Phosphoglycerate mutase 5 and necroptosis

Necroptosis, or programmed cell death, is a type of cell death with a controllable death signaling pathway and the morphological features similar to necrosis. It is mainly mediated by death receptors or pathogen pattern re-cognition receptors. Among them, tumor necrosis factor receptor 1 (TNFR1)-mediated necroptosis is the most well-studied one. Receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3) are the 2 key kinases involved in the formation of complex I & II and necrosome in the process of necroptosis. Phosphoglycerate mutase 5 (PGAM5), a member of phosphoglycerate mutase gene family, lacks PGAM activity and possesses the phosphatase activity. PGAM5 is anchored in the mitochondrial membrane and is also called mitochondrial phosphoglycerate mutase 5. It has been shown that PGAM5 involves in the formation of necrosome during necroptosis and it is able to accelerate the fission of mitochondria by dephosphorylation of dynamin-related protein 1 (DRP1), thus promoting cell necroptosis.     

Snail1/IGF-1 pathway mediates high glucose-induced EMT in renal tubular epithelial cells

AIM: To observe the expression of Snail1 and insulin-like growth factor-1 (IGF-1) in NRK-52E cells induced by high glucose, and to investigate the relationship of Snail1 and IGF-1 in the mechanism of epithelial to mesenchymal transition (EMT) in diabetic kidney disease (DKD).METHODS: The NRK-52E cells were treated with Snail1 siRNA and IGF-1 siRNA after cultured with high glucose medium for 72 h, and divided into control group, high glucose group, non-targeting (NT) siRNA group, Snail1 RNAi group and IGF-1 RNAi group. The cells were harvested at 48 h and 72 h. Real-time PCR was used to detect the mRNA expression of Snail1, IGF-1, E-cadherin and fibronectin (FN), and the protein levels were determined by immunofluorescence staining.RESULTS: Compared with control group, the expression of E-cadherin at mRNA and protein levels declined after stimulation with high glucose (P<0.01), while that of FN was elevated (P<0.01). Meanwhile, the mRNA and protein levels of Snail1 and IGF-1 were markedly increased (P<0.01).The expression of E-cadherin at mRNA and protein levels was improved in Snail1 RNAi group as compared with high glucose group(P<0.01), while that of FN, IGF-1 and Snail1 was significantly down-regulated (P<0.01). The same changes were observed in IGF-1 RNAi group (P<0.01). The protein expression of each factor in NT group had no significant change as compared with high glucose group (P>0.05). Pearson correlation analysis showed a close positive relationship between the expression of Snail1 and IGF-1 protein (r=0.852, P<0.01).CONCLUSION: Snail1 may facilitate DKD development by regulating IGF-1 in the process of EMT.     

Role of TRIM8 in mouse cardiomyocyte apoptosis induced by high glucose and high free fatty acid

AIM: To investigate the effects of tripartite motif-containing protein 8 (TRIM8) on the apoptosis of mouse cardiomyocytes (MCMs) induced by high glucose and high free fatty acid (HGHF) and the underlying mechanism. METHODS: The MCMs were divided into normal glucose (NG) group (glucose at 5.5 mmol/L), high glucose (HG) group (glucose at 33 mmol/L), high free fatty acid (HF) group (sodium palmitate at 300 μmol/L) and HGHF group (glucose at 33 mmol/L and sodium palmitate at 300 μmol/L). The expression of TRIM8 in the MCMs was knocked down by siRNA, and the MCMs was further divided into control group, scrambled siRNA (Scra-siRNA)/PBS group, TRIM8-siRNA/PBS group, Scra-siRNA/HGHF group and TRIM8-siRNA/HGHF group. To further confirm the specific mechanism of TRIM8 in the MCM injury induced by HGHF, the MCMs were subgrouped into HGHF/DMSO group, HGHF+TRIM8-siRNA+DMSO (HGHF+Ts/DMSO) group, HGHF/ML385 group and HGHF+Ts/ML385 group. Accordingly, apoptosis was analyzed by flow cytometry, and the levels of reactive oxygen species (ROS) were measured by flow cytometry and DHE staining. The expression of TRIM8, nuclear factor E2-related factor 2 (Nrf2), glutamate-cysteine ligase catalytic subunit (GCLC), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO-1) at mRNA and protein levels was determined by qPCR and Western blot. RESULTS: HGHF increased the expression of TRIM8, and suppressed the expression of Nrf2, GCLC, HO-1 and NQO-1 in the MCMs (P < 0.05). Compared with Scra-siRNA/HGHF group, the intracellular ROS content and apoptotic rate were decreased in TRIM8-siRNA/HGHF group (P < 0.05). Correspondingly, the expression of the antioxidant molecule Nrf2 and its downstream genes GCLC, HO-1 and NQO-1 was increased (P < 0.05). In contrast, the addition of Nrf2 inhibitor ML385 partially reversed the inhibitory effect of TRIM8 expression knock-down on HGHF-induced apoptosis of MCMs. CONCLUSION: TRIM8 exacerbates the HGHF-induced cardiomyocyte apoptosis by modulating Nrf2 antioxidative pathway.     

