Establishment and verification of insulin resistance model in mouse skeletal muscle cells

AIM:To establish a stable and repeatable insulin resistance model of skeletal muscle cells in vitro, so as to promote the exploration of the pathological mechanism of insulin resistance and the development and screening of related drugs. METHODS:C2C12 mouse myoblasts were used to induce differentiation in normal differentiation medium and differentiation medium containing glucose at 40 and 60 mmol/L, respectively. The effects of glucose at different concentrations on cell convergence, fusion and formation of multinucleated myotubes were observed under phase contrast microscope every day. After 1, 3, 5 and 7 d of differentiation, 2-NBDG assay was used to detect the effects of different interventions on C2C12 basal glucose uptake and insulin-stimulated glucose uptake. The effects of different interventions on the protein expression of glucose transporter 4 (GLUT4) after 5 d and 7 d of differentiation were determined by Western blot. The effects of different interventions on the distribution of GLUT4 protein after 5 d of differentiation were detected by immunofluorescence staining. RESULTS:After treated with glucose at 60 mmol/L, the morphological observation showed that high glucose treatment significantly inhibited the growth and differentiation of C2C12 cells after 3 d. High glucose treatment significantly inhibited basal glucose uptake and insulin-stimulated glucose uptake of the C2C12 cells after 5 d and 7 d (P<0.01). No difference between insulin-stimulated GLUT4 expression and basal GLUT4 expression after 5 d and 7 d of high glucose treatment was observed (P>0.05), but there was significant difference between control group and 60 mmol/L group (P<0.05) determined by Western blot. Immunofluorescence staining observation showed that the distribution of GLUT4 protein in the C2C12 cell membrane was significantly decreased after 5 d of high glucose treatment (P<0.01). Glucose treatment (40 mmol/L) also played a role to some extent, but the effect was not as obvious and stable as 60 mmol/L glucose. CONCLUSION:A stable insulin resistance model of mouse skeletal muscle cells in vitro was successfully established by high glucose stimulation. The treatment of glucose at 60 mmol/L for 5 d was the best. Morphological observation and detection of basic and insulin-stimulated glucose uptake and GLUT4 protein expression and distribution evaluates the insulin resistance level of skeletal muscle cells in vitro.     

Oxidative stress and P53 involve in fluctuant high glucose-induced apoptosis of renal tubular epithelial cells

AIM: To explore the mechanism of fluctuant high blood glucose-induced apoptosis of renal tubular epithelial cells. METHODS: Cultured human renal tubular epithelial cells (HK-2) were treated with stable high glucose or fluctuant high glucose. Antioxidant and specific inhibitor of P53 were applied for identifying the role of oxidative stress and P53 in fluctuant high glucose-induced apoptosis of renal tubular epithelial cells. Additionally, SD rats were randomly divided into normal control group (A), stable high blood glucose group (B) and fluctuant high blood glucose group (C). Diabetic rats were induced by intraperitoneal injection of streptozocin(STZ,65 mg/kg), and the fluctuant high blood glucose animal model was induced by intraperitoneal injection of ordinary insulin and glucose at different time points every day. The activity of superoxide dismutase (SOD) and the content of malonaldehyde (MDA) were detected by the method of colorimetry. The protein expression of NADPH oxidase 4(Nox4) and P53 were examined by immunohistochemistry and Western blotting. Apoptosis was assessed by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). RESULTS: The cultured HK-2 cells treated with fluctuant high glucose had significantly higher apoptotic rate and expression level of P53 protein than those in the cells treated with stable high glucose. Compared with the culture solution of the cels treated with stable high glucose, the SOD activity was decreased and the concentration of MDA was increased in the culture solution of the cells treated with fluctuant high glucose. The antioxidant and specific inhibitor of P53 significantly inhibited the p-P53 expression and decreased the apoptotic rate. After 12 experimental weeks, the cell apoptotic index and protein expression of Nox4 and p-P53 in the kidney tubular epithelial cells isolated from the diabetic rats were significantly increased in C group as compared with B group. CONCLUSION: Oxidative stress and P53 are involved in fluctuant high glucose-induced apoptosis of renal tubular epithelial cells.     

