High Glucose-induced Cell Damage Research Articles

NADPH-dependent ROS accumulation contributes to the impaired osteogenic differentiation of periodontal ligament stem cells under high glucose conditions.

Diabetes mellitus is an established risk factor for periodontal disease that can aggravate the severity of periodontal inflammation and accelerate periodontal destruction. The chronic high glucose condition is a hallmark of diabetes-related pathogenesis, and has been demonstrated to impair the osteogenic differentiation of periodontal ligament stem cells (PDLSCs), leading to delayed recovery of periodontal defects in diabetic patients. Reactive oxygen species (ROS) are small molecules that can influence cell fate determination and the direction of cell differentiation. Although excessive accumulation of ROS has been found to be associated with high glucose-induced cell damage, the underlying mechanisms remain unclear. Nicotinamide adenine dinucleotide phosphate (NADPH) is an important electron donor and functions as a critical ROS scavenger in antioxidant systems. It has been identified as a key mediator of various biological processes, including energy metabolism and cell differentiation. However, whether NADPH is involved in the dysregulation of ROS and further compromise of PDLSC osteogenic differentiation under high glucose conditions is still not known. In the present study, we found that PDLSCs incubated under high glucose conditions showed impaired osteogenic differentiation, excessive ROS accumulation and increased NADPH production. Furthermore, after inhibiting the synthesis of NADPH, the osteogenic differentiation of PDLSCs was significantly enhanced, accompanied by reduced cellular ROS accumulation. Our findings demonstrated the crucial role of NADPH in regulating cellular osteogenic differentiation under high glucose conditions and suggested a new target for rescuing high glucose-induced cell dysfunction and promoting tissue regeneration in the future.

Dietary prenylated flavonoid xanthohumol alleviates oxidative damage and accelerates diabetic wound healing via Nrf2 activation

Diabetic skin ulcer is one of the most common complications in patients suffering diabetes mellitus. Xanthohumol (XN), a hop-derived prenylated dietary flavonoid, has multiple health beneficial bioactivities. In the present study, we reported XN alleviates oxidative damage and accelerates diabetic wound healing via Nrf2 activation. In vitro, XN attenuated hydrogen peroxide (H2O2)-induced cytotoxicity, ROS production, cell apoptosis, as well as high glucose-induced cell damage. Mechanistic studies further demonstrated that XN could stabilize nuclear factor erythroid 2-related factor 2 (Nrf2) and promote its nuclear translocation, which was associated with AMPKα activation and covalent modification of Keap1 by XN. In vivo, XN increased Nrf2 expression and accelerated diabetic wound healing. Our study revealed a novel function of XN in diabetic wound healing as well as the underlying molecular mechanisms, suggesting XN is a promising lead compound and a potential food and/or drug candidate for the treatment of diabetic skin ulcers.

Inhibitory effects of Lentinus edodes mycelia polysaccharide on α-glucosidase, glycation activity and high glucose-induced cell damage

At present, diabetes and diabetic complications have become one of the serious diseases affecting human health. In this study, the inhibitory effects of Lentinus edodes mycelia polysaccharide (LMP) on α-glucosidase activity, the formation of advanced glycation end products (AGEs) and high glucose-induced human umbilical vein endothelial cells (HUVECs) damage were explored. The interaction between LMP and α-glucosidase and the inhibition against AGEs formation were investigated with spectroscopic techniques. The results revealed that LMP had a reversible inhibition on α-glucosidase activity in a mixed-type manner. When the concentration of LMP was 2.7 mM, the inhibition rate was 34.38 %. LMP quenched the fluorescence of α-glucosidase through the static quenching and formed the LMP-α-glucosidase complex. At 310 K, the number of binding sites (n) and binding constant (Kb) were 1.01 and 3.71 × 104 L mol−1, respectively. In addition, LMP could inhibit the formation of AGEs. Compared with 40 mM glucose treatment group, treatment with 0.05 mM LMP for 48 h increased the cell viability from 70.17% to 91.14% and decreased ROS production from 3.33-fold to 1.21-fold. LMP inhibited high glucose-induced activation of MAPK signaling pathways. These findings may promote the application of LMP in the functional food industry.

Ginsenoside Rb1 Attenuates High Glucose-Induced Oxidative Injury via the NAD-PARP-SIRT Axis in Rat Retinal Capillary Endothelial Cells.

