1.Metformin inhibits nuclear factor-κB activation and inflammatory cytokines expression induced by high glucose via adenosine monophosphate-activated protein kinase activation in rat glomerular mesangial cells in vitro.
Junfei GU ; Shandong YE ; Shan WANG ; Wenjia SUN ; Yuanyuan HU
Chinese Medical Journal 2014;127(9):1755-1760
BACKGROUNDThe 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).
METHODSMCs 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. Western 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.
RESULTSAfter 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 partly contribute to its reno-protection.
AMP-Activated Protein Kinases ; metabolism ; Animals ; Cells, Cultured ; Glomerular Mesangium ; cytology ; Glucose ; pharmacology ; Mesangial Cells ; drug effects ; metabolism ; Metformin ; pharmacology ; NF-kappa B ; metabolism ; Rats
2.Effect of exendin-4 on monocyte chemoattractant protein-1 expression in cultured rat glomerular mesangial cells induced by tumor necrosis factor-α in vitro.
Yingjuan JIANG ; Yaoming XUE ; Qian ZHANG ; Yanfei ZHANG ; Yuan YUAN
Journal of Southern Medical University 2013;33(6):930-933
OBJECTIVETo investigate the effect of exendin-4 on the expression of monocyte chemoattractant protein-1 (MCP-1) and fibronectin (FN) in rat glomerular mesangial cells in vitro.
METHODSRat glomerular mesangial cells were divided into 5 groups, namely control group, tumor necrosis factor-α (TNF-α) group (10 ng/ml), TNF-α (10 ng/ml)+E1 (1 nmol/L exendin-4) group, TNF-α (10 ng/ml)+E5 (5 nmol/L exendin-4) group, and TNF-α (10 ng/ml)+E10 (10 nmol/L exendin-4) group. After cultured 24 h or 48 h, RNA were extracted to determine the expression of MCP-1 with real-time PCR, the supernatant were collected to determine the expression of MCP-1 and FN with ELISA.
RESULTSCompared with control group, the cells treated with TNF-α for 24 h showed significantly increase the expression of MCP-1 and FN (P<0.01), exendin-4 significantly reduced the expression of MCP-1 and FN in TNF-α+E5 group and TNF-α+E10 group (P<0.05). After 48h incubation, the expression of MCP-1 and FN increased significantly in TNF-α group (P<0.01), which was lowered by exendin-4 in TNF-α+E1,TNF-α+E5 and TNF-α+E10 groups (P<0.05).
CONCLUSIONExendin-4 has an intrinsic capability to concentration- and time-dependently inhibit TNF-α-induced expression of MCP-1 and FN in rat mesangial cells, suggesting the beneficial effect of exendin-4 in preventing and treating diabetic nephropathy.
Animals ; Cells, Cultured ; Chemokine CCL2 ; metabolism ; Diabetic Nephropathies ; metabolism ; Glomerular Mesangium ; cytology ; Mesangial Cells ; drug effects ; metabolism ; Peptides ; pharmacology ; Rats ; Tumor Necrosis Factor-alpha ; pharmacology ; Venoms ; pharmacology
3.Effects of very low-density lipoprotein on cellular lipid accumulation and the expression of monocyte chemoattractant protein-1 in human mesangial cells.
Jing LI ; Hang LI ; Yu-Bing WEN ; Xue-Wang LI
Acta Academiae Medicinae Sinicae 2009;31(1):55-59
OBJECTIVETo investigate the effects of very low-density lipoprotein (VLDL) on cellular lipid accumulation and the expression of monocyte chemoattractant protein-1 (MCP-1) in human mesangial cells.
METHODSAn established stable human mesangial cell line (HMCL) was used in all experiments. VLDL-induced cellular lipid deposition was visualized by Oil Red O staining and analyzed quantitatively by standard enzymatic procedures. MCP-1 mRNA and protein expression levels in treated HMCLs were determined by real-time quantitative RT-PCR and enzyme-linked immunosorbent assay, respectively. For adhesion study, HMCLs were treated with VLDL for 12 hours, followed by a one-hour incubation with THP-1 cells.
RESULTSVLDL induced cellular lipid accumulation in HMCLs in a time- (0-24 h) and dose- (0-200 microg/ml) dependent manner, and the principal component of accumulated lipid is triglyceride. In HMCLs, MCP-1 mRNA expression was promoted by VLDL in a time- (0-6 h) and dose- (0-100 microg/ml) dependent manner, and VLDL also enhanced MCP-1 secretion in a dose-dependent manner. Such an effect was accompanied by increased adhesion of monocytes to HMCLs.
CONCLUSIONSVLDL can induce cellular triglyceride accumulation and upregulate the expression of MCP-1 in human mesangial cells. Hence, VLDL may be involved in the pathogenesis of lipid-mediated renal injury.
