Glucose-induced Oxidative Stress Research Articles

Oltipraz Prevents High Glucose-Induced Oxidative Stress and Apoptosis in RSC96 Cells through the Nrf2/NQO1 Signalling Pathway.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes mellitus (DM). Schwann cell (SC) apoptosis contributes to the occurrence and development of DPN. Effective drugs to prevent SC apoptosis are required to relieve and reverse peripheral nerve injury caused by DM. Oltipraz [4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione], an agonist of nuclear factor erythroid derived-2-related factor 2 (Nrf2), exerts strong effect against oxidative stress in animal models or clinical patients in certain diseases, including heart failure, acute kidney injury, and liver injury. The aim of the present study was to determine the effectiveness of oltipraz in preventing SC apoptosis induced by high glucose levels. RSC96 cells pretreated with oltipraz were cultured in high-glucose medium (50 mM glucose) for 24 h, and cells cultured in medium containing 5 mM glucose were used as the control. Flow cytometry was used to evaluate the degree of apoptosis. A Cell Counting Kit-8 assay was used to assess cell viability. The mitochondrial membrane potential was assessed using JC-1 staining, and reactive oxygen species (ROS) generation was measured using 20,70-dichlorodihydrofluorescein diacetate staining. In addition, the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) levels were also evaluated using the corresponding kits. Flow cytometry was subsequently used to detect apoptosis, and western blotting was used to measure the expression levels of nuclear factor erythroid derived-2-related factor 2 and NADPH quinone oxidoreductase 1. The results showed that high glucose concentration increased oxidative stress and apoptosis in RSC96 cells. Oltipraz improved cell viability and reduced apoptosis of RSC96 cells in the high glucose environment. Additionally, oltipraz exhibited a significant antioxidative effect, as shown by the decrease in MDA levels, increased SOD levels, and reduced ROS generation in RSC96 cells. The results of the present study suggest that oltipraz exhibits potential as an effective drug for treatment with DPN.

CircBPTF knockdown ameliorates high glucose-induced inflammatory injuries and oxidative stress by targeting the miR-384/LIN28B axis in human umbilical vein endothelial cells.

Endothelial dysfunction is a primary cause of diabetes-related vascular complications, such as atherosclerosis. Accumulated research indicates that circular RNAs (circRNAs) are involved in the pathogenesis of cardiovascular disease. This study intended to explore the function and mechanism of circBPTF in high glucose (HG)-induced vascular inflammatory models. Cell model of inflammatory injury was established in human umbilical vein endothelial cells (HUVECs) with HG treatment. The expression of circBPTF, miR-384 and lin-28 homolog B (LIN28B) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis were assessed by cell counting kit-8 (CCK-8) and flow cytometry assay, respectively. The expression of LIN28B was also examined using western blot. The release of proinflammatory cytokines was detected by enzyme-linked immunosorbent assay (ELISA). The production of ROS, SOD and MDA was detected to assess oxidative stress. The target relationship was predicted by bioinformatics analysis and verified using dual-luciferase reporter assay and RIP assay. CircBPTF was highly regulated in HG-induced HUVECs. CircBPTF knockdown increased cell viability and suppressed cell apoptosis, the release of proinflammatory cytokines and oxidative stress in HG-induced HUVECs. MiR-384 was targeted by circBPTF, and its downregulation abolished the effects of circBPTF knockdown. Moreover, circBPTF positively regulated LIN28B expression via targeting miR-384. Overall, CircBPTF knockdown protected against HG-induced inflammatory injuries and oxidative stress by mediating the miR-384/LIN28B axis in HUVECs. Our study provides a feasible theoretical strategy for preventing vascular cell dysfunction.