Genipin inhibits oxidative stress and apoptotic damage in high glucose-induced H9c2 cardiomyocytes

AIM:To explore the effects of genipin (GEN) on high glucose (HG)-induced oxidative stress injury and apoptosis in H9c2 cardiomyocytes.METHODS:H9c2 cells were cultured in vitro and HG-induced injury model was established. H9c2 cells were divided into 4 groups:normal control (NC) group (glucose at 5.6 mmol/L), HG group (glucose at 50 mmol/L), NG+GEN group and HG+GEN group. The concentration of genipin was used at 10 μmol/L. The viability of the H9c2 cells was measured by CCK-8 assay. The intracellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined by enzyme labeling and WST-1 methods, respectively. The activity of lactate dehydrogenase (LDH) in the cell culture supernatant was detected by microplate method. Fluorescent probe DCF was used to detect intracellular levels of reactive oxygen species (ROS). Nucleosome fragments was measured to evaluate cell apoptosis by ELISA. The intracellular mitochondrial membrane potential was detected by JC-1 method. The protein levels of Mn-SOD, cytochrome C (Cyt C), Bax and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with HG group, the cell viability in HG+GEN group was increased significantly (P<0.05), the levels of MDA and LDH were decreased (P<0.05), SOD activity was increased (P<0.05), the levels of ROS and nucleosome fragments in HG+GEN group were decreased (P<0.05), and the mitochondrial membranes potential was notably increased (P<0.05). Compared with NG group, the activation of Mn-SOD was decreased, but the protein levels of Cyt C, Bax and cleaved caspase-3 were increased in HG group (P<0.05). Compared with HG group, the activation of Mn-SOD was increased, and the protein levels of Cyt C, Bax and cleaved caspase-3 were decreased in HG+GEN group (P<0.05).CONCLUSION:Genipin protects HG-induced H9c2 cardiomyocytes against oxidative stress injury and apoptosis.     

Role of caveolae in high glucose-induced extracellular matrix production in rat mesangial cells

AIM：To investigate the role of caveolae in high glucose (HG)-induced extracellular matrix (ECM) production in rat mesangial cells (MCs). METHODS：Synchronized rat MCs were divided into normal glucose group, HG group, HG+methyl-β-cyclodextrin (β-MCD) group and HG+β-MCD+cholesterol (Chol) group. Western blotting was used to detect the protein expression of caveolin-1 (Cav-1), phosphorylated caveolin-1 (p-Cav-1-Y14) and collagen type 1 (Col I). The mRNA expression of Cav-1 was determined by real-time PCR. ELISA was used to measure the level of fibronectin (FN) in the supernatant. RESULTS：High glucose significantly increased the expression of FN and Col I. In HG 12, 24 and 48 h groups, the mRNA and protein levels of Cav-1 were not significantly different from those in HG 0 h group, whereas the level of p-Cav-1-Y14 was significantly increased. β-MCD significantly attenuated HG-induced elevation of p-Cav-1-Y14 and FN production, but had no effect on HG-induced Col I expression. All these responses to β-MCD were abolished by Chol. CONCLUSION：High glucose significantly increases the production of Col I and FN in rat MCs. FN production induced by high glucose is mediated by p-Cav-1-Y14.     

