proliferation and of renal functions.