Promoting angiogenesis of protein kinase D1 in vitro and in vivo

AIM: To explore the effect of protein kinase D1 (PKD1) on promoting angiogenesis in vitro and in vivo, and to furnish a new idea on targeting PKD1 for the treatment of ischemic heart disease such as myocardial infarction. METHODS: The culture, isolation and identification of endothelial progenitor cells (EPCs) were performed in vitro. The effects of PKD1 and its specific blocking agent CID755673 on expression levels of vascular endothelial growth factor (VEGF) and kinase insert domain receptor (KDR) in EPCs were determined. The rat model of myocardial infarction was established, the intervention effects of PKD1 and CID755673 on morphology, changes of microvessels and endothelial cells, and the expression of VEGF and KDR in the impaired myocardial tissue were analyzed. RESULTS: PKD1 significantly upregulated the expression of VEGF and KDR in EPCs in vitro. Meanwhile, the structure of myocardial tissue was more regular and clear, the cytomembrane of endothelial cells were more smooth and integrity, the pericytes were visible, and the expression of VEGF and KDR was significantly increased in PKD1 treatment group in vivo.CONCLUSION: PKD1 has the ability of angiogenesis obviously, which might be mediated by VEGF.     

Insulin induced LPL mRNA expression in THP-1 macrophage and acceleration of transferring the macrophage to foam cell

AIM:To investigate the effect of insulin on ox-LDL transferring the THP-1 cells to foam cells and influencing the LPL mRNA expression in THP-1 cells.METHODS:THP-1 cells were incubated with 50 mg/Lox-LDL and insulin at concentrations of 10 mU/L, 100 mU/L, 1 000 mU/L and 10 000 mU/L, respectively. The expression of LPL mRNA in cells was detected by RT-PCR. Lipoprotein lipase of THP-1 cells was presented by no-specific lipase staining. THP-1 cells were stained with oil red O. Accumulation of total cholesterol (TC) in THP-1 cells was determined with oxidase assay.RESULTS:In 100 mU/L、1 000 mU/L、10 000 mU/L insulin groups, LPL mRNA expression increased 2 times, the average cell perilength was longer, the percentage of positive oil red O staining cells was significant higher, the content of cholesterol in THP-1 cells was higher than in ox-LDL control (P＜0.05).CONCLUSION:Insulin accelerates transferring of THP-1 cells to foam cell with exposed to ox-LDL because LPL mRNA expression increased in the cells.     

Harmful factors affect glycine receptor α1 subunit mRNA expression in the neonatal rat cardiomyocytes

AIM: To investigate the effects of lipopolysaccharide, hypoxia/reoxygenation,isoproterenol and high concentration of glucose on glycine receptor α₁ subunit mRNA expression in the neonatal rat cardiomyocytes. METHODS: Isolation of cardiomyocytes from Sprague-Dawley rats aging 1～3 d were performed. Cardiomyocytes （1×10⁵～5×10⁵ cells·L^-1）were cultured in DMEM medium containing 15% fetal bovine serum at 37 ℃ in 5%CO₂ atmosphere for 72 h. Then, cultured rat cardiomyocytes were treated with lipopolysaccharide, isoproterenol or high concentration of glucose for 24 h, respectively, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell survival rate was measured using CCK-8 reactant and RT-PCR was applied to monitor the expression of glycine receptor α₁ subunit mRNA. RESULTS: Compared with the control group, no significant difference in the cell survival rate was observed （P＞0.05）. The expression of glycine receptor α₁ subunit mRNA was increased （P＜0.01） in lipopolysaccharide（5,10,20,40,80 mg/L）,isoproterenol（20,100,500 μmol/L） or hypoxia/reoxygenation, hypoxia groups, but decreased（P＜0.01）in the group treated with high concentration of glucose（25, 50 mmol/L）. CONCLUSION: Lipopolysaccharide, isoproterenol, hypoxia/reoxygenation or hypoxia upregulates the expression of glycine receptor α₁ subunit mRNA,but high concentration of glucose down-regulates the expression of glycine receptor α₁ subunit mRNA in cultured neonatal rat cardiomyocytes.     