β-Caryophyllene inhibits high glucose-induced oxidative stress, inflammation and extracellular matrix accumulation in mesangial cells

β-Caryophyllene (BCP) is a bicyclic sesquiterpene compound that has anti-diabetic activity. However, the effect of BCP on diabetic nephropathy (DN) remains unclear. Here, we aimed to evaluate the potential role of BCP in high glucose (HG)-induced glomerular mesangial cells (MCs). MCs were maintained under HG condition to simulate DN in vitro. Our results showed that BCP inhibited HG-induced cell proliferation, ROS production and NADPH oxidase (NOX) 2/4 expression. BCP exhibited anti-inflammatory activity with decreased levels of TNF-α, IL-1β, IL-6 in HG-induced MCs. Moreover, BCP treatment suppressed the HG-induced secretion of fibronectin (FN) and collagen IV (Col IV) in MCs. Furthermore, BCP suppressed the NF-κB activation and enhanced the Nrf2 activation in HG-induced MCs. However, inhibition of Nrf2 attenuated the protective effects of BCP on HG-induced MCs, while inhibition of NF-κB enhanced the nephro-protective effects of BCP on MCs. In conclusion, these findings demonstrated that BCP executed protective effects on HG-induced MCs via regulating NF-κB and Nrf2 signaling pathways.

Coloured Rice Phenolic Extracts Increase Expression of Genes Associated with Insulin Secretion in Rat Pancreatic Insulinoma β-cells.

Glucose-induced oxidative stress is associated with the overproduction of reactive oxygen species (ROS), which may dysregulate the expression of genes controlling insulin secretion leading to β-cell dysfunction, a hallmark of type 2 diabetes mellitus (T2DM). This study investigated the impact of coloured rice phenolic extracts (CRPEs) on the expression of key genes associated with β-cell function in pancreatic β-cells (INS-1E). These genes included glucose transporter 2 (Glut2), silent mating type information regulation 2 homolog 1 (Sirt1), mitochondrial transcription factor A (Tfam), pancreatic/duodenal homeobox protein 1 (Pdx-1) and insulin 1 (Ins1). INS-1E cells were cultured in high glucose (25 mM) to induce glucotoxic stress conditions (HGSC) and in normal glucose conditions (NGC-11.1 mM) to represent normal β-cell function. Cells were treated with CRPEs derived from two coloured rice cultivars, Purple and Yunlu29-red varieties at concentrations ranged from 50 to 250 µg/mL. CRPEs upregulated the expression of Glut2, Sirt1 and Pdx-1 significantly at 250 µg/mL under HGSC. CRPEs from both cultivars also upregulated Glut2, Sirt1, Tfam, Pdx-1 and Ins1 markedly at 250 µg/mL under NGC with Yunlu29 having the greatest effect. These data suggest that CRPEs may reduce β-cell dysfunction in T2DM by upregulating the expression of genes involved in insulin secretion pathways.

Up-regulation of miR-135b expression induced by oxidative stress promotes the apoptosis of renal tubular epithelial cells under high glucose condition.

This study aimed to investigate the role and underlying mechanism of miR-135b in high glucose-induced oxidative stress of renal tubular epithelial cells. Here, in vivo experiments found that compared to the control group, miR-135b expression was significantly up-regulated in the diabetes group, whereas BMP7 mRNA and protein levels were down-regulated. In high glucose-treated renal tubular epithelial cells (HK-2) in vitro, oxidative stress was induced, which up-regulated miR-135b expression. In addition, the regulation of miR-135b on BMP7 expression was confirmed in HK-2 cells. Under high glucose conditions, oxidative stress promoted the apoptosis of HK-2 cells through the up-regulation of miR-135b expression. In vivo experiments indicated that interference with miR-135b improved renal function in mice with diabetic nephropathy. In conclusion, these results indicated that the up-regulation of miR-135b expression induced by oxidative stress promotes the apoptosis of HK-2 cells under high glucose conditions.

Heat shock protein 60 (HSP60) modulates adiponectin signaling by stabilizing adiponectin receptor

Adiponectin, an adipokine produced and secreted by adipocytes, is involved in regulating the development and progression of insulin resistance, diabetes, and diabetic complications. Heat shock protein 60 (HSP60) is a molecular chaperone, most commonly presenting in mitochondria and participating in the maintenance of protein homeostasis. Accumulating studies have demonstrated that the elevated circulating HSP60 and the decreased intracellular HSP60 are closely associated with diabetic complications such as diabetic cardiomyopathy. However, the underlying mechanism remains poorly understood. In the present study, we reported that HSP60 interacted directly with adiponectin receptors. Its abundance was positively associated with adiponectin action. Furthermore, HSP60 depletion markedly mitigated the protective impacts of adiponectin on high glucose-induced oxidative stress and cell apoptosis in rat cardiac H9c2 cells. In addition, HSP60 knockdown significantly enhanced proteasome activity leading to the degradation of adiponectin receptor 1. Taken together, we showed for the first time that HSP60 interacted with adiponectin receptors and mediated adiponectin signaling through stabilizing adiponectin receptor. This in vitro study also provides an alternative explanation for mechanism by which adiponectin exerts its action.B94avigycLYsoGgop984zWVideo abstract