Cell Line ; Chemokine CCL2 ; genetics ; metabolism ; Humans ; Lipoproteins, VLDL ; pharmacology ; toxicity ; Mesangial Cells ; cytology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Triglycerides ; metabolism
4.Effect of Chinese herbal compounds for blood activating stasis removing, qi benefiting Shen invigorating on high glucose stimulated proliferation of renal mesangial cells and expressions of fibronectin.
Dan-Dan LIU ; Wen-Xia HAN ; Yu-Wen SONG ; Kai LOU ; Chun-Xiao YU ; Ling GAO ; Jia-Jun ZHAO
Chinese Journal of Integrated Traditional and Western Medicine 2013;33(10):1400-1403
OBJECTIVETo study effects of Chinese Herbal Compounds (CHC) for blood activating stasis removing (BASR), qi benefiting Shen invigorating (QBSI) on high glucose stimulated proliferation of renal mesangial cells (RMCs) and expressions of fibronectin (FN).
METHODSRats' RMCs were dealt with high glucose and different concentrations of Chinese medicine for 24 and 48 h respectively. The proliferation of RMCs was detected with 4-A thiazolyl blue. mRNA expressions of FN was detected by real time quantitative PCR. The protein expression of FN was detected by ELISA.
RESULTSCompared with the control group, the proliferation obviously increased (P < 0.05, P < 0.01) after 24 and 48 h of treatment in the high glucose group, mRNA and protein expressions of FN also increased (P < 0.01). There was no statistical difference in the proliferation of RMCs or expressions of FN at 24 h between each CHC group and the high glucose group (P > 0.05). Compared with the high glucose group, the proliferation of RMCs and expressions of FN at 24 h each obviously decreased in the CHC group (P < 0.05, P < 0.01).
CONCLUSIONSHigh glucose could promote the proliferation of RMCs and induce expressions of FN. No obvious effect could be stimulated by CHC treatment for 24 h. The proliferation of RMCs, protein and mRNA expressions of FN could be reversed by CHC treatment for 48 h.
Animals ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Fibronectins ; metabolism ; Glucose ; adverse effects ; Kidney Tubules ; cytology ; Male ; Mesangial Cells ; drug effects ; metabolism ; RNA, Messenger ; genetics ; Rats
5.HMGB1/SREBP-1 mediated IFN-gamma-induced lipid deposition in mouse mesangial cells.
Yu-Jun ZHANG ; Yan-Ning CHEN ; Shu-Shen ZHENG ; Qing-Juan LIU ; Jun HAO ; Yan HAN ; Bao-Wei YANG ; Shu-Xia LIU
Chinese Journal of Applied Physiology 2013;29(1):6-10
OBJECTIVETo explore the possible mechanism of lipid deposition induced by interferon-gamma (IFN-gamma).
METHODSThe mouse mesangial cells (MMC) were randomly divided into control group, stimulation group, stimulation + control vector group (sh-HMGB1) and stimulation+ specific sh-vector group (sh-SREBP-1). RT-PCR was used to detect the expression of HMGB1, SREBP-1 and fatty acid synthetase (FAS) mRNA; the protein expression was determined by Western blot.
RESULTSThe Oil Red O staining revealed that the mouse mesangial cells showed significant lipid droplet in IFN-gamma group. IFN-gamma up-regulated the expression of HMGB1, SREBP-1, FAS mRNA and protein time-dependently; Transfection of MMC with HMGB1 siRNA resulted in the suppression of SREBP-1, FAS protein levels induced by IFN-gamma, following with decrease of lipid deposition. Stimulation with HMGB1 markedly induced expression of SREBP-1, FAS expression and peaked at 8 h, decreased at 12 h compared with that at 8 h. Sh-SREBP-1 decreased the lipid deposition induced by HMGB1 in MMC.
CONCLUSIONIFN-gamma might induce lipid deposition in mouse mesangial cells partly by up-regulating the expression of HMGB1/SREBP-1/FAS.
Animals ; Cells, Cultured ; Fatty Acid Synthases ; metabolism ; HMGB1 Protein ; metabolism ; Interferon-gamma ; pharmacology ; Kidney Tubules ; cytology ; Lipid Metabolism ; Male ; Mesangial Cells ; drug effects ; metabolism ; Mice ; Sterol Regulatory Element Binding Protein 1 ; metabolism
6.Changes of fibronectin and type IV collagen expression in cultured rat mesangial cells transfected with connective tissue growth factor expression vector.