(1) Aims: The present study aimed to observe the effects of Ginsenoside Rb1 on high glucose-induced endothelial damage in rat retinal capillary endothelial cells (RCECs) and to investigate the underlying mechanism. (2) Methods: Cultured RCECs were treated with normal glucose (5.5 mM), high glucose (30 mM glucose), or high glucose plus Rb1 (20 μM). Cell viability, lactate dehydrogenase (LDH) levels, the mitochondrial DNA copy number, and the intracellular ROS content were measured to evaluate the cytotoxicity. Superoxide dismutase (SOD), catalase (CAT), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), poly(ADP-ribose) polymerase (PARP), and sirtuin (SIRT) activity was studied in cell extracts. Nicotinamide adenine dinucleotide (NAD+)/NADH, NADPH/NADP+, and glutathione (GSH)/GSSG levels were measured to evaluate the redox state. The expression of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), SIRT1, and SIRT3 was also evaluated after Rb1 treatment. (3) Results: Treatment with Rb1 significantly increased the cell viability and mtDNA copy number, and inhibited ROS generation. Rb1 treatment increased the activity of SOD and CAT and reduced the activity of NOX and PARP. Moreover, Rb1 enhanced both SIRT activity and SIRT1/SIRT3 expression. Additionally, Rb1 was able to re-establish the cellular redox balance in RCECs. However, Rb1 showed no effect on NMNAT1 expression in RCECs exposed to high glucose. (4) Conclusion: Under high glucose conditions, decreases in the reducing power may be linked to DNA oxidative damage and apoptosis via activation of the NMNAT-NAD-PARP-SIRT axis. Rb1 provides an advantage during high glucose-induced cell damage by targeting the NAD-PARP-SIRT signaling pathway and modulating the redox state in RCECs.

Elevated Fibroblast Growth Factor-21 Protect Endothelial Function in Type 2 Diabetes Patients

Background: Fibroblast growth factor 21 (FGF-21) is a metabolic regulator with multiple effects on improving glucose metabolism and energy utilization. Recent researches pointed that FGF-21 protect endothelial function against high glucose induced cell damage. Our research tried to detect the relationship of FGF-21 and endothelial dysfunction in newly diagnosed type 2 diabetes (T2D) patients. Methods: Fifty newly diagnosed T2D patients and fifty healthy controls were recruited in the research. Clinical and biological data, FGF-21 were measured. Endothelial function was tested by reactive hyperaemia index (RHI) approved by American Food and Drug Administration. Results: T2DM patients had higher FGF-21 levels (155.57±85.3 vs. 117.39±68.9ug/l,P<0.05) and more serious endothelial dysfunction (1.73±0.51 vs. 2.05±0.52,P<0.05) than controls. RHI was negatively related with low density lipoprotein cholesterol (LDL-C) (r=-0.322, P<0.05) and positively with FGF-21 (r=0.543, P<0.01). In the multivariate linear analysis, FGF-21 (β=0.551,P<0.01) and LDL-C (β=-0.188,P<0.01) were independently predictors of RHI. FGF-21 was the protective factor of endothelial function in the T2D patients. Conclusions: T2D patients had endothelial dysfunction even in the early period. Elevated FGF-21 might protect endothelial dysfunction. Disclosure Y. Hu: None. Y. Xu: None. G. Wang: None.

Ginsenoside Rb1 prevents high glucose-induced Schwann cell injury through the mitochondrial apoptosis pathway

ObjectiveTo investigate the effects of ginsenoside Rb1 on high glucose-induced neurotoxicity and the underlying molecular mechanism in primary cultured Schwann cells (SCs). MethodsCultured SCs were divided into six groups that received (a) normal glucose, (b) osmotic control, (c) high glucose, (d) high glucose plus 1 μM ginsenoside Rb1, (e) high glucose plus 10 μM ginsenoside Rb1, or (f) high glucose plus 100 μM alpha lipoic acid (ALA). Intracellular reactive oxygen species (ROS) generation and mitochondrial transmembrane potential (ΔΨm) were detected by flow cytometric analyses. Apoptosis was confirmed by the annexin V-FITC/propidium iodide (PI) method, and the concentration of 8-hydroxy-2-deoxy guanosine (8-OHdG) was detected by an enzyme-linked immunosorbent assay. Western blotting was performed to analyze the expression levels of important transcription factors such as cytochrome c, bcl-2, bax, activated caspase-3, and activated poly (ADP-ribose) polymerase (PARP). ResultsGinsenoside Rb1 inhibited high glucose-induced oxidative stress by decreasing ROS and 8-OHdG levels as well as mitochondrial depolarization in SCs. 3-(4,5)-dimethylthiahiazo(-z-y1)-3, 5-di-phenytetrazoliumromide and annexin V-FITC/PI assays showed that incubating SCs with high glucose decreased cell viability and increased the number of apoptotic cells, whereas treatment with ginsenoside Rb1 protected SCs against high glucose-induced cell damage. Furthermore, ginsenoside Rb1 down-regulated the expression of high glucose-induced bax and cytochrome c release but up-regulated bcl-2 expression. In addition, ginsenoside Rb1 attenuated high glucose-induced activation of caspase-3 and minimized cleavage of PARP in SCs. ConclusionThese results suggest that ginsenoside Rb1 antagonizes high glucose-induced oxidative stress and activation of the mitochondrial apoptosis pathway in SCs.