Effects of atorvastatin on expression of PAPP-A induced by TNF-α and IL-1β in rat aortic endothelial cells

AIM: To investigate the effects of atorvastatin on the expression of pregnancy-associated plasma protein A(PAPP-A)induced by TNF-α and IL-1β in endothelial cells. METHODS: The rat aortic endothelial cells were isolated from thoracic aortas and cultured by the tissue explant method. The cells in passage 3-4 were used in the experiment and were randomly divided into 4 groups: blank control group: the cells were treated without any intervention; atorvastatin concentration groups: the cells were incubated with atorvastatin at the concentrations of 0.1, 1 and 10 μmol/L for 24 h; atorvastatin time groups: the cells were incubated with atorvastatin at the concentration of 10 μmol/L for 6 h,12 h and 24 h; atorvastatin+inflammatory factors groups: the cells were pre-incubated with 60 μg/L TNF-α or 20 μg/L IL-1β for 1 h, then different concentrations of atorvastatin (0.1, 1.0, 10 μmol/L) were added for 6 h,12 h and 24 h. MTT reduction assay was used to observe the cell proliferation. The mRNA expression of PAPP-A was detected by RT-PCR. The protein level of PAPP-A in the supernatants of cultured cells was measured by ELISA. RESULTS: Compared with blank control group, no significant change of cell proliferation was observed after the intervention of atorvastatin and TNF-α/IL-1β for 3 h, 6 h, 12 h, 24 h and 48 h, indicating that the drugs had no toxic effects on the cells. No significant difference of PAPP-A expression between atorvastatin groups and blank control groups was found. Compared with TNF-α groups and IL-1β groups, PAPP-A expressions in atorvastatin intervention groups significantly decreased. The protein level of PAPP-A was gradually decreased with the raised concentration of atorvastatin and the prolonged time in a concentration- and time-dependent manner. CONCLUSION: Atorvastatin doesn't influence the PAPP-A expression, but inhibits the expression of PAPP-A activated by inflammatory factors in a concentration- and time-dependent manner in primary cultured rat aortic endothelial cells.     

Antagonistic effect of captopril on activation and injury of vascular endothelial cells induced by lipopolysaccharide

AIM: To investigate the antagonistic action of Captopril (Cap) on the activation and injury of human umbilical vascular endothelial cells (HUVECs) induced by lipopolysaccharide(LPS) and the possible mechanisms. METHODS: After 18 h exposure of the cultured HUVECs to LPS (1 mg/L), or LPS (1 mg/L) plus Cap at the concentration of 10^-7mol/L, 10^-5mol/L and 10^-3mol/L, the expression of vWF protein in the conditioned media was tested by enzyme-linked immunosorbent assay (ELISA), the expression of ICAM-1 protein in HUVECs was determined by indirect immunofluorescence technique with flow cytometry as well. In addition, the expression of TNFα mRNA was determined by in situ hybridization. RESULTS: The results of ELISA and indirect immunofluorescence technique showed that exposure to LPS at a concentration of 1 mg/L led to a significant increase in the vWF and ICAM-1 expression in HUVECs as compared to the control (P＜0. 05), whereas they were somewhat decreased when exposed to Cap at three increasing concentrations mentioned above, especially in the Cap (10^-3mol/L) plus LPS group (P＜0.05). Cap inhibited vWF secretion and ICAM-1 expression of HUVECs caused by LPS in a concentration-dependent manner. In situ hybridization revealed that the expression of TNFα mRNA was inhibited by Cap both in a concentration of 10^-3mol/L, and in a lower concentration of 10^-5mol/L. CONCLUSION: Cap antagonizes the activation and injury of HUVECs induced by LPS, which may be related to the decrease in TNFα mRNA expression.     

Role of nitric oxide in proliferation and secretion effect of vascular endothelial cells induced by vascular endothelial growth factor

AIM: To investigate the role of nitric oxide in proliferation and secretion of vascular endothelial cells induced by vascular endothelial growth factorr (VEGF). METHODS: The in vitro cultured vascular endothelial cells of rabbit aorta were divided into control group, VEGF-treated group and VEGF+L-NAME treated group, the absorbance (A) value of vascular endothelial cells, endothelin-1(ET-1) and von Willebrand factor (vWF) in the supernatant were examined by WST-1 assay, radioimmunoassay and ELISA. RESULTS: The A value in VEGF and VEGF+L-NAME treated group were higher than that in control group (P＜0.01). A value in VEGF group was higher than that in VEGF+L-NAME group the ET-1 and vWF were markedly decreased in VEGF group compared with the control and VEGF+L-NAME treated group (P＜0.05, P＜0.01). These results indicated that VEGF promoted the proliferation and inhibited the secretion of ET-1 and vWF in vascular endothelial cells, and L-NAME inhibited the effect of VEGF. CONCLUSION:Nitric oxide is an important mediator in the process of stimulating proliferation and regulating secretion of vascular endothelial cells by VEGF.     

