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
Objective: To investigate the effect of Zao Huang Mixture (藻黄合剂ZHM) on expressions of growth factor-β1 (TGF-β1) and collagen IV (Col IV) in human glomerular mesangial cells (GMC) cultured in high-glucose environ...Objective: To investigate the effect of Zao Huang Mixture (藻黄合剂ZHM) on expressions of growth factor-β1 (TGF-β1) and collagen IV (Col IV) in human glomerular mesangial cells (GMC) cultured in high-glucose environment. Methods: After primary culture of GMC, in vitro culture was carried out in normal group, high glucose group and high glucose medium with ZHM of different concentrations, and the expressions of TGF-β1 and Col IV in the GMC group and in ZHM group were detected at 24 and 48 h respectively. Results: Compared with the normal group, expressions of TGF-β1 and Col IV significantly increased at 24 h, 48 h in the high glucose group (all P<0.01); Compared with the high glucose group, the expressions of TGF-β1 and Col IV in all the ZHM groups significantly decreased at 24 h, 48 h (P<0.05 or P<0.01). Conclusion: ZHM may modulate the process of diabetic nephropathy by changing the expression of TGF-β1 and Col IV in glomerular mesangial cells.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
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 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-P...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: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
Objective To study the expressions of MMP-2 and TIMP-2 mRNA on cultured rat mesangial cells (MsC) and in human diseased glomeruli, and to explore their significance in the development of glomerulosclerosis. Methods Th...Objective To study the expressions of MMP-2 and TIMP-2 mRNA on cultured rat mesangial cells (MsC) and in human diseased glomeruli, and to explore their significance in the development of glomerulosclerosis. Methods The expressions of MMP-2, TIMP-2, and Col ⅣmRNA on cultured rat MsC stimulated by IL-1 or/and TGF-β1were investigated through Northern blot analysis. The levels of MMP-2 and TIMP-2 mRNA expressions and immunoreacti-vity of PCNA and Col Ⅳin human diseased glomeruli from renal biopsies of lupus nephritis (LN) patients were examined by insituhybridization and immunohistochemistry, respectively. Results The levels of MMP-2, TIMP-2, and Col ⅣmRNA expressions were markedly increased on cultured rat MsC stimulated by IL-1 or/and TGF-β1. Meanwhile, upregulation of MMP-2 and TIMP-2 mRNA expressions was confirmed in diseased glomeruli from patients with various subtypes of LN, and was closely related to the positive cell number of PCNA presentation and deposition of Col Ⅳin glomeruli. Conclusion The results suggest that the over-expressions of MMP-2 and TIMP-2 mRNA on glomerular cells might play a critical role in the development of glomerulosclerosis.展开更多
The proliferation of glomerular mesangial cells (GMC) and secretion of the extracellular matrix (ECM) in rat with Thy-1 nephritis (Thy-1N) resembling human mesangioproliferative glomerulonephritis have been explored f...The proliferation of glomerular mesangial cells (GMC) and secretion of the extracellular matrix (ECM) in rat with Thy-1 nephritis (Thy-1N) resembling human mesangioproliferative glomerulonephritis have been explored for many years; however, the molecular mechanisms of GMC proliferation and ECM production remain unclear. Our previous studies have demonstrated that the thrombospondin-1 (TSP-1) gene was involved in mediating rat GMC proliferation and ECM synthesis induced by sublytic C5b-9 in vitro. In the present study, the roles of the TSP-1 gene in GMC proliferation, ECM production, and urinary protein secretion in Thy-1N rats were determined by using TSP-1 small hairpin RNA, and the results revealed that silencing of the TSP-1 gene in rat renal tissues could diminish GMC proliferation (P < 0.01) and ECM secretion (P < 0.01) as well as urinary protein secretion (P < 0.05) in Thy-1N rats. Together, the current findings suggested that TSP-1 gene expression was required for GMC proliferation and ECM production in Thy-1N rats.展开更多
Objective To investigate the effects of rapamycin on cholesterol homeostasis of glomerular mesangial cells and the underlying mechanisms. Methods Intracellular cholesterol accumulation was measured by Oil Red O staini...Objective To investigate the effects of rapamycin on cholesterol homeostasis of glomerular mesangial cells and the underlying mechanisms. Methods Intracellular cholesterol accumulation was measured by Oil Red O staining and high performance liquid chromatography. The effects of rapamycin on interleukin-1β (IL-1β)-induced mRNA and protein changes of low-density lipoprotein receptor (LDLR) and ATP-binding cassette transporter A1 (ABCA1) were assayed by quantitative real-time PCR and Western blot. Transient expressions of 3 types of mammalian target of rapamycin (mTOR), including mTOR-WT (wild type), mTOR-RR (rapamycin resistant, with kinase activity), and mTOR-RR-KD (rapamycin resistant, without kinase activity), were obtained by plasmid transfection. Results Rapamycin had no significant influence on intracellular cholesterol concentration under normal condition, but it significantly decreased the intracellular cholesterol concentration in the presence of IL-1β. Rapamycin dose-dependently suppressed the increased expression of LDLR induced by IL-1β and up-regulated the suppressed expression of ABCA1 caused by IL-1β. Transient expression of 3 types of mTOR all reduced ABCA1 mRNA expression significantly, which all could be overroded by rapamycin. Conclusions Rapamycin may contribute to the maintaining of glomerular mesangial cell intracellular cholesterol homeostasis under inflammatory state by both reducing cholesterol uptake and increasing cholesterol efflux. And the effect may be not completely mediated by mTOR.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
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Objective: To investigate the effect of Zao Huang Mixture (藻黄合剂ZHM) on expressions of growth factor-β1 (TGF-β1) and collagen IV (Col IV) in human glomerular mesangial cells (GMC) cultured in high-glucose environment. Methods: After primary culture of GMC, in vitro culture was carried out in normal group, high glucose group and high glucose medium with ZHM of different concentrations, and the expressions of TGF-β1 and Col IV in the GMC group and in ZHM group were detected at 24 and 48 h respectively. Results: Compared with the normal group, expressions of TGF-β1 and Col IV significantly increased at 24 h, 48 h in the high glucose group (all P<0.01); Compared with the high glucose group, the expressions of TGF-β1 and Col IV in all the ZHM groups significantly decreased at 24 h, 48 h (P<0.05 or P<0.01). Conclusion: ZHM may modulate the process of diabetic nephropathy by changing the expression of TGF-β1 and Col IV in glomerular mesangial cells.