Inhibitory effect of oxymatrine on high glucose-induced rat renal tubular epithelial-mesenchymal transition

AIM:To investigate the inhibitory effect of oxymatrine (OM) on high glucose-induced rat renal tubular epithelial-mesenchymal transition (EMT). METHODS:The rat renal tubular epithelial NRK52E cells were cultured in vitro. The cells were divided into control group, high glucose group, high glucose+different concentrations of OM groups and high glucose+0.50 g/L OM dynamic observation group. The expression of TGF-β1, Smad7, α-SMA and E-cadherin at mRNA and protein levels was detected by real-time PCR and Western blotting. The viability of NRK52E cells was determined by MTT assay. RESULTS:(1) Compared with control group, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose group gradually increased, and Smad7 protein and E-cadherin mRNA and protein gradually reduced, but the mRNA expression of Smad7 gradually increased. (2) Compared with high glucose group, as increases in OM doses, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose+different concentrations of OM groups gradually reduced, and Smad7 protein and E-cadherin mRNA and protein gradually increased, but the mRNA expression of Smad7 had no significant change. (3) Compared with high glucose group, the expression of TGF-β1 and α-SMA at mRNA and protein levels was significantly reduced, the expression of E-cadherin at mRNA and protein levels significantly increased, and the protein expression of Smad7 significantly increased, but the mRNA expression of Smad7 had no significant change in high glucose+0.50 g/L OM dynamic observation group. CONCLUSION: In NRK52E cells, oxymatrine inhibits high glucose induced EMT by down-regulating TGF-β1 and up-regulating Smad7, thus preventing the fibrosis effect of TGF-β1/Smads signaling.     

Effects of fluctuant high blood glucose on apoptosis in glomerular endothelial cells and renal tubular epithelial cells in diabetic rats

AIM:To explore the effects of fluctuant high blood glucose and stable high blood glucose on apoptosis and the expression of Bax and Bcl-2 in glomerular endothelial cells and renal tubular epithelial cells in diabetic rats. METHODS: 24 SD rats were divided into 3 groups: control group, stable high blood glucose group and fluctuant high blood glucose group. Diabetic rats were induced by intraperitoneal injection of STZ, and the fluctuant high blood glucose animal model was induced by intraperitoneal injection of aspart and glucose at different time points every day. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), and immunohistochemistry was used to detect apoptosis associated gene bax and bcl-2 expression in kidney. RESULTS:After 4 experimental weeks, a significant increase in cell apoptosis, up-regulation of Bax protein expression in kidney tubular epithelial cell and down-regulation of Bcl-2 in glomerular endothelial cell in fluctuant high blood glucose rats were observed compared with stable high blood glucose rats.CONCLUSION: Fluctuant high blood glucose induces more apoptosis in renal tubular epithelial cells than that in stable high blood glucose diabetic rats.     

Up-regulation of Snail1 expression in renal tubular epithelial cells through Akt/GSK-3β pathway under high-glucose condition

AIM: To examine the effects of high glucose (HG) on the expression of Snail1 and protein kinase B (Akt)/glycogen synthase kinase 3β (GSK-3β) in primary renal tubular epithelial cells (RTECs). METHODS: The primary RTECs were randomly treated with normal glucose, high glucose or D-mannitol for 30 min~72 h. RT-PCR and Western blotting were used to observe the expression of Snail1, Akt and GSK-3β at mRNA and protein levels in these cells. The primary cultured RTECs were pretreated with LY294002 (a PI3K inhibitor, 25 μmol/L) to observe the specific inhibitory effects of phosphatidylinositol 3-kinase (PI3K) on HG-induced expression of Snail1 protein. RESULTS: Treatment of RTECs with HG resulted in increased mRNA and protein levels of Snail1, Akt1, and phosphorylation of Akt and GSK-3β. LY294002 blocked the HG-induced up-regulation of p-Akt, p-GSK-3β and Snail1 expression at protein level, but no effect of LY294002 was seen on the total protein expression of Akt1 and GSK-3β. HG did not affect the expression of GSK-3β at mRNA and protein levels. CONCLUSION: HG-induced up-regulation of Snail1 may be regulated by Akt/GSK-3β pathway in RTECs.     

