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
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 effects of emodin（EMD） on cell proliferation and correlative cytokines secretion of glomerular mesangial in rats. Methods：The effects of EMD on cell proliferation and IL-6, TGF-β1 sec...Objective：To investigate the effects of emodin（EMD） on cell proliferation and correlative cytokines secretion of glomerular mesangial in rats. Methods：The effects of EMD on cell proliferation and IL-6, TGF-β1 secretion of glomerular mesangial in rats were observed. Cell proliferation was measured by MTT method. IL-6 and TGF-β1 secretion was detected with ELISA. Results：EMD was able to inhibit the cell proliferation and down-regulate the IL-6 and TGF-β 1 secretion of glomerular mesangial, as compared to the model group in rats （P 〈 0.05）. Conclusion：EMD could significantly inhibit the cell proliferation, and reduce the creation of extracellular matrix（ECM）, this indicated that it could play an important role in alleviation and prevention of glomerular sclerosis. The mechanism may be that EMD can reduce the IL-6 and TGF-β1 secretion ofglomerular mesangial cell in rats.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-lN） resembling human mesangioproliferative glomerulonephritis have been expl...The proliferation of glomerular mesangial cells （GMC） and secretion of the extracellular matrix （ECM） in rat with Thy-1 nephritis （Thy-lN） 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. 111 the present study, the roles of the TSP-1 gene in GMC proliferation, ECM production, and urinary protein secretion in Thy-lN 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-lN rats. Together, the current findings suggested that TSP-1 gene expression was required for GMC proliferation and ECM production in Thy-lN 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
Sublytic complement C5b-9 complexes can cause cell apoptosis, but the mechanism of glomerular mesangial cell （GMC） apoptosis mediated by these complexes has not been well defined. The activating transcription factor...Sublytic complement C5b-9 complexes can cause cell apoptosis, but the mechanism of glomerular mesangial cell （GMC） apoptosis mediated by these complexes has not been well defined. The activating transcription factor 3 （ATF3） gene is an immediate early gene for the cell to cope with a variety of stress signals and can promote apoptosis of some cells. In this study, ATF3 expression and cell apoptosis in GMCs induced by sublytic C5b-9 were measured, and then the effects of ATF3 gene over-expression or knockdown on GMC apoptosis induced by sublytic C5b-9 were examined at a fixed time. The results showed that both ATF3 expression and GMC apoptosis were markedly increased and ATF3 over-expression obviously increased sublytic C5b-9-induced GMC apoptosis, whereas ATF3 gene silencing had a significant opposite effect. Collectively, these findings indicate that upregulation of ATF3 gene expression is involved in regulating GMC apoptosis induced by sublytic C5b-9 complexes.展开更多
Background The renoprotective mechanisms of adenosine monophosphate （AMP）-activated protein kinase （AMPK） agonist-metformin have not been stated clearly.We hypothesized that metformin may ameliorate inflammation v...Background The renoprotective mechanisms of adenosine monophosphate （AMP）-activated protein kinase （AMPK） agonist-metformin have not been stated clearly.We hypothesized that metformin may ameliorate inflammation via AMPK interaction with critical inflammatory cytokines The aim of this study was to observe the effects of metformin on expression of nuclear factor-κB （NF-κB）,monocyte chemoattractant protein-1 （MCP-1）,intercellular adhesion molecule-1 （ICAM-1） and transforming growth factor-beta 1 （TGF-β1） induced by high glucose （HG） in cultured rat glomerular mesangial cells （MCs）.Methods MCs were cultured in the medium with normal concentration glucose （group NG,5.6 mmol/L）,high concentration glucose （group HG,25 mmol/L） and different concentrations of metformin （group M1,M2,M3）.After 48-hour exposure,the supernatants and MCs were collected.The expression of NF-κB,MCP-1,ICAM-1,and TGF-β1 mRNA was analyzed by real time polymerase chain reaction.Westem blotting was used to detect the expression of AMPK,phospho-Thr-172 AMPK （p-AMPK）,NF-κB p65,MCP-1,ICAM-1,and TGF-β1 protein.Results After stimulated by HG,the expression of NF-κB,MCP-1,ICAM-1,TGF-β1 mRNA and protein of MCs in group HG increased significantly compared with group NG （P ＜0.05）.Both genes and protein expression of NF-κB,MCP-1,ICAM-1,TGF-β1 of MCs induced by high glucose were markedly reduced after metformin treatment in a dose-dependent manner （P ＜0.05）.The expression of p-AMPK increased with the rising of metformin concentration,presenting the opposite trend,while the level of total-AMPK protein was unchanged with exposure to HG or metformin.Conlusion Metformin can suppress the expression of NF-κB,MCP-1,ICAM-1 and TGF-β1 of glomerular MCs induced by high glucose via AMPK activation,which may partlv contribute to its reno-protection.展开更多
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 effects of emodin（EMD） on cell proliferation and correlative cytokines secretion of glomerular mesangial in rats. Methods：The effects of EMD on cell proliferation and IL-6, TGF-β1 secretion of glomerular mesangial in rats were observed. Cell proliferation was measured by MTT method. IL-6 and TGF-β1 secretion was detected with ELISA. Results：EMD was able to inhibit the cell proliferation and down-regulate the IL-6 and TGF-β 1 secretion of glomerular mesangial, as compared to the model group in rats （P 〈 0.05）. Conclusion：EMD could significantly inhibit the cell proliferation, and reduce the creation of extracellular matrix（ECM）, this indicated that it could play an important role in alleviation and prevention of glomerular sclerosis. The mechanism may be that EMD can reduce the IL-6 and TGF-β1 secretion ofglomerular mesangial cell in rats.