Xiao-gang WANG ; Hui-juan WU ; Qi CHEN ; Zhong-hua ZHAO ; Zhi-gang ZHANG ; Mu-yi GUO
Chinese Journal of Pathology 2006;35(9):555-558
OBJECTIVETo study the role of connective tissue growth factor (CTGF) in the development of glomerulosclerosis by experimental alteration of fibronectin (FN) and Type IV collagen (Col IV) expression in cultured rat mesangial cells (MsC).
METHODSCTGF expression vector was transfected into MsC by Lipofectimine method. Protein and mRNA expression levels of CTGF, FN and Col IV were studied by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) respectively.
RESULTSTwo of MsC clones (MCT-1 and MCT-2) with CTGF overexpression were successfully established and found to have significant increases of FN and Col IV at both protein and mRNA levels. Compared with the controls, the expression of FN protein and mRNA in the two clones were 3.2 times (P < 0.05) and 2.9 times (P < 0.05) higher respectively. The expression of Col IV protein and mRNA was 3.8 times (P < 0.01) and 2.4 times (P < 0.01) higher respectively.
CONCLUSIONCTGF up-regulates FN and Col IV expression in MsC and may play an important role in the development of glomerulosclerosis.
Animals ; Blotting, Western ; Cells, Cultured ; Collagen Type IV ; genetics ; metabolism ; Connective Tissue Growth Factor ; genetics ; metabolism ; Fibronectins ; genetics ; metabolism ; Genetic Vectors ; genetics ; Mesangial Cells ; cytology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection
7.Effect of Modified Hangqi Chifeng Decoction Containing Serum on the Expression of Col IV, MMP-2, and TIMP-2 in Glomerular Mesangial Cells Induced by LPS.
Hong-xia LIU ; Yu ZHANG ; Peng LI ; Yan-hong GAO ; Shuang LI ; Zi-kai YU
Chinese Journal of Integrated Traditional and Western Medicine 2016;36(5):592-596
OBJECTIVETo explore the effect of Modified Hangqi Chifeng Decoction (MHCD) on levels of collagen type IV (Col IV), matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2) in extracellular matrix (ECM) of glomerular mesangial cells (GMCs) in LPS induced mice.
METHODSNormal serum and telmisartan, high, medium, low dose MHCD containing serums were prepared by using serum pharmacology method. GMCs were cultured in vitro. The proliferation of mesangial cells were induced using LPS as stimulating factor. GMCs were divided into six groups, i.e., the normal group, the model group, the telmisartan group, high, medium and low dose MHCD groups. Col IV content in the supernatant of mesangial cells was detected using ELISA. Protein expressions of MMP-2 and TIMP-2 were detected using Western blot.
RESULTSCompared with the normal group, Col IV content obviously increased in the model group after 72-h LPS stimulation; protein expressions of MMP-2 and TIMP-2 were obviously up-regulated, and MMP-2/TIMP-2 ratio was down-regulated in the model group (P < 0.01). Compared with the model group, Col IV content obviously decreased in high and medium dose MHCD groups and the telmisartan group (P < 0.01); protein expressions of MMP-2 were obviously down-regulated in medium and low dose MHCD groups (P < 0.01, P < 0.05); the protein expression of TIMP-2 was obviously down-regulated in high, medium, low dose MHCD groups and the telmisartan group (P < 0.01). The pro- tein expression of TIMP-2 was obviously lower in the high dose MHCD group than in the low dose MHCD group (P < 0.01). MMP-2/TIMP-2 ratio was obviously up-regulated in the telmisartan group, high and medium dose MHCD groups (P < 0.01).
CONCLUSIONMHCD could regulate disordered MMP-2/TIMP-2 ratio in LPS induced ECM, inhibit excessive production of Col IV in ECM, promote the degradation of ECM, reduce the accumulation of ECM, thereby, delaying the process of glomerular sclerosis.
Animals ; Cells, Cultured ; Collagen Type IV ; metabolism ; Extracellular Matrix ; metabolism ; Kidney Glomerulus ; cytology ; Matrix Metalloproteinase 2 ; metabolism ; Mesangial Cells ; drug effects ; Mice ; RNA, Messenger ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism
8.Effect of tissue factor pathway inhibitor on apoptosis of rat mesangial cells and Fas and bcl-2 expression.
Yi-feng LIN ; Duan MA ; Wang LIANG ; Tao JIANG ; Zhong-hua ZHAO ; Nong ZHANG
Chinese Journal of Pathology 2008;37(11):754-759
OBJECTIVETo study the biological impact and mechanism of recombinant tissue factor pathway inhibitor (rTFPI) on apoptosis of rat kidney mesangial cells (MsC).