Notoginsenoside R1 attenuates high glucose-induced endothelial damage in rat retinal capillary endothelial cells by modulating the intracellular redox state.

The aim of this study was to examine whether Notoginsenoside R1 (NR1) attenuates high glucose-induced cell damage in rat retinal capillary endothelial cells (RCECs) and to explore the mechanisms involved. The exposure of rat RCECs to high concentration of glucose (30 mM) for 72 h led to significant cytotoxicity, including decreased cell viability, reduced mitochondrial DNA copy number, increased lactate dehydrogenase release and elevated apoptosis. NR1, when present in the culture medium, markedly attenuated the high glucose-induced cytotoxicity in rat RCECs. Moreover, high glucose also induced a significant increase in intracellular reactive oxygen species and subsequently increased the activity of NADPH oxidase and poly-ADP (ribose) polymerase, whereas the activity of catalase decreased. The addition of NR1 to the medium significantly reduced the generation of reactive oxygen species, inhibited NADPH oxidase and poly-ADP (ribose) polymerase activities and increased catalase activity in RCECs, accompanied by a reduced cellular nitrotyrosine level. To explore the underlying mechanisms involved, the cellular redox status was monitored. Both the cellular NAD+ and NADPH levels decreased significantly in high glucose medium, which resulted in a marked decrease in the NAD+/NADH and NADPH/NADP+ ratios. High glucose stimulation also enhanced the accumulation of GSSG, maintaining the GSH/GSSG ratio lower than that in the control group with 5.5 mM glucose. When treated with NR1, the cellular NAD+, NADPH and GSH concentrations increased, and the ratios of NAD+/NADH, NADPH/NADP+ and GSH/GSSG increased, similar to the control group. These results demonstrate that NR1 attenuates high glucose-induced cell damage in RCECs. Therefore, NR1 may exert its protective effects via mechanisms that involve changes in the cellular redox state.

Sugar Composition Analysis of Fuzi Polysaccharides by HPLC-MSn and Their Protective Effects on Schwann Cells Exposed to High Glucose

Fuzi has been used to treat diabetic complications for many years in china. In a previous study, we have shown that Fuzi aqueous extract can attenuate Diabetic peripheral neuropathy (DPN) in rats and protect Schwann cells from injury. Thus, the protective effect of Fuzi polysaccharides (FPS) on high glucose-induced SCs and the preliminary mechanism were investigated. Firstly, the FPS were obtained and their monose composition was analyzed by the combination of pre-column derivatization and high performance liquid chromatography coupled with electrospray ionization multi-tandem mass spectrometry (HPLC/ESI-MSn). The results witnessed the efficiency of this method and seven monosaccharides were tentatively identified, among which fucose was first reported. Simultaneously, m/z 215 can be considered as diagnostic ions to confirm the number of monosaccharides. Next, high glucose-induced SC model was applied and divided into model group, treated group of FPS, normal and osmotic control group. After treatment for 48 h, the data showed FPS could significantly decrease the intracellular ROS and apoptosis, which were determined by the corresponding fluorescent probes. Then, the expression of oxidative stress-related proteins in SCs were measured by Western blot. Furthermore, the protein tests found that FPS markedly up-regulated superoxide dismutase (SOD), catalase (CAT) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein level, but down-regulated NADPH oxidase-1 (Nox1) protein level. Moreover, FPS could also increase AMP-activated protein kinase (AMPK) activation significantly. Hence, we preliminary deduced that AMPK-PGC-1α pathway may play an important role in the protective effect of FPS against high glucose-induced cell damage.

