Effects of Shikonin on Proliferation, Apoptosis, and Extracellular Matrix of Human Mesangial Cells

Objective:To investigate the effect of shikonin on the proliferation, apoptosis and extracellular matrix (ECM) of human mesangial cells (MC). Methods: MC was cultured in vitro with different concentrations of glucose (30, 50, 80 mmol/L). The cell growth was observed by using MTT method and apoptosis by using an aunexin-V-Fluos. Immunohistochemical studies for Laminin (LN), Fibronectin (FN) and type Ⅳ Collagens (Col Ⅳ) were measured. Results: Shikonin inhibited their growth (P<0.05) and apoptosis in the glycated cultured cells. Shikonin 0.05 mmol/L significantly reduced the secretion of LN, FN and Col Ⅳ from MC (P<0.05) cultured in 30, 50 and 80 mmol/L glucose. Conclusion: Shikonin could prevent or treat diabetic nephropathy (DN) and glomerulosclerosis (GS).

Akt1/p27^(kip1) Pathway Mediates Inhibition of LXA_4 on TNF-α-induced Proliferation of Rat Mesangial Cells

Objective:To examine whether lipoxin A 4(LXA 4) has an inhibitory effect on tumor necrosis factor-α(TNF-α)-induced proliferation of glomerular mesangial cells of rat, and explore the molecular mechanisms of signal pathway in LXA 4 actions. Methods: Glomerular mesangial cells of rat were cultured and treated with TNF-α(10 ng/ml), with or without preincubation with LXA 4 at different concentrations. Cell proliferation was evaluated by monotetrazolium (MTT) colorimetric assay. The expression of cyclin E mRNA was measured by RT-PCR. Phosphorylated Akt1(Thr308) and p27 kip1 were analyzed by Western blotting. Results: TNF-α-stimulated proliferation of mesangial cells was inhibited by LXA 4 in a dose-dependent manner. The marked increments in cyclin E mRNA expression induced by TNF-α during proliferation of mesangial cells were down-regulated by LXA 4. Threonine phosphorylated Akt1 proteins at 308 site stimulated by TNF-α was reduced by LXA 4. TNF-α-induced decrements in expression of p27 kip1 proteins was ameliorated by LXA 4 in a dose-dependent manner. Conclusion: TNF-α-induced proliferation of rat mesangial cells can be inhibited by TXA 4 through the mechanism of Akt 1/p27 kip1 pathway-dependent signal transduction.

Effects of emodin on the proliferation of the glomerular mesangial cell and correlative cytokines in rats

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.

Effect of L158,809 and Cilazapril on the Expression of TGF-β_1 and Secretion of Extracellular Matrix Proteins in Cultured Human Mesangial Cells

Objective: To explore the effect of L158, 809 (angiatensin Ⅱ receptorMockers, ARBs) and Cilazapril (Angiotensin converting enzyme inhibitor, ACEI) on the expression oftransforming growth factor-β_1 (TGF-β_1) and secretion of fibronectin, laminin and type Ⅳcollagen from the cultured human mesangial cells . Methods: Human mesangial cells were cultured indifferent glucose (5.6 mmol/L and 30 mmol/L) and agents (1, 10, 100 and 500 μmol/L) concentrations. The proliferation of mesangial cells were detected at 24, 48 and 72 h . Then the mesangial cellsare divided into four groups, low glucose (5.6 mmol/L) control group, high glucose (30 mmol/L)control group , L158, 809 (10 μmol/L) group and cilazapril (10 μmol/L) group. Forty- eight hourslater, the expression of TGF-β_1 were detected by RT-PCR. Concentrations of TGF-β_1 ,fibronection, laminin and type Ⅳ collagen in the su-pematants of the, mesangial cells weredetermined by EUSA and radioimmunoassay methods. Results: Compared with low glucose control group,the mesangial cells under high glucose medium show excessive proliferation and more TGF-β_1,fibronectin, laminin and type Ⅳ collagen in the supernatant. The expression of TGF-β_1 mRNA wasalso significantly increased under high glucose. The levels of TGF-β_1 and ECM (extracellularmatrix) proteins in the L158, 809 group and cilazapril group are obviously lower than that of thehigh glucose control group. The expression of TGF-β_1 mRNA was markedly decreased in the L158, 809group and cilazapril group compared with that of high glucose control group . Conclusion: Highglucose stimulated the cultured human mesangial cells to excessively proliferate, express TGF-β_1and secrete ECM proteins, and the high glucose-indeced changes were suppressed by either L158, 809and cilazapril.