Proteomic analysis of oxidative stress response in human umbilical vein endothelial cells (HUVECs): role of heme oxygenase 1 (HMOX1) in hypoxanthine-induced oxidative stress in HUVECs.

BackgroundErectile dysfunction (ED) is a well-known complication of diabetes, affecting up to 75% of diabetic men. Although the etiology of diabetic ED is multifactorial, endothelial dysfunction is considered to be a pillar of its pathophysiology. Endothelial dysfunction is caused by the harmful effects of high glucose levels and increased oxidative stress on the endothelial cells that comprise the vascular endothelium. The aim of this study was to identify the proteomic changes caused by high glucose-induced oxidative stress and explore the role of heme oxygenase 1 (HMOX1) in it.MethodsThe cellular proteomic response to hypoxanthine-induced oxidative stress in human umbilical vein endothelial cells (HUVECs) was analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins (DEPs) were analyzed through Network and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Further validation assays was performed to validate the role of HMOX1.ResultsThe results showed that 66 and 76 DEPs were markedly upregulated and downregulated, respectively, for HUVECs oxidative stress. Among these proteins, we verified eight dysregulated genes by quantitative reverse transcription PCR, including nucleolin (NCL), X-ray repair cross-complementing protein 6 (XRCC6), ubiquinol-cytochrome C reductase binding protein (UQCRB), non-POU domain containing octamer binding (NONO), heme oxygenase 1 (HMOX1), nucleobindin 1 (NUCB1), DEK, and chromatin target of prmt1 (CHTOP). Further, using overexpression and genetic knockdown approaches, we found that HMOX1 was critical for the oxidative stress response in HUVECs.ConclusionsWe found that HMOX1 was closely related to the oxidative stress response induced by hypoxanthine. To the best of our knowledge, this study is the first overview of the responses of HUVECs to oxidative stress. The findings will contribute to analyses of the detailed molecular mechanisms involved in the pathogenesis of endothelial dysfunction and related molecular mechanisms in ED patients.

Knockdown of CTRP6 inhibits high glucose-induced oxidative stress, inflammation and extracellular matrix accumulation in mesangial cells through regulating the Akt/NF-κB pathway.

C1qTNF-related protein 6 (CTRP6) is a member of the CTRP family and exerts a key role in the progression of diabetes mellitus. However, the role of CTRP6 in diabetic nephropathy remains unknown. The present study was designed to examine the roles of CTRP6 in diabetic nephropathy and explore the potential molecular mechanisms. Our results showed that the expression level of CTRP6 was significantly increased in high glucose (HG)-stimulated glomerular mesangial cells (MCs). The following loss/gain-of-function assays demonstrated that CTRP6 knockdown significantly inhibited HG-induced reactive oxygen species (ROS) production in MCs. CTRP6 knockdown caused significant decreases in tumournecrosisfactor-α (TNF-α), interleukin (IL)-1β and IL-6 production levels in HG-induced MCs. Moreover, knockdown of CTRP6 inhibited HG-stimulated extracellular matrix (ECM) accumulation in MCs characterized by decreased expression and production levels of fibronectin (FN) and collagen IV (Col IV). Furthermore, CTRP6 knockdown suppressed HG-induced the activation of Akt/NF-κB pathway in MCs, while overexpression of CTRP6 exhibited the opposite effects. Treatment with LY294002, an inhibitor of Akt, reversed the induction effects of CTRP6 overexpression on ROS production, inflammation and ECM accumulation in MCs. In conclusion, these findings demonstrated that CTRP6 knockdown inhibits HG-induced ROS production, inflammation and ECM accumulation in MCs, which were mediated by the inactivation of the Akt/NF-κB pathway. The roles of CTRP6 in diabetic nephropathy provided evidence for its therapeutic potential for the treatment of diabetic nephropathy.