Aflibercept, bevacizumab and ranibizumab prevent glucose-induced damage in human retinal pericytes in vitro, through a PLA2/COX-2/VEGF-A pathway

Diabetic retinopathy, a major cause of vision loss, is currently treated with anti-VEGF agents. Here we tested two hypotheses: (i) high glucose damages retinal pericytes, the cell layer surrounding endothelial cells, via VEGF induction, which may be counteracted by anti-VEGFs and (ii) activation of PLA2/COX-2 pathway by high glucose might be upstream and/or downstream of VEGF in perycites, as previously observed in endothelial cells. Human retinal pericytes were treated with high glucose (25mM) for 48h and/or anti-VEGFs (40μg/ml aflibercept, 25μg/ml bevacizumab, 10μg/ml ranibizumab). All anti-VEGFs significantly prevented high glucose-induced cell damage (assessed by LDH release) and improved cell viability (assessed by MTT and Evans blue). High glucose-induced VEGF-A expression, as detected both at mRNA (qPCR) and protein (ELISA) level, while receptor (VEGFR1 and VEGFR2) expression, detected in control condition, was unaffected by treatments. High glucose induced also activation of PLA2/COX-2 pathway, as revealed by increased phosphorylation of cPLA2, COX-2 expression and PGE2 release. Treatment with cPLA2 (50μM AACOCF3) and COX-2 (5μM NS-392) inhibitors prevented both cell damage and VEGF-A induced by high glucose. Finally, challenge with exogenous VEGF-A (10ng/ml) induced VEGF-A expression, while anti-VEGFs reduced VEGF-A expression induced by either high glucose or exogenous VEGF-A. These data indicate that high glucose directly damages pericytes through activation of PLA2/COX-2/VEGF-A pathway. Furthermore, a kind of feed-forward loop between cPLA2/COX-2/PG axis and VEGF appears to operate in this system. Thus, anti-VEGFs afford protection of pericytes from high glucose by inhibiting this loop.

Pyrroloquinoline quinone protects mouse brain endothelial cells from high glucose-induced damage in vitro.

To investigate the effects of pyrroloquinoline quinone (PQQ), an oxidoreductase cofactor, on high glucose-induced mouse endothelial cell damage in vitro. Mouse brain microvascular endothelial bEND.3 cells were exposed to different glucose concentrations (5.56, 25 and 40 mmol/L) for 24 or 48 h. The cell viability was examined using MTT assay. Flow cytometry was used to analyze the apoptosis and ROS levels in the cells. MitoTracker Green staining was used to examine the mitochondria numbers in the cells. Western blot analysis was used to analyze the expression of HIF-1α and the proteins in JNK pathway. Treatment of bEND.3 cells with high glucose significantly decreased the cell viability, while addition of PQQ (1 and 10 μmol/L) reversed the high glucose-induced cell damage in a concentration-dependent manner. Furthermore, PQQ (100 μmol/L) significantly suppressed the high glucose-induced apoptosis and ROS production in the cells. PQQ significantly reversed the high glucose-induced reduction in both the mitochondrial membrane potential and mitochondria number in the cells. The high glucose treatment significantly increased the expression of HIF-1α and JNK phosphorylation in the cells, and addition of PQQ led to a further increase of HIF-1α level and a decrease of JNK phosphorylation. Addition of JNK inhibitor SP600125 (10 μmol/L) also significantly suppressed high glucose-induced apoptosis and JNK phosphorylation in bEND.3 cells. PQQ protects mouse brain endothelial cells from high glucose damage in vitro by suppressing intracellular ROS and apoptosis via inhibiting JNK signaling pathway.

Dihydromyricetin attenuates high glucose‐induced oxidative stress in human umbilical vascular endothelial cells (1146.2)

Objective To investigate the effects of Dihydromyricetin on high glucose‐induced cell damage in human umbilical vascular endothelial cells (HUVECs).Methods HUVECs were cultured in 5.56 mmol/L glucose as normal control, 5.56mmol/L glucose plus 27.8mmol/L mannitol as an osmotic control, or 33.36 mmol/L high glucose for 48 hours in the absence and presence of Dihydromyricetin, cell viability and apoptosis were detected by CCK‐8 assays and annexin V‐FITC/PI double staining using flow cytometry, respectively. In addition, the intracellular reactive oxygen species (ROS), malondialdehyde (MDA) content and the activity of superoxide dismutase (SOD) and catalase (CAT) in the culture supernatants were measured.Results High concentration of glucose significantly decreased cell viability and the activity of SOD and CAT, while increased the apoptotic rate, the intracellular ROS and the MDA content in HUVECs (P<0.05). Dihydromyricetin pretreatment significantly inhibited the decrease in cell viability and the increase in cell apoptosis induced by high glucose. The ROS and MDA levels were reduced while the SOD and CAT levels were increased after pretreatment with Dihydromyricetin following high glucose stimulation.Conclusion Dihydromyricetin attenuated high glucose‐induced oxidative stress in HUVECs.Grant Funding Source: Supported by The National Natural Science Foundation of China (81102129)