HBV X Gene Transfection Upregulates IL-1β and IL-6 Gene Expression and Induces Rat Glomerular Mesangial Cell Proliferation

The X gene of HBV encodes a 17-kD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin （IL）-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

Upregulation of MiR-126 Delays the Senescence of Human Glomerular Mesangial Cells Induced by High Glucose via Telomere-p53-p21-Rb Signaling Pathway

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.

THE LOCALIZATION OF ADRENOMEDULLIN IN RAT KIDNEY TISSUE AND ITS INHIBITORY EFFECT ON THE GROWTH OF CULTURED RAT MESANGIAL CELLS

OBJECTIVE: To observe the localization of adrenomedullin (AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC). METHODS: A monoclonal antibody against AM developed by our laboratory was used to detect the localization of AM protein in rat kidney tissue by avidin-biotin complex immunohistochemistry. The expressions of AM and its receptor CRLR mRNA on cultured glomerular epithelial cells (GEC) and MsC were investigated by Northern blot assay, and the possible effect of AM secreted by GEC on MsC proliferation was observed using [3H]thymidine incorporation as an index. RESULTS: A specific monoclonal antibody against AM was succesfully developed. AM was immunohistochemically localized mainly in glomeruli (GEC and endothelial cells), some cortical proximal tubules, medullary collecting duct cells, interstitial cells, vascular smooth muscle cells and endothelial cells. Northern blot assay showed that AM mRNA was expressed only on cultured GEC, but not on MsC, however, AM receptor CRLR mRNA was only expressed on MsC. GEC conditioned medium containing AM can inhibit MsC growth and AM receptor blocker CGRP8-37 may partially decreased this inhibitory effect. CONCLUSION: AM produced by GEC inhibits the proliferation of MsC, which suggests that AM as an important regulator is involved in glomerular normal physiological functions and pathologic processes.

Isolation and Purification of Polysaccharides from Cordyceps minlitaris and Its Inhibition on the Proliferation of Rat Glomerular Mesangial Cells

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.

High Glucose Promotes the CTGF Expression in Human Mesangial Cells via Serum and Glucocorticoid-induced Kinase 1 Pathway

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.

Effect of Colquhoumia Root on the Expression of Transforming Growth Factor-β in Mesangial Proliferation Glomerulonephritis Model

Summary： To study the efficacy and the mechanism of Colquhoumia root （ Tripterygium hypoglaucure （Le,vL） Hutch） in the treatment of mesangial proliferation glomerulonephritis （MsPGN）, SD rats were injected with anti-thymoeyte serum （ATS） to make MsPGN model （anti-Thyl model）. The rats were then divided into 3 groups： normal control group, anti-Thyl model group and treatment group. Histopathologieal （HE, PAS）, immunohistoehemieal, RT-PCR technique and computer imaging analysis system were used to evaluate mesangial matrix production, the expression of TGF-β protein and mRNA in the tissues of kidney. Our result showed that proteinuria and the ratio of extraeellular matrix/glomerular capillaries area （ECM/CA） were increased significantly in model group. The expression of both TGF-β protein and mRNA in glomeruli was much higher in model group than in control group （P〈0.01）. After the treatment with Colquhoumia root, proteinuria, ECM/CA and the expression of both TGF-β1 protein and mRNA in glomeruli were significantly decreased in treatment group as compared with those in model group. It is concluded that Colquhoumia root is effective in reducing proteinuria and mesangial matrix proliferation in MsPGN and it may achieve these effects by inhibiting the expressions of TGF-β1 protein and mRNA of mesangial cells.