Effects of Hirudin on High Glucose-Induced Oxidative Stress and Inflammatory Pathway in Rat Dorsal Root Ganglion Neurons.

To investigate protective effects of hirudin on oxidative stress and apoptosis of spinal dorsal root ganglion cells in high-glucose rats at the cellular and molecular level. Dorsal root ganglion neurons (DRGn) were harvested from embryonic day in 15 SD rats, purified and identificated after primary culture. They were divided into the normal control group, high-glucose (HG) group, positive control (alpha-lipoic acid, ALA) group, low-dose hirudin group (H1), medium-dose hirudin group (H2) and high-dose hirudin group (H3). The control group was cultured by neuron specific culture medium, while the HG group was cultured by neuron specific culture medium and 20 mmol/L glucose (HG medium). The hirudin groups were cultured by HG medium+0.25 IU/mL hirudin (H1), HG medium+0.5 IU/mL hirudin (H2) and HG medium+1 IU/mL hirudin (H3). The ALA group was cultured by HG medium+100 µ mol/L ALA. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenylt etrazolium bromide (MTT) assay was used to explore the optimum concentration and intervention time. Flow cytometry assay was used to detect the level of reactive oxygen series (ROS). Western blot and quantificational realtime polymerase chain reaction (qRT-PCR) were used to detect the expression of protein and mRNA of nuclear factor erythroid 2-related factor 2 (Nrf-2), hemeoxygence-1 (HO-1), nuclear factor-κ B (NF-κ B) and Caspase-3. TUNEL assay was used to test the apoptosis rate of different groups. After 24 h of culture, the cell activity of hirudin and ALA groups were higher than that of HG group, and there was a statistical difference between the H1 group and HG group (P<0.05). In hirudin groups, the apoptosis rate of cells, the expression of activated Caspase-3 protein and Caspase-3 mRNA were lower than those of HG group (P<0.01), higher than those of ALA group (P<0.01 or P<0.05). The ROS level of hirudin groups was higher than that of ALA group (P<0.01), lower than that of HG group (P<0.01 or P<0.05). The expression of NF-κ B (P65) protein in H3 group were lower than those of HG group (P<0.05). The expression of Nrf-2 protein in hirudin groups was higher than that of HG group (P<0.01), lower than that of ALA group (P<0.01 or P<0.05). The expression of HO-1 protein in hirudin groups was lower than that of ALA group (P<0.01 or P<0.05), higher than that of HG group (P<0.01 or P<0.05). The activity of DRGn cells can be promoted by hirudin under HG conditions. The effects of hirudin on the inhibition of HG on DRGn cells damage mainly include scavenging ROS, up-regulating Nrf-2/HO-1 pathway, inhibiting activation of NF-κ B pathway, down-regulating the expression of and Caspase-3 and reducing DRGn cell apoptosis.

DJ-1 protects retinal pericytes against high glucose-induced oxidative stress through the Nrf2 signaling pathway

Oxidative stress has been associated with the etipathogenesis of Diabetic retinopathy (DR). Studies have shown that DJ-1 plays an important role in regulating the reactive oxygen species (ROS) production and resistance to oxidative stress-induced apoptosis. This study aimed to investigate whether DJ-1 upregulates oxidative stress and prevents damage to retinal capillary pericytes by increasing antioxidant capacity through the Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Nrf2 is a redox-sensitive transcription factor that encode antioxidant enzymes and phase II metabolic enzymes, activation of Nrf2 functions is one of the critical defensive mechanisms against oxidative stress in many tissues. Our results showed after DJ-1 overexpression, apoptosis of rat retinal pericytes (RRPs) decreased, the ratio of B-cell lymphoma-2 (Bcl-2) to BCL2-Associated X Protein (BAX) increased, the production of ROS decreased, and the protein expression and activity of manganese superoxide dismutase (MnSOD, also called SOD2) and catalase (CAT) increased. DJ-1 overexpression activated Nrf2 expression, however, after Nrf2 silencing, apoptosis of RRPs increased, the ratio of Bcl-2 to BAX decreased, the production of ROS increased, the protein expression of MnSOD and CAT decreased, and the expression of heme oxygenase-1 (HO-1), NADP(H) quinone oxidoreductase (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) decreased. These data suggest that enhancement of the Nrf2 pathway is a potential protective strategy for the treatment of DR. Therefore, DJ-1 may prevent high glucose-induced oxidative stress and RRPs apoptosis through the Nrf2 signaling pathway, thereby preventing the early onset and progression of DR.