METHODSTFPI expression in human glomerular minor lesion (GML), mesangial proliferative glomerulonephritis (MPGN) and cultured rat MsC was detected using immunohistochemistry and immunofluorescence, respectively. Rat MsC were incubated with rTFPI and its variant peptides. Morphological changes of apoptosis were investigated by Hoechst 33258 and the apoptotic rate was assessed by flow cytometry. DNA fragmentation and effect of rTFPI on expression of caspase-3, Fas and bcl-2 were studied using gel electrophoresis and Western blot respectively.
RESULTSThe expression of TFPI in MPGN was higher than that in GML. TFPI was expressed in cultured rat mesangial cells. Apoptosis of MsC was induced by rTFPI, especially by its C-termianl, in a dose- and time-dependent manner. Apoptosis ratios of MsC treated with rTFPI were 2.1, 3.0 and 4.9 times more than control, respectively. Expression of gene caspase-3 and Fas was up-regulated in a dose-dependent manner wherease bcl-2 expression did not show any changes.
CONCLUSIONrTFPI induces apoptosis in cultured rat mesangial cells by its C-terminal possibly via Fas/FasL pathway.
Animals ; Apoptosis ; drug effects ; physiology ; Caspase 3 ; metabolism ; Cells, Cultured ; drug effects ; DNA Fragmentation ; Flow Cytometry ; Humans ; Lipoproteins ; metabolism ; pharmacology ; Mesangial Cells ; cytology ; drug effects ; enzymology ; metabolism ; Peptides ; pharmacology ; Rats ; bcl-Associated Death Protein ; metabolism ; pharmacology
9.Effects of beraprost sodium on extracellular matrix metabolism in cultured rat mesangial cells induced by high glucose.
Qian ZHANG ; Yaoming XUE ; Yuan YUAN ; Yingjuan JIANG ; Ling WANG
Journal of Southern Medical University 2013;33(12):1819-1822
OBJECTIVETo explore effects of beraprost sodium (BPS) on the metabolism of extracellular matrix (ECM) in rat mesangial cells cultured in the presence of high glucose and the possible mechanism.
METHODSRat mesangial cells were cultured in the presence of high glucose with or without BPS for 24 or 48 h. The levels of transforming growth factor β1 (TGFβ1), fibronectin (FN) and matrix metalloproteinase-2 (MMP-2) protein in the culture supernatants were measured by enzyme-linked immunosorbent assay, and photoshop-Smad3 was detected by Western blotting.
RESULTSCompared with the cells in normal glucose, the cells cultured in the presence of high glucose for 24 and 48 h showed significantly increased TGFβ 1 and FN protein expression and lowered MMP-2 protein expression (P<0.01). Compared with the cells cultured in high glucose, BPS exposure at the concentration of 1, 2, and 5 µmol/L for 24 and 48 h significantly lowered TGFβ 1 protein expression (P<0.01), and at 2 and 5 µmol/L, BPS significantly decreased FN protein expression and increased MMP-2 protein expression in high glucose-induced cells (P<0.05). High glucose exposure also significantly increased the expression phosphorylated Smad3 (P<0.01), which was lowered by BPS treatment at 2 and 5 µmol/L (P<0.01).
CONCLUSIONBPS can regulate ECM metabolism in rat mesangial cells cultured in high glucose by inhibiting TGFβ 1/Smad3 pathway, suggesting the beneficial effects of BPS in preventing and treating diabetic nephropathy.
Animals ; Cell Line ; Cells, Cultured ; Diabetic Nephropathies ; Enzyme-Linked Immunosorbent Assay ; Epoprostenol ; analogs & derivatives ; pharmacology ; Extracellular Matrix ; metabolism ; Fibronectins ; metabolism ; Glomerular Mesangium ; cytology ; Glucose ; Matrix Metalloproteinase 2 ; metabolism ; Mesangial Cells ; drug effects ; Rats ; Transforming Growth Factor beta1 ; metabolism
10.High glucose promotes the CTGF expression in human mesangial cells via serum and glucocorticoid-induced kinase 1 pathway.
Quansheng, WANG ; Ali, ZHANG ; Renkang, LI ; Jianguo, LIU ; Jiwen, XIE ; Anguo, DENG ; Yuxi, FENG ; Zhonghua, ZHU
Journal of Huazhong University of Science and Technology (Medical Sciences) 2008;28(5):508-12
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.
Cells, Cultured
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Connective Tissue Growth Factor/genetics
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Connective Tissue Growth Factor/*metabolism
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Glucose/*pharmacology
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Immediate-Early Proteins/metabolism
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Immediate-Early Proteins/*physiology
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Mesangial Cells/cytology
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Mesangial Cells/*metabolism
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Protein-Serine-Threonine Kinases/metabolism
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Protein-Serine-Threonine Kinases/*physiology
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Signal Transduction/drug effects