Role of calreticulin-induced mitochondrial damage in high glucose-induced apoptosis of myocardial cells

AIM: To observe the effect of high glucose on the protein expression of calreticulin (CRT) and its association with cell apoptosis and mitochondrial dysfunction in the cardiomyocytes. METHODS: AC-16 cardiomyocytes were randomly divided into normal glucose group, high glucose group, high glucose+ CRT siRNA group and isotonic control group. The cell apoptotic rate, reactive oxygen species (ROS), mitochondrial membrane potential level, respiratory enzyme activity, and protein expression of CRT were observed. RESULTS: Compared with the cardiomyocytes in normal glucose group, the apoptotic rate and ROS production of cardiomyocytes increased in high glucose group, accompanying with the decreases in the mitochondrial membrane potential level and enzyme activitiy of the respiratory chain. The protein expression of CRT was significantly increased in high glucose group. However, compared with high glucose group, high glucose+ CRT siRNA decreased the expression of CRT and attenuated the damage of mitochondria, but CRT siRNA did not reduce the ROS level in cardiomyocytes. CONCLUSION: High glucose brings about CRT over-expression to induce mitochondrial injury, thus increasing myocardial apoptosis.     

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.     

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.     

Activation of TGR5 reduces high glucose-induced cardiomyocyte hypertrophy by inhibiting CaN/NFAT3 signaling

AIM: To investigate the role of G-protein-coupled bile acid receptor 1(GPBR1; also known as TGR5) activation in high glucose-induced cardiomyocyte hypertrophy and calcineurin (CaN)/nuclear factor of activated T-cells 3 (NFAT3) signaling.METHODS: Primarily cultured mouse cardiomyocytes were used in the study. The cell surface areas of the cardiomyocytes were measured by an image analysis system. The cell protein content was detected by BCA method. The expression of TGR5, CaN and NFAT3 at mRNA and protein levels was determined by RT-PCR and Western blot.RESULTS: The mouse cardiomyocytes were successfully cultured. High glucose significantly induced the increases in the cell surface area, the cell protein content and the expression of CaN and NFAT3 (P<0.05) in the cardiomyocytes. TGR5 activation or a CaN antagonist cyclosporin A inhibited high glucose-induced cardiomyocyte hypertrophy and the expression of CaN and NFAT3 (P<0.05). These effects of TGR5 activation were abolished by TGR5 gene interference (P<0.05).CONCLUSION: TGR5 activation reduces high glucose-induced cardiomyocyte hypertrophy by inhibiting CaN/NFAT3 signaling.     

Expression and significance of thrombospondin-1 in myocardium of type 2 diabetic cardiomyopathy rats

AIM: To investigate the expression and significance of thrombospondin-1 (TSP-1) in left ventricular myocardium of type 2 diabetic cardiomyopathy (DCM).METHODS: The rat model of DCM was established by eating a high-fat diet together with injection of low dose streptozocin (30 mg/kg) intrapertoneally.After 12 weeks,the content of collagen was quantified by Masson staining.The mRNA level of TSP-1 was determined by quantification real-time RT-PCR,while the protein level of TSP-1 was analyzed by Western blotting and immunohistochemistry.RESULTS: Compared with the control group,the content of collagen in the DCM group was increased greatly (11.01±3.05 vs 16.92±3.18,P<0.01).The mRNA and protein expressions of TSP-1 were significantly higher than those in control group (0.0089±0.0034 vs 0.0141±0.0037,P<0.05；96.38±16.80 vs 129.98±16.96,P<0.05).In DCM group,the mRNA and protein expressions of TSP-1 showed significantly positive correlations with the levels of fasting blood glucose and collagen (r=0.762,P<0.01; r=0.717,P<0.05; r=0.735,P<0.01; r=0.750,P<0.01).There was a significantly positive correlation of TSP-1 mRNA level with LVEDP (r=0.658,P<0.05).In contrast,there was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.605,P<0.05; r=-0.694,P<0.05).There was a significantly positive correlation of TSP-1 protein with LVEDP (r=0.716,P<0.05).There was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.633,P<0.05; r=-0.669,P<0.05).CONCLUSION: The increased expression of TSP-1 may play an important role in the development of myocardial interstitial fibrosis in DCM.     

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.     

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.     