Glucose-induced oxidative stress and accelerated aging in endothelial cells are mediated by the depletion of mitochondrial SIRTs.

Diabetic complications cause significant morbidity and mortality. Dysfunction of vascular endothelial cells (ECs), caused by oxidative stress, is a main mechanism of cellular damage. Oxidative stress accelerates EC senescence and DNA damage. In this study, we examined the role of mitochondrial sirtuins (SIRTs) in glucose‐induced oxidative stress, EC senescence, and their regulation by miRNAs. Human retinal microvascular endothelial cells (HRECs) were exposed to 5 mmol/L (normoglycemia; NG) or 25 mmol/L glucose (hyperglycemia; HG) with or without transfection of miRNA antagomirs (miRNA‐1, miRNA‐19b, and miRNA‐320; specific SIRT‐targeting miRNAs). Expressions of SIRT3, 4 and 5 and their targeting miRNAs were examined using qRT‐PCR and ELISAs were used to study SIRT proteins. Cellular senescence was investigated using senescence‐associated β‐gal stain; while, oxidative stress and mitochondrial alterations were examined using 8‐OHdG staining and cytochrome B expressions, respectively. A streptozotocin‐induced diabetic mouse model was also used and animal retinas and hearts were collected at 2 months of diabetes. In HRECs, HG downregulated the mRNAs of SIRTs, while SIRT‐targeting miRNAs were upregulated. ELISA analyses confirmed such downregulation of SIRTs at the protein level. HG additionally caused early senescence, endothelial‐to‐mesenchymal transition and oxidative DNA damage in ECs. These changes were prevented by the transfection of specific miRNA antagomirs and by resveratrol. Retinal and cardiac tissues from diabetic mice also showed similar reductions of mitochondrial SIRTs. Collectively, these findings demonstrate a novel mechanism in which mitochondrial SIRTs regulate glucose‐induced cellular aging through oxidative stress and how these SIRTs are regulated by specific miRNAs. Identifying such mechanisms may lead to the discovery of novel treatments for diabetic complications.

Knockdown of GCN2 inhibits high glucose-induced oxidative stress and apoptosis in retinal pigment epithelial cells.

Increasing evidence suggests that general control nonderepressible 2 (GCN2) is a critical regulator of oxidative stress and cell apoptosis in response to various stimuli. However, the role of GCN2 in diabetic retinopathy remains unclear. The aim of the present study was to investigate the effects of GCN2 on oxidative stress and apoptosis in ARPE-19 cells exposed to high glucose. The results showed that GCN2 was highly expressed in high glucose-induced ARPE-19 cells. Moreover, knockdown of GCN2 greatly improved ARPE-19 cell viability in response to high glucose. In addition, GCN2 knockdown significantly suppressed the production of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as increased superoxide dismutase (SOD) activity in high glucose-stimulated ARPE-19 cells. Furthermore, GCN2 knockdown reduced cell apoptosis and enhanced the activation of nuclear factor E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway in high glucose-stimulated ARPE-19 cells. However, knockdown of Nrf2 reversed the effects of GCN2 on oxidative stress and cell apoptosis. Taken together, our findings suggest that knockdown of GCN2 inhibits high glucose-induced oxidative stress and apoptosis in ARPE-19 cells through activation of the Nrf2/HO-1 pathway.

Inhibition of NADPH Oxidase 5 (NOX5) Suppresses High Glucose-Induced Oxidative Stress, Inflammation and Extracellular Matrix Accumulation in Human Glomerular Mesangial Cells.