High glucose induces down-regulation of BMP-7 expression in renal tubular epithelial cells through p38 MAPK pathway

AIM: To observe the BMP-7 expression in primary renal tubular epithelial cells cultured with high glucose and to investigate the role of p38MAPK signaling pathway.METHODS: The primary renal tubular epithelial cells were randomly treated with normal glucose, high glucose, co-incubation of high glucose with specific p38MAPK inhibitor SB202190 or D-mannitol for 72 h. The protein expression of BMP-7 and fibronectin (FN) in all the cells was assessed by the method of immunocytochemistry. The protein expression of p38MAPK and p-p38MAPK was determined by Western blotting. The mRNA expression of BMP-7 and FN was detected by RT-PCR.RESULTS: In normal glucose group, BMP-7 was highly expressed in the cytoplasm of tubular epithelial cells, and only small amounts of p38MAPK and FN, but not p-p38MAPK, were observed. High glucose was able to activate p38MAPK, and therefore the protein of p-p38MAPK increased remarkably in high glucose-treated cells. High glucose also enhanced FN production. Meanwhile, the expression of BMP-7 decreased. Co-incubation of high glucose with SB202190 for 72 h reduced the activity of p38MAPK by 80% and the FN expression was also decreased, while the BMP-7 expression significantly increased. No significant difference of the BMP-7 or FN expression between control group and D-mannitol group was observed.CONCLUSION: The expression of BMP-7 at mRNA and protein levels in renal tubular epithelial cells is decreased under the condition of high-glucose cultivation. Suppression of p38MAPK signaling pathway stimulates endogenous BMP-7 expression, indicating that p38MAPK pathway may be involved in the down-regulation of BMP-7 in renal tubular epithelial cells by high glucose.     

Retinoid X receptor agonists antagonize high-glucose-induced proliferation of rat vascular smooth muscle cells by inactivation of protein kinase C

AIM: To explore the effect of retinoid X receptor (RXR) agonists on high-glucose-induced proliferation of rat aortic smooth muscle cells (RASMCs). METHODS: RASMCs were cultured in DMEM containing glucose at normal concentration (5.5 mmol/L). For high glucose treatment, glucose solution was added up to a final concentration of 25 mmol/L. The proliferation of RASMCs was detected by WST-1 assay. DNA synthesis was measured by the method of BrdU incorporation. Cell cycle progression was determined by flow cytometry. Phosphorylated protein kinase C (PKC) and the expression levels of cyclin-dependent kinase 2(CDK2) and p27Kip1 were detected by immunoblotting. RESULTS: High glucose increased DNA synthesis, cell cycle progression, the expression of CDK2 and the proliferation of RASMCs. Meanwhile, the expression of p27Kip1 was decreased by high glucose. Treatment of RASMCs with RXR natural ligand 9-cis-retinoic acid (9-cis-RA) resulted in significant inhibition of high-glucose-induced proliferation, DNA synthesis, cell cycle progression and the expression of CDK2 in a concentration-dependent manner. 9-cis-RA also reversed the effect of high glucose on the expression of p27Kip1. RXR specific ligand SR11237 demonstrated the same effect as the effect of 9-cis-RA at the same concentration. PKC inhibitor showed the similar effect on high-glucose-induced proliferation and the expression of CDK2 and p27Kip1 as the RXR agonists did. Furthermore, 9-cis-RA and SR11237 rapidly inhibited high-glucose-induced activation of PKC. CONCLUSION: PKC is involved in high-glucose-induced proliferation of RASMCs. RXR agonists inhibit high-glucose-induced proliferation by depressing PKC activation in vascular smooth muscle cells.     

Effects of platelet-derived growth factor and angiotensinⅡ on the expression of cell cycle related genes in vascular smooth muscle cells

AIM: To investigate the different expressions of cell cycle related genes in hyperplastic and hypertrophic vascular smooth muscle cells caused by platelet-derived growth factor (PDGF-BB) and angiotensinⅡ(AngⅡ). METHODS: Rat aorta media smooth muscle cells were cultured. PDGF-BB and AngⅡ were added into serum-free medium at a concentration of 20 μg/L and 10^-6 mol/L , respectively. Vascular smooth muscle cells (VSMCs) were harvested after stimulated for 24 hours. The expression of cell cycle related genes was measured by DNA chips(Atlas cDNA Expression Arrays, Clontech Laboratories, Inc.). RESULTS: The expression of cyclin D3 mRNA ,cyclin G1 mRNA,p57 mRNA,p16 mRNA,E2F-3 mRNA and DP2 mRNA were higher in PDGF-BB than those in AngⅡstimulated VSMCs. p15 mRNA,p19 mRNA,E2F-1 mRNA, E2F-5mRNA,and N-myc mRNA were only detected in PDGF-BB stimulated group. But the expression of p53-associated protein mRNA were higher in AngⅡstimulation group. The expression of PCNA mRNA, c-myc binding protein mRNA,p53-dependent cell growth regulater mRNA,cyclinC mRNA, cyclinB1 mRNA, E2F-3mRNA were similar in the two groups. CONCLUSION: The procession of cell cycle relys on the coordination of many regulater molecules expressed in different phases. Our study preliminarily definite the genes that express during PDGF-stimulated VSMC's hyperplasia and Ang II-stimulated VSMC's hypertrophy.     