Baicalin inhibits high glucose-induced apoptosis of mouse glomerular mesangial cells via miR-141/Sirt1 signaling pathway

AIM:To investigate the therapeutic mechanism of baicalin for diabetic nephropathy involving microRNA-141 (miR-141)/silent information regulator 1 (Sirt1) signaling pathway. METHODS:Mouse glomerular mesan-gial cell line SV40-MES-13 was treated with high glucose (HG, 25 mmol/L glucose) to establish diabetic nephropathy cell model. Baicalin at 100 μmol/L was used to treat glomerular mesangial cells. qPCR and Western blot were performed to determine the expression levels of miR-141 and Sirt1. The regulatory relationship between miR-141 and Sirt1 was detected by dual-luciferase assay. The apoptosis of glomerular mesangial cells was analyzed by flow cytometry. RESULTS:Compared with control group, the cells treated with HG showed increased levels of miR-141 and apoptosis, and Sirt1 expression was decreased (P<0.01). Baicalin and miR-141 inhibitor suppressed the HG-induced effect on the levels of miR-141, Sirt1 and apoptosis. Knockdown of Sirt1 expression reversed the effect of miR-141 inhibitor on the levels of miR-141, Sirt1 and apoptosis. Over-expression of miR-141 reversed the effect of baicalin on the glomerular mesangial cells treated with HG. Up-regulation of Sirt1 abolished the effect of miR-141 over-expression on the glomerular mesangial cells. CONCLUSION:Baicalin inhibits the apoptosis of mouse glomerular mesangial cells via miR-141/Sirt1 signaling pathway, thus attenuating diabetic nephropathy.     

Changes and clinical implications of plasma ANP,BNP and CNP levels in type 2 diabetic patients with vascular complications

AIM:To examine the changes and clinical implications of the plasma atrial natriuretic peptide (ANP), brain natriuretic peptide(BNP) and C-type natriuretic peptide(CNP) levels in type 2 diabetic patients with vascular complications. METHODS:9 subjects without diabetes, 34 diabetic patients without vascular complications and 23 diabetic patients with vascular complications were enrolled in the study and categorized into three groups. Plasma proANP, BNP fragment and N-terminal pro-CNP (NT-proCNP) were measured by enzyme linked immunosorbent assay (ELISA). The changes and associations of the plasma natriuretic peptide levels among three groups were analyzed. RESULTS:Compared with those in the controls and diabetics without vascular complications, the plasma proANP and BNP fragment levels in diabetic patients with vascular complications increased significantly (P<0.01). However, the plasma NT-proCNP level in this group decreased significantly (P<0.01). Nevertheless, no significant differences in the plasma proANP, BNP fragment and NT-proCNP levels among three subgroups (diabetics patients with macrovascular and/or microvascular complications) were observed (P>0.05). The plasma ANP level of diabetic patients with vascular complications was corelated significantly with the BNP levels (r=0.309, P<0.05). Furthermore, the ANP and BNP levels were correlated significantly with the CNP level (r=-0.374, P<0.05; r=-0.653, P<0.01). CONCLUSION:Measurement of the plasma ANP, BNP and CNP levels together may be a simple, cost-effective and reliable way to screen for diabetic patients with vascular complications.     

Effects of uric acid on mitochondrial damage and Pgam5/Drp1 expression in rat kidney cells

AIM To investigate the effects of uric acid on mitochondrial damage and the expression of phosphoglycerate mutase family member 5 (Pgam5) and dynamin-related protein 1 (Drp1) in rat kidney cells. METHODS Normal rat kidney NRK-52E cells were treated with uric acid at 0.6 mmol/L. The cell viability was measured by MTT assay. The cell morphological change was observed by Hoechst 33258 staining. The apoptosis of the cells was analyzed by flow cytometry. The morphological structure of mitochondria was observed under transmission electron microscope. The expression of Pgam5 and Drp1 was examined by immunofluorescence staining. The mRNA expression of Pgam5 in mitochondria was detected by RT-qPCR. The protein expression of Pgam5 and Drp1 in the cytoplasmic matrix was determined by Western blot. RESULTS Uric acid significantly decreased the viability of NRK-52E cells, and significantly increased the apoptotic rate of the cells (P<0.01). Mitochondrial swelling, vacuolation and cristal rupture were observed after the NRK-52E cells were treated with uric acid, and the mRNA expression of Pgam5 in mitochondria was decreased significantly, while the protein expression of Pgam5 and Drp1 in the cytoplasmic matrix was increased significantly (P<0.01). CONCLUSION Uric acid intervention destroys the mitochondrial structure of rat renal cells, up-regulates the expression of Pgam5 and Drp1 in the cytoplasmic matrix, and induces apoptosis of the cells.     