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.
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.
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.
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Supported by Shanghai Municipal Science and Technology Develop-ment Funds (01JC14018).
文摘Objective To study the expressions of MMP-2 and TIMP-2 mRNA on cultured rat mesangial cells (MsC) and in human diseased glomeruli, and to explore their significance in the development of glomerulosclerosis. Methods The expressions of MMP-2, TIMP-2, and Col ⅣmRNA on cultured rat MsC stimulated by IL-1 or/and TGF-β1were investigated through Northern blot analysis. The levels of MMP-2 and TIMP-2 mRNA expressions and immunoreacti-vity of PCNA and Col Ⅳin human diseased glomeruli from renal biopsies of lupus nephritis (LN) patients were examined by insituhybridization and immunohistochemistry, respectively. Results The levels of MMP-2, TIMP-2, and Col ⅣmRNA expressions were markedly increased on cultured rat MsC stimulated by IL-1 or/and TGF-β1. Meanwhile, upregulation of MMP-2 and TIMP-2 mRNA expressions was confirmed in diseased glomeruli from patients with various subtypes of LN, and was closely related to the positive cell number of PCNA presentation and deposition of Col Ⅳin glomeruli. Conclusion The results suggest that the over-expressions of MMP-2 and TIMP-2 mRNA on glomerular cells might play a critical role in the development of glomerulosclerosis.
基金supported by grants from National Natural Science Foundations of China (No. 31000396, and No.81072402)grants from Natural Science Foundations of Jiangsu Province in China (No. BK2009417, No. 10KJB310006, and No. 09hx43)
文摘The proliferation of glomerular mesangial cells (GMC) and secretion of the extracellular matrix (ECM) in rat with Thy-1 nephritis (Thy-1N) resembling human mesangioproliferative glomerulonephritis have been explored for many years; however, the molecular mechanisms of GMC proliferation and ECM production remain unclear. Our previous studies have demonstrated that the thrombospondin-1 (TSP-1) gene was involved in mediating rat GMC proliferation and ECM synthesis induced by sublytic C5b-9 in vitro. In the present study, the roles of the TSP-1 gene in GMC proliferation, ECM production, and urinary protein secretion in Thy-1N rats were determined by using TSP-1 small hairpin RNA, and the results revealed that silencing of the TSP-1 gene in rat renal tissues could diminish GMC proliferation (P < 0.01) and ECM secretion (P < 0.01) as well as urinary protein secretion (P < 0.05) in Thy-1N rats. Together, the current findings suggested that TSP-1 gene expression was required for GMC proliferation and ECM production in Thy-1N rats.
文摘Objective To investigate the effects of rapamycin on cholesterol homeostasis of glomerular mesangial cells and the underlying mechanisms. Methods Intracellular cholesterol accumulation was measured by Oil Red O staining and high performance liquid chromatography. The effects of rapamycin on interleukin-1β (IL-1β)-induced mRNA and protein changes of low-density lipoprotein receptor (LDLR) and ATP-binding cassette transporter A1 (ABCA1) were assayed by quantitative real-time PCR and Western blot. Transient expressions of 3 types of mammalian target of rapamycin (mTOR), including mTOR-WT (wild type), mTOR-RR (rapamycin resistant, with kinase activity), and mTOR-RR-KD (rapamycin resistant, without kinase activity), were obtained by plasmid transfection. Results Rapamycin had no significant influence on intracellular cholesterol concentration under normal condition, but it significantly decreased the intracellular cholesterol concentration in the presence of IL-1β. Rapamycin dose-dependently suppressed the increased expression of LDLR induced by IL-1β and up-regulated the suppressed expression of ABCA1 caused by IL-1β. Transient expression of 3 types of mTOR all reduced ABCA1 mRNA expression significantly, which all could be overroded by rapamycin. Conclusions Rapamycin may contribute to the maintaining of glomerular mesangial cell intracellular cholesterol homeostasis under inflammatory state by both reducing cholesterol uptake and increasing cholesterol efflux. And the effect may be not completely mediated by mTOR.
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.