Shp-2／NF-κB Pathway Mediates the Inhibition of Lipoxin A4 onIL-1β-induced Synthesis of IL-6 in Glomerular Mesangial Cells

Objective: To examine whether lipoxin A 4 (LXA 4) has an antagonistic effect on IL-1β-induced synthesis of IL-6 in glomerular mesangial cells, and to explore the molecular mechanisms of signal pathway in LXA 4 actions. Methods: The glomerular mesangial cells of rat were cultured and treated with IL-1β, with or without preincubation with LXA 4 at different concentrations. The amount of IL-6 in the supernatant of cells was analyzed by enzyme-linked immunosorbent assay(ELISA). The expressions of mRNA of IL-6 were determined by RT-PCR. The expressions of Src homology 2(SH 2) containing protein-tyrosine phosphatase 2(Shp-2) were assessed by immunoprecipitation and immunoblotting. Activities of DNA-binding of nuclear factor-kappa B(NF-κB) were measured by electrophoretic mobility shift assay(EMSA). Results: IL-1β-stimulated secretion of protein and expression of mRNA of IL-6 in mesangial cells were inhibited by LXA 4 in a dose-dependent manner. LXA 4 antagonizes the phosphorylation of Shp-2 and activities of NF-κB induced by IL-1β. Conclusion: LXA 4 antagonists IL-1β-induced synthesis of IL-6 in glomerular mesangial cells through the mechanism of Shp-2/NF-κB pathway-dependent signal transduction.

Effect of heparin on high glucose induced proliferation and expression of matrix metalloproteinases in normal human mesangial cells

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.

TSP-1 promotes glomerular mesangial cell proliferation and extracellular matrix secretion in Thy-1 nephritis rats

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.

Interleukin-13 inhibits cytokines synthesis by blocking nuclear factor-κB and c-Jun N-terminal kinase in human mesangial cells

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.

Effects of Cyclosporin A on Proliferation of Cultured Rat Mesangial Cells

<Abstrat>The proliferation of mesangial cells on cyclosporin (CsA) test mediumwas studied by MTT assay and TNF-Q in cultured supernatant was examined byusing ELISA. The results showed that cyclosporin A significantly inhibited theproliferation of mesangial cells at the concentration between 0. 25 - 15 μg/ml(IC50 1μg/ml). This action appeared to be dose-dependent. Release of TNF-αfrom mesangial cells stimulated by LPS was also dose-dependently suppressed. Itis suggested that cyclosporin A play an important role in antiproliferation mecha-nism of mesangial cells in vitro.

PKCα signaling pathway involves in TNF-α-induced IP_3R1 expression in human mesangial cells

BACKGROUND： This study aimed to explore the effects of TNF-α on the expression of IP3R1 mRNA and protein in human mesangial cells （HMCs）, and to elucidate the mechanism of TNF-α relating to IP3R1 expression in the occurrence of hepatorenal syndrome （HRS）.METHODS： HMCs were stimulated by tumor （TNF-α） with 100 ng/mL for different hours （2, 4, 8, and 24 hours）. The expression changes of IP3R1 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 of TNF-α-regulated IP3R1 in HMCs.RESULTS： The levels of IP3R1 mRNA at 2 hours after TNF-α 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 IP3R1 protein at 4 hours after TNF-α exposure were obviously increased and peaked at 24 hours after TNF-α exposure （P〈0.01）. Compared to the control group, safingol （PKCa inhibitor） and D609 （phosphatidylcholine-specific phospholipase C inhibitor） significantly blocked the TNF-α- induced expression of IP3R1 mRNA （3.30±0.81 vs. 1.95±0.13, P〈0.05; 2.10±0.49, P〈0.01） and IP3R1 protein （3.09±0.13 vs. 1.86±0.39, P〈0.01; 1.98±0.02, P〈0.01）. TNF-α 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-α increased the expression of IP3R1 and this was mediated, at least in part, through the PC-PLC/PKCa signaling pathways in HMCs.