Effect of cell cycle regulator p21WAF1 on hypertrophy of peritoneal mesothelial cells

AIM: To investigate the effect of cell cycle regulator p231^WAF1 on hypertrophy of peritoneal mesothelial cells affected by high concentrated glucose. METHODS: RT-PCR and Western Blot method were used to detect p21^WAF1 expression of rat peritoneal mesothelial cells in high glucose concentration medium (containing 1.86%, 3.86% glucose) after 24 hours. Flow cytometer technique was used to analyze the cell cycle distribution. RESULTS: In high glucose medium, most of the cells became hypertrophy, and were arrested in G1 phase of the cell cycle, which was obvious in 3.86% glucose group. Glucose increased p21 mRNA and protein expression in a dose-dependent manner, and the levels of p21^WAF1 mRNA and protein in 3.86% glucose group were higher than those in 1.38% glucose group (P＜0.05). p21 ^WAF1 mRNA and protein expression were absent in the serum-free normal medium and D-mannitol groups which had the same osmolarity as the glucose groups. CONCLUSION: p21^WAF1 may be pivotal in the hypertrophy and arrest in the G1 phase of mesothelial cells induced by high concentrated glucose.     

Effects of different oxygen concentrations on differentiation of marrow stroma cells into osteoblasts

AIM: To investigate the effect of different oxygen concentrations on the differentiation of marrow stroma cells into osteoblasts and to evaluate the expression of Cbfα1/Runx2, bone-morphogenesis protein 2 (BMP2) and peroxisome proliferator-activated receptor γ2 (PPAR-γ2) in bone marrow stromal cells. METHODS: The bone marrow stomal cells obtained from 4-month-old female SD rats were cultured in growth medium and were used between passages 3 to 5. The cells were divided randomly into 4 groups, each group has 8 samples. The cells in all 4 groups were used for the following experiments after cultured with different oxygen concentrations for 3 d in osteoblastic differentiation medium: total cellular RNA was isolated using total RNA kit; RT-PCR was performed to detect the mRNA expression of Cbfα1/Runx2, BMP2 and PPARγ2. The protein expression of Cbfα1/Runx2 and BMP2 was assayed by Western blotting. RESULTS: Compared to the cells in normoxia condition (20%), the mRNA and protein expressions of Runx2 were enhanced significantly, the mRNA expression of BMP2 was also enhanced significantly, the protein expression of BMP2 increased and the mRNA expression of PPARγ2 decreased significantly in the cells cultured with lower oxygen concentrations. The lower oxygen concentration was in the culture, the more Runx2 mRNA, BMP2 mRNA, BMP2 and Runx2 protein were expressed. On the contrary, hypoxia significantly decreased the expression of PPARγ2 mRNA in bone marrow stronmal cells and the lower the oxygen concentration was used, the less expression of PPARγ2 mRNA was achieved. CONCLUSION: Hypoxia promotes the mRNA and protein expressions of Runx2 and BMP2, also significantly decreases the expression of PPARγ2 mRNA in bone marrow stronmal cells in an oxygen concentration dependent manner, indicating that hypoxia significantly stimulates the differentiation of bone marrow stromal cells into osteoblasts instead of lipocytes.     

Effects of high glucose and homocysteine on expression of MMP-9 in rat fibroblasts

AIM: To observe the effect of different concentrations of glucose and homocysteine on the expression of MMP-9 in fibroblasts, and to investigate the relationship between homocysteine and diabetic foot.METHODS: Fibroblasts were obtained from the dermis of 1-day-old normal SD rat and cultured in DMEM containing 10% FBS. After deprivation of serum (using 0.5% FBS) for 24 h, the fibroblasts in 2-4 passages were cultured for 6 h under the conditions of normal glucose, high glucose and high glucose with high concentration of homocysteine, respectively. The expression of MMP-9 at mRNA and protein levels was assessed by RT-PCR and Western blotting, and the activity of MMP-9 was determined by gelatin zymography analysis. RESULTS: The expression of MMP-9 at mRNA and protein levels and activity of MMP-9 in high glucose (22 mmol/L) group were 1.15 folds, 1.59 folds and 1.34 folds of those in normal glucose group (P<0.05), respectively. The effect of high glucose became more pronounced when co-treated with high concentration of homocysteine (100 μmol/L), which were 1.25 folds, 2.63 folds and 2.52 folds of those in normal glucose group (P<0.05), respectively.CONCLUSION: The increase in the expression of MMP-9 is concentration-dependen with glucose content in the culture. High concentration of homocysteine promotes this process, suggesting that hypercysteinemia might play an important role in the expression of MMP-9 in fibroblast.     