Role of sphingosine 1-phosphate on high glucose-induced vascular endothelial cell dysfunction

AIM: To explore the role of sphingosine 1-phosphate (S1P) in the dysfunction of vascular endothelial cells exposed to high glucose. METHODS: In human aortic endothelial cells cultured under high-glucose (22 mmol/L glucose) medium, nitric oxide (NO) level, polymorphonuclear neutrophil-endothelial cell adhesion rate, protein level of intercellular adhesion molecule-1 (ICAM-1), migration of endothelial cells and Akt/endothelial nitric oxide synthase (eNOS) pathway activation were observed after S1P, sphingosine kinase-1 inhibitor and/or Akt inhibitor treatments. RESULTS: S1P decreased NO level, increased polymorphonuclear neutrophil adhesive rate, enhanced ICAM-1 protein level, and inhibited migration of endothelial cells and activation of Akt/eNOS pathway in endothelial cells cultured under high-glucose condition. Sphingosine kinase-1 inhibitor, which reduced S1P content, significantly improved the above endothelial cell function indexes and restored the activation of Akt/eNOS pathway. CONCLUSION: S1P promoted high glucose-induced dysfunction of endothelial cells probably by inhibiting the activation of Akt/eNOS signal pathway. Targeting S1P is expected to become one of potential treatment strategies to reduce endothelial cell dysfunction.     

Effects of fucoidan on angiogenesis of human multiple myeloma RPMI 8226 cells

AIM: To investigate the effects of fucoidan on the angiogenesis of multiple myeloma cells in vitro, and its related mechanisms. METHODS: The human multiple myeloma RPMI 8226 cells and human endothelial cells were cultured in vitro. The growth inhibition rate of RPMI 8226 cells was examined by MTT assay. The cell cycle and apoptosis rate were measured by flow cytometry. RPMI 8226 cells were treated with fucoidan for 72 h, and the cell culture supernatant was collected. The VEGF concentration was examined by ELISA, and the tube formation assay was applied to assess the angiogenic activity. After treatment with fucoidan for 72 h at different concentrations, the protein levels of HIF-1α, VEGF, p-AKT and p-ERK1/2 were detected by Western blot. RESULTS: Fucoidan inhibited the growth of RPMI 8226 cells in a dose- and time-dependent manner. After treatment with fucoidan for 72 h, the cell cycle was arrested at G₁ phase, and the apoptotic rate of RPMI 8226 cells was increased with the increasing concentration of fucoidan, which was much higher than that in control group (P<0.05). The VEGF concentration was significantly decreased with the increa-sing concentration of fucoidan. The numbers and areas of the capillary-like structures decreased while the concentration of fucoidan increased, and those at 100 mg/L were less than those in the control (P<0.05). The protein levels of HIF-1α, VEGF, p-AKT and p-ERK1/2 in fucoidan group were significantly lower than those in control group (P<0.05). CONCLUSION: Fucoidan inhibits the secretion of VEGF in multiple myeloma cells, and reduces angiogenesis induced by multiple myeloma cells. It inhibits the protein expression of HIF-1α and VEGF, which may be related to inhibiting the phosphorylation of AKT and ERK1/2.     

Transforming growth factor α promotes the proliferation,migration and adhesion of human endothelial progenitor cells