Effects of hydrogen sulfide on high glucose-induced injury of mouse glomerular podocytes

AIM: To investigate the effect of hydrogen sulfide (H2S) on high glucose （HG）-induced injury of the mouse podocyte cell line MPC5. METHODS: The cultured MPC5 cells were randomly divided into 4 groups: HG group, normal glucose (NG) group, NG+DL-propargylglycine (PPG) group, and HG+NaHS group. After treated for a certain time, the cells were collected for further detection. The expression of zonula occludens-2 (ZO-2), nephrin, β-catenin and cystathionine γ-lyase (CSE) was determined by Western blotting. RESULTS: High glucose significantly reduced the expression of nephrin, ZO-2 and CSE (P<0.05), while the level of β-catenin was elevated obviously (P<0.05), all in a time-dependent manner. NG+PPG inhibited the levels of ZO-2 and nephrin significantly (P<0.05), and increased the level of β-catenin (P<0.05), all in a PPG concentration-dependent manner. HG+NaHS induced a more significant increase in the levels of ZO-2 and nephrin as compared with HG group (P<0.01), whereas a severe reduction of β-catenin in HG+NaHS group was observed as compared with HG group. Compared with NG group, the expression of ZO-2 and nephrin was decreased obviously, and the level of β-catenin was increased in HG+NaHS group. CONCLUSION: Down-regulation of CSE contributes to hyperglycemia-induced podocyte injury. Exogenous H2S protects against hyperglycemia-induced podocyte injury, possibly through up-regulation of ZO-2 and subsequent suppression of Wnt/β-catenin pathway.     

Role of ubiquitin E3 ligase TRIM10 in regulating cardiomyocyte hypertrophy

AIM: To explore the role of ubiquitin E3 ligase tripartite motif 10 (TRIM10) in the development of cardiomyocyte hypertrophy. METHODS: Primary cultured neonatal rat cardiomyocytes (NRCMs) were infected with siRNA-TRIM10, siRNA-control, Ad-TRIM10 or Ad-GFP for 24 h respectively, and then stimulated with phenylephrine (PE) for additional 24 h. The protein levels of TRIM10, AKT and ERK1/2 were determined by Western blot. The size of the NRCMs was measured by immunofluorescence staining. The mRNA expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was detected by RT-qPCR. RESULTS: Compared with the control, PE treatment significantly increased the protein expression of TRIM10. Moreover, transfection of NRCMs with siRNA-TRIM10 markedly inhibited cardiomyocyte size, the mRNA expression of ANP and BNP, and the phosphorylation levels of AKT and ERK as compared with siRNA-control after PE treatment. In contrast, overexpression of TRIM10 significantly enhanced PE-induced hypertrophic effect on NRCMs above. CONCLUSION: TRIM10 regulates cardiomyocyte hypertrophy partially through AKT and ERK signaling pathways.     

Vitamin D prevents high glucose-induced human umbilical vein endothe-lial cell apoptosis by inhibition of prolyl isomerase 1-mediated mitochon-drial oxidative stress

AIM: To observe the effects of vitamin D on the apoptosis, prolyl isomerase 1 (Pin1) protein expression and activity, mitochondrial translocation of p66Shc, and reactive oxygen species (ROS) production in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and to explore the role of vitamin D receptor (VDR) in these processes. METHODS: HUVECs were treated with high glucose (33 mmol/L) in the presence or absence of vitamin D or Pin1 inhibitor juglone. The cell apoptosis was measured by flow cytometry and TUNEL staining. Intracellular ROS levels were examined by flow cytometry and fluorescence microscopy. The protein levels of Pin1, p66Shc, p-p66Shc, mitochondria to cytoplasm ratio of p66Shc, and caspase-3 in HUVECs were measured by Western blot. Pin1 activity in HUVECs lysate was assessed by a commercial kit. Knockdown of VDR by siRNA was conducted to evaluate the role of VDR in the regulatory effects of vitamin D on Pin1 protein expression and activity in HUVECs under high-glucose condition. RESULTS: Vitamin D suppressed the apoptosis and intracellular ROS generation of HUVECs induced by high glucose (P<0.05). Vitamin D inhibited high glucose-induced upregulation of Pin1 protein expression and activity (P<0.05). Vitamin D inhibited the phosphorylation and mitochondrial translocation of p66Shc and caspase-3 protein expression induced by high glucose (P<0.05). Knockdown of VDR by siRNA abolished the inhibitory effects of vitamin D on high glucose-induced upregulation of Pin1 protein expression and activity. CONCLUSION: Vitamin D alleviates high glucose-induced endothelial cell apoptosis by inhibition of Pin1 protein expression and activity, and attenuation of p66Shc-mediated mitochondrial oxidative stress, which are dependent on VDR activation.     