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.     

Protective effect of SIRT1 on rat mesangial cells by decreasing high glucose-induced acetylation of NF-κB p65

AIM: To investigate the effects of silent information regulator 1 (SIRT1) on high glucose-induced acetylation of NF-κB p65 subunit and its protective role in rat mesangial cells. METHODS: Rat mesangial cells were cultured in DMEM supplemented with 10% FBS and were divided into control group, mannitol group, high glucose group, resveratrol group and SIRT1 RNAi group. The cell viability was determined by MTT assay. The mRNA expression of SIRT1, monocyte chemoattratant protein 1 (MCP-1), vascular cell adhesion molecule 1 (VCAM-1-1), tumor necrosis factor α (TNF-α), transforming growth factor β1 (TGF-β1) was analyzed by real-time quantitative PCR. The protein expression of SIRT1 and the acetylation of NF-κB p65 subunit were determined by Western blotting. The protein concentrations of MCP-1, VCAM-1, TNF-α, TGF-β1 and malondialdehyde (MDA) were detected by ELISA. RESULTS: The cell viability, superoxide dismutase (SOD) activity, and the expression of SIRT1 at mRNA and protein levels were decreased by high glucose treatment as compared with control group. The acetylation of NF-κB p65 subunit was significantly increased after interfered with high glucose, resulting in the increase in the secretion of MCP-1, VCAM-1, TNF-α and TGF-β1. Resveratrol decreased high glucose-induced acetylation of NF-κB p65 subunit. However, silencing SIRT1 significantly enhanced the acetylation of NF-κB p65 subunit and the expression of MCP-1, VCAM-1, TNF-α and TGF-β1. CONCLUSION: SIRT1 remarkably inhibits the inflammatory reactions by deacetylating NF-κB p65, suggesting that SIRT1 is a possible target for preventing diabetic nephropathy.     

Quercetin inhibits endothelin-1-induced T-type Ca2+ channel expression in human umbilical arterial smooth muscle cells

AIM: To investigate the effect of quercetin on endothelin-1-induced T-type calcium channel(TCC) expression in primary cultured human umbilical arterial smooth muscle cells for exploring the protective role of quercetin in cardiovascular system. METHODS: Human umbilical arterial smooth muscle cells were verified by immunocytochemistry. The cells in 2-3 passages were used and randomly divided into control group, quercetin alone group, model group and experimental group. The cells in control group were cultured without any drugs for 24 h. The cells in quercetin alone group were cultured with 80 μmol/L quercetin for 24 h. The cells in model group were cultured with ET-1 at the concentration of 100 nmol/L for 24 h. The cells in experimental groups were pretreated with quercetin for 1 h, then coincubated with 100 nmol/L ET-1 for 24 h. The concentrations of quercetin used in this study were 20, 40and 80 μmol/L, respectively. The expression of α_1G, a TCC major subunit, was assayed at mRNA and protein levels by RT-PCR and Western blotting, respectively. The TCC currents(I_caT) were detected by the technique of whole-cell patch-clamp. RESULTS: Compared with control and experimental group, I_CaT density (P<0.01) and the expression of α_1G at mRNA (P<0.05) and protein (P<0.01) levels in model group were significantly increased. No significant difference in the results of quercetin alone group and control group was observed. CONCLUSION: The protective roles of quercetin in cardiovascular functions are related to the depressive effects of quercetin on ET-1-induced increase in both I_CaT density and the expression of α_1G at mRNA and protein levels in cultured human vascular smooth muscle cells.     