EFFECTS OF PDGF-BB ON INTRACELLULAR CALCIUM CONCENTRATION AND PROLIFERATION IN CULTURED GLOMERULAR MESANGIAL CELLS

Objective To investigate the relationship between the alteration of intracellular calcium concentration and proliferation in cultured glomerular mesangial cells. Methods Rat mesangial cells were cultured. lntracellular calcium concentrations were measured by confocal Laser Scanning Microscopy and Fura-3 fluorescence dyeing techniques. Cell growth was measured by MTT assay. Results PDGF-BB increased intracellular calcium concentrations in a dose-dependent manner, and at the same time promote the proliferation of mesangial cells. After preincubation with calcium channel blocker nifedipine or angiotensin converting enzyme inhibitor captopril, both the increase of intracellular calcium concentrations and cell proliferations induced by PDGF-BB were inhibited. Tripteriglum Wilfordii Glycosides （TMG） significantly inhibited the mesangial cell proliferations, but it had no significant effect on intracellular calcium concentrations. Conclusion There was a positive relationship between the elevation of intracellular calcium concentration and cell proliferation in glomerular mesangial cells, but the increase of intracellular calcium concentrations wasn＇t the only way for proliferation.

EFFECTS OF RAPAMYCIN ON INTRACELLULAR CHOLESTEROL HOMEOSTASIS OF GLOMERULAR MESANGIAL CELL IN THE PRESENCE OF INTERLEUKIN-1β

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（1L-1β）-induced mRNA and protein changes of low-density lipoprotein receptor （LDLR） and ATP-binding cassette transporter Al （ABCAl） 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 trader 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 ABCAl caused by IL-1β Transient expression of 3 types of mTOR all reduced ABCAl 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 effiux. And the effect may be not completely mediiated by mTOR.

Effects of Mycophenolic Acid on High Glucose-induced Expression of TGF-β and CTGF in Mesangial Cells

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.

PI3-K/PKB/NF-κB and p42/44 MAPK pathway mediates inhibition of lipoxin A_4 on CTGF-induced production of RANTES in mesangial cells

In order to investigate the regulatory role of connective tissue growth factor （CTGF） on production of RANTES （regulated on activation, normal T cell expressed and secreted） in rat glomerular mesangial cells, and the modulatory effect of lipoxin A4 （ LXA4 ） on action of CTGF, and to explore the mechanisms of action of CTGF and LXA4, cultured rat mesangial cells were treated with CTGF, with or without preincubation with LXA4. Expression of mRNA was analyzed by RT-PCR. Protein of RANTES in the supematants was determined by ELISA. Monocyte transmigration was assessed by in vitro chemotaxis assay. Expression of p42/44 mitogen-activated protein kinase （MAPK）, phosphoinositide 3-kinase （ PI3- K） and protein kinase B （PKB） was assessed by Western blotting. DNA-binding activity of nuclear factor-roB （NF-kB） was determined by electrophoretic mobility shift assay （EMSA）. To observe whether transfection of LXA4 receptor homologue gene （LRHg） into mesangial cells intensified these modulatory effects of LXA4, mesangial cells were transfected with pcDNA3.1/LRHG vector. The results showed that CTGF enhanced the mRNA expression and protein release of RANTES, and the expression of phospho （P）-p42/44 MAPK, P-PI3-K, P-PKB and NF-kB. P-p42/44 MAPK blockade inhibited the CTgF-induced expression of P-p42/44 MAPK and partially decreased the level of RANTES in supematants. P- PI3-K blockade downregulated the CTGF-stimulated expression of P-PI3-K, P-PKB and NF-kB, and partially decreased the release of RANTES. NF-kB blockade abrogated the CTGF-activated NF-kB and partially decreased the secretion of RANTES. LXA4 dose-dependently inhibited the CTGF-stimulated above action. Transfection of LRHG into mesangial cells intensified these inhibitory effects of LXA4 on CTGFinduced release of RANTES and expression of the P-p42/44 MAPK. In conclusion, LXA4 inhibits CTGFinduced production of RANTES via PI3-K/PKB/NF-kB and p42/44 MAPK-dependent signal pathway, which is mediated by LRHG in rat mesangial cells.