AIM: To explore the effects of transforming growth factor-α (TGF-α) in the monoclonal formation, proliferation, migration and adhesiveness of human endothelial progenitor cells (EPCs). METHODS: The isolated and cultured EPCs were treated with various concentrations of TGF-α (final concentrations of 1, 5, 10 μg/L, respectively). At the same time, the PBS control and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) group (10 μg/L TGF-α plus 1: 1 000 EGFR-TKI) were set. The effects of TGF-α on monoclonal formation, proliferation, migration and adhesiveness of EPCs were determined by clone formation experiment, thiazolyl blue tetrazolium bromide (MTT), EdU, Transwell and adhesion assays, respectively. The expression of epithelial growth receptor (EGFR) and vascular endothelial growth factor (VEGF) were measured by Western blotting. RESULTS: Different concentrations of TGF-α all significantly induced the monoclonal formation, proliferation, migration and adhesiveness of EPCs (P<0.01), which were inhibited by EGFR-TKI. The results of Western blotting showed that TGF-α also induced the expression of EGFR and VEGF with a certain concentration effect (P<0.01). CONCLUSION: By combining with EGFR induced the expression of VEGF, TGF-α significantly promotes the related cell function of monoclonal formation, proliferation, migration, adhesiveness in EPCs.     

Overexpression of Jagged1 promotes aged rat-derived endothelial proge-nitor cells differentiating into mature endothelial cells

AIM: To investigate the effect of Jagged1 overexpression on endothelial cell-directional differentiation of aged rat-derived endothelial progenitor cells (EPC).METHODS: Mononuclear cells were obtained from bone marrow of young (1 to 2 months old) or aged (19 to 26 months old) Sprague-Dawley rats and cultured in DMEM/F12 medium supplemented with 10% FBS. EPC were characterized as double positive for DiI-ac-LDL uptake and lectin binding. The experiments were divided into control group, PIRES2-EGFP transfection group, PIRES2-EGFP-Jagged1 transfection group and young rat-derived EPC group in which transfection was not performed. The GFP expression positive cell number was acquired by fluorescence microscopy and the transfection efficiency was calculated. Immunofluorescence, RT-PCR and Western blotting were used to detect the mRNA and protein expression. In vitro vasculogenesis kit was used to test the tube formation ability of EPC.RESULTS: EGFP-Jagged1 transfection induced a significant increase in the expression of Jagged1 in aged rat-derived EPC (P<0.01). Compared with the control, Jagged1 overexpression markedly enhanced the mRNA expression of von Willebrand factor (vWF) and kinase insert domain receptor (KDR) of vascular endothelial grouth factor vWF in aged rat-derived EPC (P<0.01) and improved the EPC-related tube formation (P<0.01). No significant difference between Jagged1 transfection and young rat-derived EPC groups in vWF and KDR mRNA expression and the ability of tube formation was found. CONCLUSION: In endothelial cell-conditioning medium, Jagged1 overexpression significantly promotes aged rat-derived EPC differentiation into mature endothelial cells.     

miR-126 regulates expression of vascular endothelial growth factor in EA.hy926 cells

AIM：To explore the effects of miR-126 on the expression of vascular endothelial growth factor (VEGF) in vascular endothelial cell line EA.hy926. METHODS：EA.hy926 cells were cultured in vitro and transfected with miR-126 mimics or miR-126 inhibitor by cation-mediated transfection method. The total RNA was extracted from the culture cells 36 h after transfection of miR-126 mimics or miR-126 inhibitor. The expression of VEGF at mRNA and protein levels was detected by real-time PCR and Western blotting. RESULTS：Thirty-six hours after transfection of miR-126 inhibitor at concentration of 50 nmol/L, the expression of VEGF at mRNA and protein levels increased significantly as compared with the negative control (P<001). However, transfection of miR-126 mimics at concentration of 50 nmol/L for 36 h significantly decreased the expression of VEGF at mRNA and protein levels as compared with the negative control. CONCLUSION：miR-126 inhibits the expression of VEGF. VEGF may be one of the target genes regulated by miR-126.     

Inhibitory effect of Shenci capsule in combination with DDP on angiogenesis in mice with Lewis lung cancer