Effect of p53 agonist nutlin-3 on proliferation of mesangial cells cultured in high glucose

AIM:To observe the effect of p53 agonist nutlin-3 on the proliferation, apoptosis and expression of extracellular matrix in the glomerular mesangial cells (MCs) cultured in high glucose, and to explore its possible mechanism. METHODS:After successful modeling of diabetic nephropathy (DN), PAS staining was performed on the kidney tissues to observe pathological changes. In addition, the p53 expression in the kidney tissue was detected by immunohistochemical staining. The mesangial cell SV40 was cultured in vitro and divided into mannitol (Motl) group, normal glucose (NG) group, high glucose (HG) group, high glucose plus nutlin-3 (HG+Nut) group and nutlin-3 control (Nut) group. The cell viability was detected by CCK-8 assay, and the living cell counting was performed by the method of Trypan blue exclusion. The apoptosis was analyzed by flow cytometry. The protein levels of collagen type IV (Col-IV), p53, p-p53, Bcl-2 and Bax were determined by Western blot. RESULTS:The significant increases in the proliferation of mesangial cells and the levels of p53 in renal tissue of diabetic nephropathy mice were observed. The results of CCK-8 assay showed that high glucose promoted the viability of mesangial cells, and nutlin-3 at 40 μmol/L inhibited the viability of mesangial cells in high glucose environment. Western blot analysis showed that the protein levels of p53, Bax and p-p53 were significantly increased (P<0.05), and the protein levels of Bcl-2 and Col-IV was decreased in HG+Nut group compared with HG group (P<0.05). Furthermore, the ratio of Bax/Bcl-2 was greater than 1. The results of flow cytometry showed that compared with HG group, nutlin-3 promoted the apoptosis of mesangial cells in high glucose environment, the apoptotic rate was significantly increased (P<0.05).CONCLUSION:High glucose promotes the proliferation of mesangial cells. p53 agonist inhibits the viability and promotes apoptosis of mesangial cells under high glucose.     

The expression of heme oxygenase-1 in vascular smooth muscle cells induced by lipopolysaccharide

AIM:To study the alterations of heme oxygenase-1 mRNA in vascular smooth muscle cells(VSMC) induced by lipopolysaccharide(LPS) and the role of heme oxygenase(HO)/carbon monoxide(CO)pathway in the disorders of regulation of cardiovascular system by LPS. METHODS: LPS (final concentrations 10 mg/L,30 mg/L and 50 mg/L) was added in cultured VSMCs for 6 h respectively or 10 mg/L LPS for 9 h and 18 h. MDA content, LDH release and the rate of trypan blue uptake of VSMC were measured. HO-1 mRNA expression was examined by Northern Blot. RESULTS:VSMC HO-1 mRNA expression was increased gradually with the increasing of LPS concentration. When final concentration of LPS was 50 mg/L, the HO-1 mRNA expression of VSMC was increased by 176.7% compared with control. When LPS final concentration was 10 mg/L, the HO-1 mRNA expression increased gradually along with the culture time. When cultured for 18 h, the HO-1 mRNA expression of VSMC was increased by 195.6% compared with control. Only at LPS 50 mg/L for 6 h and 10 mg/L for 18 h, the rate of trypan blue uptake,MDA content and LDH release were significantly increased. CONCLUSION: LPS can induce the HO-1 mRNA expression of VSMC and that were dose-dependent and time-dependent. The inducible HO may play an important role in the pathogenesis of vascular system under LPS.     

Over-expression of SIRT1 gene inhibits high glucose-stimulated H9c2 cardiomyocyte apoptosis and ROS level through PI3K/AKT signaling pathway

AIM: To investigate the effects of silent information regulator 1 (SIRT1) over-expression on the apoptosis and the level of reactive oxygen species (ROS) in high glucose-induced H9c2 cardiomyocytes. METHODS: H9c2 cardiomyocytes were transfected with empty plasmid (pcDNA3.1-NC) and SIRT1 over-expression plasmid (pcDNA3.1-SIRT1), and then stimulated by high glucose. The H9c2 cells were divided into control group, high glucose group, high glucose + pcDNA3.1-NC group and high glucose + pcDNA3.1-SIRT1 group. The expression of SIRT1 at mRNA and protein levels in each group was determined by qPCR and Western blot. The viability of the cells was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The protein levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K, AKT and phosphorylated AKT were examined by Western blot. RESULTS: SIRT1 was significantly decreased in high glucose-induced H9c2 cardiomyocytes, the cell viability was significantly decreased compared with control group, while the ROS levels and apoptotic rate were increased, and the phosphorylated PI3K and AKT protein levels were down-regulated (P<0.05). Over-expression of SIRT1 significantly promoted the viability of H9c2 cardiomyocytes induced by high glucose, decreased the ROS levels and apoptotic rate, and up-regulated phosphorylated PI3K and AKT protein levels (P<0.05). CONCLUSION: SIRT1 over-expression reverses the decrease in the viability of high glucose-stimulated H9c2 cardiomyocytes, and the increases in apoptotic rate and oxidative stress by regulating PI3K/AKT signaling pathway.