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-lN） 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. 111 the present study, the roles of the TSP-1 gene in GMC proliferation, ECM production, and urinary protein secretion in Thy-lN 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-lN rats. Together, the current findings suggested that TSP-1 gene expression was required for GMC proliferation and ECM production in Thy-lN 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.
文摘Sublytic complement C5b-9 complexes can cause cell apoptosis, but the mechanism of glomerular mesangial cell （GMC） apoptosis mediated by these complexes has not been well defined. The activating transcription factor 3 （ATF3） gene is an immediate early gene for the cell to cope with a variety of stress signals and can promote apoptosis of some cells. In this study, ATF3 expression and cell apoptosis in GMCs induced by sublytic C5b-9 were measured, and then the effects of ATF3 gene over-expression or knockdown on GMC apoptosis induced by sublytic C5b-9 were examined at a fixed time. The results showed that both ATF3 expression and GMC apoptosis were markedly increased and ATF3 over-expression obviously increased sublytic C5b-9-induced GMC apoptosis, whereas ATF3 gene silencing had a significant opposite effect. Collectively, these findings indicate that upregulation of ATF3 gene expression is involved in regulating GMC apoptosis induced by sublytic C5b-9 complexes.
基金This work was supported by grants from the Natural Science Foundation （No. 11040606M 159） and Natural Science Research Project （No. K J2011A157） of Anhui Province, China.
文摘Background The renoprotective mechanisms of adenosine monophosphate （AMP）-activated protein kinase （AMPK） agonist-metformin have not been stated clearly.We hypothesized that metformin may ameliorate inflammation via AMPK interaction with critical inflammatory cytokines The aim of this study was to observe the effects of metformin on expression of nuclear factor-κB （NF-κB）,monocyte chemoattractant protein-1 （MCP-1）,intercellular adhesion molecule-1 （ICAM-1） and transforming growth factor-beta 1 （TGF-β1） induced by high glucose （HG） in cultured rat glomerular mesangial cells （MCs）.Methods MCs were cultured in the medium with normal concentration glucose （group NG,5.6 mmol/L）,high concentration glucose （group HG,25 mmol/L） and different concentrations of metformin （group M1,M2,M3）.After 48-hour exposure,the supernatants and MCs were collected.The expression of NF-κB,MCP-1,ICAM-1,and TGF-β1 mRNA was analyzed by real time polymerase chain reaction.Westem blotting was used to detect the expression of AMPK,phospho-Thr-172 AMPK （p-AMPK）,NF-κB p65,MCP-1,ICAM-1,and TGF-β1 protein.Results After stimulated by HG,the expression of NF-κB,MCP-1,ICAM-1,TGF-β1 mRNA and protein of MCs in group HG increased significantly compared with group NG （P ＜0.05）.Both genes and protein expression of NF-κB,MCP-1,ICAM-1,TGF-β1 of MCs induced by high glucose were markedly reduced after metformin treatment in a dose-dependent manner （P ＜0.05）.The expression of p-AMPK increased with the rising of metformin concentration,presenting the opposite trend,while the level of total-AMPK protein was unchanged with exposure to HG or metformin.Conlusion Metformin can suppress the expression of NF-κB,MCP-1,ICAM-1 and TGF-β1 of glomerular MCs induced by high glucose via AMPK activation,which may partlv contribute to its reno-protection.