BackgroundThe aim of this study was to explore the effects of NADPH oxidase 5 (NOX5) in high glucose-stimulated human glomerular mesangial cells (HMCs).Material/MethodsCells were cultured under normal glucose (NG) or high glucose (HG) conditions. Then, NOX5 siRNA was transfected into HG-treated HMCs. A Cell Counting Kit-8 assay, colony formation assay and 5-ethynyl-20-deoxyuridine (EDU) incorporation assay were applied to measure cell proliferative ability. In addition, the levels of oxidative stress factors including reactive oxygen species (ROS), malonaldehyde (MDA), NADPH, superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX), inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1β, and monocyte chemoattractant protein-1 (MCP-1) in HMCs were detected by kits. Moreover, the expression of TLR4/NF-κB signaling and extracellular matrix (ECM) associated genes were evaluated by western blotting.ResultsThe results revealed that the NOX5 was overexpressed in HG-treated HMCs. Silencing of NOX5 decreased proliferation of HMCs induced by HG. And NOX5 silencing alleviated the production of MDA and NADPH accompanied by an increase of SOD and GSH-PX levels. Additionally, the contents of TNF-α, IL-6, IL-1β, and MCP-1 were reduced after transfection with NOX5 siRNA. Furthermore, silencing of NOX5 deceased the expression of collagen I, collagen IV, TGF-β1, and fibronectin induced by HG stimulation. TLR4, MyD88, and phospho-NF-κB p65 expression were downregulated notably in NOX5 silencing group.ConclusionsTaken together, these findings demonstrated that inhibition of NOX5 attenuated HG-induced HMCs oxidative stress, inflammation, and ECM accumulation, suggesting that NOX5 may serve as a potential therapeutic target for diabetic nephropathy (DN) treatment.

Phelligridin D maintains the function of periodontal ligament cells through autophagy in glucose-induced oxidative stress.

PurposeThe objective of this study was to investigate whether phelligridin D could reduce glucose-induced oxidative stress, attenuate the resulting inflammatory response, and restore the function of human periodontal ligament cells (HPDLCs).MethodsPrimary HPDLCs were isolated from healthy human teeth and cultured. To investigate the effect of phelligridin D on glucose-induced oxidative stress, HPDLCs were treated with phelligridin D, various concentrations of glucose, and glucose oxidase. Glucose-induced oxidative stress, inflammatory molecules, osteoblast differentiation, and mineralization of the HPDLCs were measured by hydrogen peroxide (H2O2) generation, cellular viability, alkaline phosphatase (ALP) activity, alizarin red staining, and western blot analyses.ResultsGlucose-induced oxidative stress led to increased production of H2O2, with negative impacts on cellular viability, ALP activity, and calcium deposition in HPDLCs. Furthermore, HPDLCs under glucose-induced oxidative stress showed induction of inflammatory molecules (intercellular adhesion molecule-1, vascular cell adhesion protein-1, tumor necrosis factor-alpha, interleukin-1-beta) and disturbances of osteogenic differentiation (bone morphogenetic protein-2, and -7, runt-related transcription factor-2), cementogenesis (cementum protein-1), and autophagy-related molecules (autophagy related 5, light chain 3 I/II, beclin-1). Phelligridin D restored all these molecules and maintained the function of HPDLCs even under glucose-induced oxidative stress.ConclusionsThis study suggests that phelligridin D reduces the inflammation that results from glucose-induced oxidative stress and restores the function of HPDLCs (e.g., osteoblast differentiation) by upregulating autophagy.

Peptides from walnut (Juglans mandshurica Maxim.) protect hepatic HepG2 cells from high glucose-induced insulin resistance and oxidative stress

Oxidative stress is an important factor in the pathogenesis of insulin resistance (IR) in type 2 diabetes mellitus (T2DM). Bioactive peptides from nuts have been shown to alleviate oxidative stress and IR. However, the specific mechanisms underlying their activity remain unclear. This study investigated the protective effects of three novel peptides derived from Juglans mandshurica Maxim., LVRL, LRYL, and VLLALVLLR, against high glucose-induced IR and oxidative stress in HepG2 cells. The possible mechanisms underlying these effects were also elucidated. The walnut-derived peptides improved glucose consumption, glucose uptake, and glucose transporter type 4 (GLUT4) translocation, and elevated the phosphorylation of insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (Akt). They also increased the activities of glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD), the nuclear transport of nuclear factor E2-related factor 2 (Nrf2), and the protein expression of heme oxygenase-1 (HO-1). Furthermore, the walnut-derived peptides reduced high glucose-induced ROS overproduction and the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. These results suggested that walnut-derived peptides protect HepG2 cells from high glucose-induced IR and oxidative stress by activating IRS-1/PI3 K/Akt and Nrf2/HO-1 signaling pathways.