Role of CRIF1 in cardiomyocyte apoptosis induced by palmitic acid

AIM: To investigate the effects of CR6-interacting factor 1 (CRIF1) on the apoptosis of mouse cardiomyocytes (MCMs) induced by palmitic acid. METHODS: The MCMs cultured with medium containing palmitic acid at 0 and 300 μmol/L for 24 h were divided into control group and palmitic acid group, respectively. In order to explore the effects of CRIF1 on MCMs injuries induced by high fat, MCM exposed to palmitic acid at 300 μmol/L for 24 h were divided into vehicle group, scrambled (Scra) siRNA group and CRIF1 siRNA group. The cells in vehicle group were only treated with transfection reagent, the cells in Scra siRNA group were given a treatment with transfection reagent and scrambled RNA, and the cells in CRIF1 siRNA group were given a treatment with transfection reagent and CRIF1 specific siRNA. In order to further confirm the specific mechanism of CRIF1 in high fat-induced MCM injuries, MCMs in CRIF1 siRNA group were divided into DMSO group and N-acetyl cysteine (NAC) group, and were given the same intervention of palmitic acid. RT-qPCR, Western blot and immunofluorescence staining were used to detect the mRNA and protein expression of CRIF1. The apoptotic rate was analyzed by flow cytometry, and the level of intracellular reactive oxygen species (ROS) was tested by DHE staining and ELISA. RESULTS: The expression of CRIF1 was significantly increased after exposure to palmitic acid (P<0.05). Compared with control group, the apoptotic rate was increased significantly in vehicle group and Scra siRNA group (P<0.05). The apoptotic rate was significantly increased in CRIF1 siRNA group (P<0.05). Compared with control group, the intracellular ROS content was significantly increased in vehicle group and Scra siRNA group (P<0.05). Compared with vehicle group and Scra siRNA group, the intracellular ROS content was significantly increased in CRIF1 siRNA group (P<0.05). Compared with DMSO group, the intracellular ROS content and the apoptotic rate were remarkably decreased in NAC group (P<0.05). CONCLUSION: With the inhibition of oxidative stress, CRIF1 may reduce the apoptosis of MCMs induced by high fat.     

Importance of mitochondrial calcium uniporter in high glucose-induced cardiac myocyte apoptosis

AIM: To investigate the role of mitochondrial calcium uniporter (MCU) in high glucose(HG)-induced apoptosis of cardiac myocytes. METHODS: Cardiac myocytes were exposed to normal glucose (5.5 mmol/L glucose+ 19.5 mmol/L mannitol), HG (25 mmol/L glucose), or HG combined with 5 μmol/L spermine for 72 h. Mitochondrial free Ca²⁺ concentration ([Ca²⁺]_m), MCU at mRNA and protein levels, pyruvate dehydrogenase (PDH) activity, mitochondrial membrane potential (Δψ_m), the levels of ATP and reactive oxygen species (ROS), and apoptosis were determined. RESULTS: The [Ca²⁺]_m, the mRNA and protein levels of MCU, PDH activity, ATP levels, and Δψ_m were reduced (P<0.05), while ROS content and the protein levels of caspase-9 and caspase-3 were increased in HG group (P<0.05). Adding 5 μmol/L spermine returned these parameters toward control levels (P<0.05). Moreover, apoptosis was reduced by adding spermine and HG treatment (P<0.05). CONCLUSION: HG-induced cardiac myocyte apoptosis may be associated with the decreased MCU expression and activity, abnormal mitochondrial Ca²⁺ handling, deviant mitochon-drial respiratory chain, and mitochondrial dysfunction.