AIM: To investigate the inhibitory effect of Shenci capsule, a Chinese medicine, in combination with cisplatin (DDP) on the tumor growth and angiogenesis in mouse Lewis lung cancer.METHODS: Forty mice with Lewis lung cancer were randomly divided into 4 groups: Shenci capsule group, Shenci capsule in combination with DDP group, DDP group and control group. Shenci capsule, Shenci capsule in combination with DDP,DDP and sodium chloride wene given, respectively. After 21 days, the weight of subcutaneous tumors was measured, and the tumor inhibition rate was calculated. The microvessel density (MVD) and the protein level of vascular endothelial growth factor (VEGF) were detected by immunohistochemical(IHC) staining. The mRNA expression levels of kinase insert domain-containing receptor (KDR) and the receptor of VEGF were detected by FQ-PCR. RESULTS: The tumor growth inhibitory rate in Shenci capsule in combination with DDP group (63.99%) was higher than that in Shenci capsule group, DDP group and control group (P<0.01). The tumor MVD, VEGF protein and KDR mRNA expression level in Shenci capsule in combination with DDP group were lower than those in the above three groups (P<0.01). CONCLUSION: Shenci capsule in combination with DDP have addition or synergistic effects to inhibit the tumor growth and angiogenesis in mouse Lewis lung cancer. Down-regulation of VEGF and its receptor KDR may be one of the mechanisms that combined use of these two drugs for inhibiting angiogenesis.     

Effects of NBP on expression of VEGF and HIF-1α in HUVECs under the condition of oxygen-glucose deprivation

AIM: To investigate the mechanism that dl-3-n-butylphthalide (NBP) protects cells from the induced by oxygen-glucose deprivation (OGD).METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to OGD to induce endothelial damage. Endothelial injury was assessed by measuring the changes of chromatin morphology and MTT method. The protein levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) were determined by immunofluorescence and quantitatively analyzed with the software IPP. Western blotting was applied to verify the results.RESULTS: NBP at the concentrations of 0.01 to 100 μmol/L dose-dependently protected against OGD-induced cell damage. Compared with OGD group, NBP enhanced OGD-induced the expression of VEGF and HIF-1α, and the difference was statistically significant. The expression of VEGF and HIF-1α reached to the peak at the time points of 6 h and 8 h after OGD, respectively.CONCLUSION: Under the condition of OGD, NBP enhances the expression of HIF-1α in HUVECs, subsequently promotes the expression of downstream VEGF, and eventually elevates the survival of the cells.     

Effect of shikonin on high glucose-induced apoptosis and oxidative stress in vascular endothelial cells

AIM:To investigate the effect of shikonin on the apoptosis and oxidative stress induced by high concentration of glucose in vascular endothelial cells. METHODS:Rat thoracic aortic endothelial cells were randomly divided into 5 groups:normal control group (with glucose at concentration of 5.5 mmol/L in cell culture medium), high glucose group (with glucose at concentration of 33 mmol/L in cell culture medium), high glucose+low shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 0.1 μmol/L in cell culture medium), high glucose+medium shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 1 μmol/L in cell culture medium), and high glucose+high shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 10 μmol/L in cell culture medium). After treatments, the cell viability was measured by CCK-8 assay and cell apoptotic rate was analyzed by flow cytometry. In addition, the status of oxidative stress was evaluated by determining the levels of malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The activation of Nrf2/HO-1 signaling pathway was determined by Western blot. RESULTS:Compared with high glucose group, shikonin reversed high glucose-induced decrease in cell viability and increase in apoptosis in a concentration-dependent manner. High concentration of glucose induced high levels of MDA and ROS, while decreased the levels of SOD and GSH-Px. However, after treatment with shikonin, the contents of MDA and ROS were decreased, while the activities of SOD and GSH-Px were increased as compared with high glucose group. Furthermore, the high concentration of glucose up-regulated the protein levels of cleaved caspase-3, HO-1 and Nrf2 (nuclear). Compared with high glucose group, the protein levels of cleaved caspase-3, HO-1 and Nrf2 (nuclear) were partly decreased after treatment with shikonin. CONCLUSION:Shikonin alleviates high glucose-induced vascular endothelial cell apoptosis. Its mechanism may be related to activation of Nrf2/HO-1 signaling pathway and down-regulation of oxidative stress in vascular endothelial cells.     

Effects of insulin on VEGF expression in cultured retinal Müller cells

AIM: To investigate the effect of insulin on the expression of vascular endothelial growth factor(VEGF) in cultured rabbit retinal Müller cells. METHODS: Immunocytochemical method and ELISA were used to assay the change of VEGF expression in cultured Müller cells in vitro at different insulin concentrations in qualitative and quantitative ways. RESULTS: VEGF expression in retinal Müller cells was enhanced markedly by insulin. CONCLUSION: Insulin at high concentration enhances VEGF expression in cultured Müller cells, which may be one of factors that insulin accelerates the progress of diabetic retinopathy.     