Inhibition of miR-383 suppresses oxidative stress and improves endothelial function by increasing sirtuin 1.

Previous research has shown that suppression of miR-383 can prevent inflammation of the endothelium, as well as postpone the development of atherosclerosis. However, the role of miR-383 in endothelial cell apoptosis in diabetes remains unclear. The aim of this study was to investigate the function of miR-383 in high glucose-induced apoptosis and oxidative stress in endothelial cells. A series of experiments involving qualitative polymerase chain reaction, cell transfection, luciferase assay, assessment of cell death, detection of catalase and superoxide dismutase concentrations, detection of intracellular reactive oxygen species (ROS), and western blot analysis were performed in this study. We found that miR-383 expression was promoted, while NAD+-dependent deacetylase and sirtuin 1 (SIRT1) expressions were suppressed in the endothelium of the aorta in db/db mice as well as in human umbilical vein endothelial cells, which were treated with high glucose (HG). Increased expression of miR-383 decreased expression of SIRT1, while suppression of miR-383 promoted expression of SIRT1 in human umbilical vein endothelial cells (HUVECs). Furthermore, suppression of miR-383 following transfection with miR-383 suppressor repressed cell death and generation of ROS in HUVECs. SIRT1 knockdown by siRNA-SIRT1 reversed the suppressive effect of miR-383 inhibition on ROS production and cell apoptosis induced by HG treatment. Overall, the findings of our research suggested that suppression of miR-383 repressed oxidative stress and reinforced the activity of endothelial cells by upregulation of SIRT1 in db/db mice, and targeting miR-383 might be promising for effective treatment of diabetes.

Wheat germ-derived peptide ADWGGPLPH abolishes high glucose-induced oxidative stress via modulation of the PKCζ/AMPK/NOX4 pathway.

This study explores the antioxidative effect of a specific wheat germ-derived peptide on high glucose-induced oxidative stress in vascular smooth muscle cells (VSMCs) and the underlying mechanisms. The peptide ADWGGPLPH was identified by LC-MS/MS. The effects of this peptide on the production of ROS and the expression of oxidative stress signaling proteins in VSMCs were determined. STZ-induced mice were utilized to confirm the anti-oxidative and anti-diabetic cardiovascular disease effects of this peptide in vivo. The results showed that ADWGGPLPH significantly prevented high glucose-induced cell proliferation, decreased intracellular ROS generation, stimulated AMPK activity, inhibited the PKCζ, AKT and Erk1/2 phosphorylation, and suppressed NOX4 protein expression. In addition, ADWGGPLPH enhanced the antioxidant abilities and attenuated inflammatory cytokine generation in STZ-induced diabetic mice. Therefore, ADWGGPLPH prevents high glucose-induced oxidative stress in VSMCs by modulating the PKCζ/AMPK/NOX4 pathway.

Evaluation of apocynin in vitro on high glucose-induced oxidative stress on tenocytes.

AimsThe purpose of this study was to evaluate the in vitro effects of apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase (NOX) and a downregulator of intracellular reactive oxygen species (ROS), on high glucose-induced oxidative stress on tenocytes.MethodsTenocytes from normal Sprague-Dawley rats were cultured in both control and high-glucose conditions. Apocynin was added at cell seeding, dividing the tenocytes into four groups: the control group; regular glucose with apocynin (RG apo+); high glucose with apocynin (HG apo+); and high glucose without apocynin (HG apo–). Reactive oxygen species production, cell proliferation, apoptosis and messenger RNA (mRNA) expression of NOX1 and 4, and interleukin-6 (IL-6) were determined in vitro.ResultsExpression of NOX1, NOX4, and IL-6 mRNA in the HG groups was significantly higher compared with that in the RG groups, and NOX1, NOX4, and IL-6 mRNA expression in the HG apo+ group was significantly lower compared with that in the HG apo– group. Cell proliferation in the RG apo+ group was significantly higher than in the control group and was also significantly higher in the HG apo+ group than in the HG apo– group. Both the ROS accumulation and the amounts of apoptotic cells in the HG groups were greater than those in the RG groups and were significantly less in the HG apo+ group than in the HG apo– group.ConclusionApocynin reduced ROS production and cell death via NOX inhibition in high-glucose conditions. Apocynin is therefore a potential prodrug in the treatment of diabetic tendinopathy.Cite this article: Bone Joint Res 2020;9(1):23–28.