Effect of cladribine on growth inhibition and autocrining cytokines in human umbilical vein endothelial cell line EA.hy926

AIM: To study the effects of cladribine on growth and secretion activity of human umbilical vein endothelial cell line EA.hy926, and to investigate the mechanism of its anti-tumor effect by inhibiting endothelial cells. METHODS: The effects of cladribine at different concentrations on the cell viability were detected by CCK-8 assay. Apoptosis and cell cycle distribution were examined by flow cytometry. The protein expression levels were determined by Western blot. The levels of tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) secreted by EA.hy926 cells with cladribine treatment for 48 h were analyzed by ELISA. The nitric oxide (NO) production was measured by Gries method. RESULTS: Cladribine at 0.4~1 μmol/L inhibited the viability of EA.hy926 cells in time-and dose-dependent manners. The IC₅₀ was about 3.644 μmol/L. The results showed 43.74% cells in S phase when the concentration of cladribine was 0.4 μmol/L, and 77.23% cells in S phase when the concentration of cladribine was 1 μmol/L. The apoptosis was not induced by cladribine at 0.4~10 μmol/L. The protein expression of Bax and caspase-3 did not change. The expression of p21 increased and the p53 decreased (P<0.05). The levels of TNF-α and TGF-β1 secreted by EA.hy926 cells increased after cladribine treatment for 48 h. The levels of VEGF and NO decreased. CONCLUSION: Cladribine obviously inhibits the viability of EA.hy926 cells. The mechanism is related to the cell cycle arrest. Cladribine promotes the secretion of TNF-α and TGF-β1 by EA.hy926 cells and inhibits the secretion of VEGF and NO.     

Minocycline inhibits BV-2 cell activation by regulating P2X7 receptor

AIM：To explore the role of P2X7 receptor in inhibition of lipopolysaccharide (LPS)-stimulated BV-2 cell activation by minocycline. METHODS：BV-2 cells were divided into 5 groups: control group, LPS group, LPS+0.1 μmol/L Mino group, LPS+1 μmol/L Mino group and LPS+10 μmol/L Mino group. The expression of P2X7 receptor was determined by real-time PCR and Western blotting. The levels of TNF-α and IL-1β in the microglia culture supernatants were measured by ELISA. The morphological changes of the cells were also observed. RESULTS：After exposed to LPS, the expression of P2X7 receptor increased in BV-2 cells at mRNA and protein levels. The concentrations of TNF-α and IL-1β in the microglia culture supernatants also increased. Meanwhile, 0.1~10 μmol/L minocycline inhibited those changes in a dose-dependent manner. CONCLUSION：Minocycline inhibits the activation of microglia. The mechanism may be related to the P2X7 receptor.     

Effect of insulin on the SGK1 expression and extracellular matrix synthesis in human mesangial cells cultured in high glucose

AIM:To study the effect of insulin on the serum and glucocorticoid-inducible kinase 1 (SGK1) expression and extracellular matrix synthesis in human glomerular mesangial cells (HMC) cultured in high glucose. METHODS:The HMCs were cultured in the presence of 5.5 or 25 mmol/L glucose with or without 100 nmol/L insulin (i.e. NG, HG, NI and HI groups). 4 h latter, expressions of SGK1, insulin receptor substrate-1 (IRS1) and IRS2 in corresponding groups were detected by immunofluorescence or examined by Western blotting. The phosphorylation of IRS1 and IRS2 was measured by immunoprecipitation. 24 h latter, connective tissue growth factor(CTGF) and fibronectin (FN) were also examined by RT-PCR and ELISA, respectively. RESULTS:Compared with NG, the SGK1 protein expression in HG, NI and HI groups was significantly higher (P<0.01). High glucose mainly caused IRS2 protein and its phosphorylation level increase (P<0.01). When treated with 100 nmol/L insulin, IRS1 protein and its phosphorylation in HI group apparently elevated while slight inhibition of IRS2 protein expression and its phosphorylation were observed (HI vs HG, P<0.05). High glucose enhanced the expression of CTGF and FN, and insulin strengthened this effect.CONCLUSION:Insulin and high glucose up-regulate the expression of SGK1 in mesangial cells through different target molecular pathways and ultimately enhance ECM synthesis. The effect of insulin is highly associated with IRS1 signaling cascades.