Empagliflozin reduces high glucose-induced oxidative stress and miR-21-dependent TRAF3IP2 induction and RECK suppression, and inhibits human renal proximal tubular epithelial cell migration and epithelial-to-mesenchymal transition

Proximal tubular epithelial cells (PTEC) in the S1 segment of the kidney abundantly express sodium-glucose co-transporters (SGLT) that play a critical role in whole body glucose homeostasis. We recently reported suppression of RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane anchored endogenous MMP inhibitor and anti-fibrotic mediator, in the kidneys of db/db mice, a model of diabetic kidney disease (DKD), as well as in high glucose (HG) treated human kidney proximal tubule cells (HK−2). We further demonstrated that empagliflozin (EMPA), an SGLT2 inhibitor, reversed these effects. Little is known regarding the mechanisms underlying RECK suppression under hyperglycemic conditions, and its rescue by EMPA. Consistent with our previous studies, HG (25 mM) suppressed RECK expression in HK-2 cells. Further mechanistic investigations revealed that HG induced superoxide and hydrogen peroxide generation, oxidative stress-dependent TRAF3IP2 upregulation, NF-κB and p38 MAPK activation, inflammatory cytokine expression (IL-1β, IL-6, TNF-α, and MCP-1), miR-21 induction, MMP2 activation, and RECK suppression. Moreover, RECK gain-of-function inhibited HG-induced MMP2 activation and HK-2 cell migration. Similar to HG, advanced glycation end products (AGE) induced TRAF3IP2 and suppressed RECK, effects that were inhibited by EMPA. Importantly, EMPA treatment ameliorated all of these deleterious effects, and inhibited epithelial-to-mesenchymal transition (EMT) and HK-2 cell migration. Collectively, these findings indicate that hyperglycemia and associated AGE suppress RECK expression via oxidative stress/TRAF3IP2/NF-κB and p38 MAPK/miR-21 induction. Furthermore, these results suggest that interventions aimed at restoring RECK or inhibiting SGLT2 have the potential to treat kidney inflammatory response/fibrosis and nephropathy under chronic hyperglycemic conditions, such as DKD.

Effect of High Glucose-Induced Oxidative Stress on Paraoxonase 2 Expression and Activity in Caco-2 Cells.

(1) Background: Hyperglycemia leads to several biochemical and physiological consequences, such as the generation of advanced glycation end products (AGEs) and reactive oxygen species (ROS), which are involved in the development of several human diseases. Intestinal cells are continuously exposed to pro-oxidants and lipid peroxidation products from ingested foods, and also to glyco-oxidative damage. It has been reported that free radical generation may be linked to the development of inflammation-related gastrointestinal diseases. (2) Methods: The effects of high glucose (HG) treatment (50 mM) were assessed in terms of free radical production, lipid peroxidation, and AGEs formation. Furthermore, the expression and the antiapoptotic and antioxidant activity of the paraoxonase-2 (PON2) enzyme in intestinal cells has been investigated. (3) Results: Caco-2 cells treated with media supplied with high glucose (HG) (50 mM) showed, with respect to physiological glucose concentration (25 mM), an increase in ROS production, lipid peroxidation, and AGEs formation. Moreover, a lower PON2 expression and activity in HG-treated cells was related to activation of the apoptotic pathways. (4) Conclusions: Our results demonstrated that high glucose concentrations triggered glyco-oxidative stress in intestinal cells; the downregulation of PON2 could result in a higher oxidative stress and might contribute to intestinal dysfunction.