Ginsenoside Rb1 protects hippocampal neurons from high glucose-induced neurotoxicity by inhibiting GSK3β-mediated CHOP induction

Ginsenoside Rb1 is generally recognized as one of the principal bioactive ingredients in ginseng and shows neuroprotective effects in various neurons. Endoplasmic reticulum (ER) stress is considered to play an important role in numerous neurodegenerative disorders. Recently, glucogen synthase kinase 3β (GSK3β) was reported to regulate ER stress-induced C/EBP homologous protein (CHOP) in neuronal cells. Therefore, in this study, we investigated the effects of ginsenoside Rb1 on GSK3β-mediated ER stress in high glucose-treated hippocampal neurons. Results from the MTT assay showed that treatment with 1 µM Rb1 for 72 h protected neurons from high glucose-induced cell injury. Using western blot analysis, we found that treatment with Rb1 effectively inhibited the phosphorylation of the high glucose-induced protein kinase RNA-like ER kinase (PERK) and of GSK3β, and reduced the level of the CHOP protein. The levels of these proteins were also decreased by treatment with the GSK3β inhibitor Licl. Rb1 also significantly decreased the mRNA expression of the gene CHOP, as shown by quantitative RT-PCR analysis. Taken together, the present results suggested that Rb1 may protect neurons from high glucose-induced cell damage by inhibiting GSK3β‑mediated CHOP induction, providing a potentially new strategy for preventing and treating cognitive impairment caused by diabetes.

Fibroblast Growth Factor 21 Protects Against High Glucose Induced Cellular Damage and Dysfunction of Endothelial Nitric-Oxide Synthase in Endothelial Cells

Aims: Fibroblast growth factor 21 (FGF21) is a powerful endocrine hormone modulating glucose and lipid metabolism and represents a promising drug for type 2 diabetes. The present study was to determine the effect of FGF21 on high glucose-induced damage and dysfunction in endothelial cells. Methods: The protein expression of β-klotho was examined in human umbilical vein endothelial cell (HUVECs) using immunofluorescence and Western blotting. HUVECs were cultured in medium with normal glucose (NG), high glucose (HG) and HG + FGF21 (30 nM). Cell viability, migration, reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase, nitric oxide (NO) production, intracellular cyclic guanosine monophosphate (cGMP) and endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177/Ser-633 sites were measured. Results: β-klotho, the anchor protein of FGF21, is expressed in HUVECs. Administration of FGF21 prevented HG-induced impairment of cell viability, migration, oxidant stress, NO production and intracellular cGMP levels in HUVECs. FGF21 also rescued HG-induced decrease of eNOS phosphorylation at Ser-1177 and Ser-633. HG and FGF21 had no effects on eNOS phosphorylation at Ser-617 and Thr-495. Inhibition of AMP-activated protein kinase (AMPK), but not Akt or Ca²⁺/calmodulin-dependent protein kinase II, abolished the protective effect of FGF21 on eNOS phosphorylation at Ser-1177. The protective effect of FGF21 on eNOS phosphorylation at Ser-633 was also abolished by inhibition of AMPK but not by Akt or cAMP-dependent protein kinase A. Conclusion: Our results provide the first evidence that FGF21 protects against high glucose induced cell damage and eNOS dysfunction in an AMPK-dependent manner in HUVECs, and suggest that FGF21 may be a promoting therapeutic agent for vascular complications in diabetes.

Neuroprotective Effects of Ginsenoside Rb1 on High Glucose-Induced Neurotoxicity in Primary Cultured Rat Hippocampal Neurons

Ginsenoside Rb1 is one of the main active principles in traditional herb ginseng and has been reported to have a wide variety of neuroprotective effects. Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, so the present study aimed to observe the effects of ginsenoside Rb1 on ER stress signaling pathways in high glucose-treated hippocampal neurons. The results from MTT, TUNEL labeling and Annexin V-FITC/PI/Hoechst assays showed that incubating neurons with 50 mM high glucose for 72h decreased cell viability and increased the number of apoptotic cells whereas treating neurons with 1 μM Rb1 for 72h protected the neurons against high glucose-induced cell damage. Further molecular mechanism study demonstrated that Rb1 suppressed the activation of ER stress-associated proteins including protein kinase RNA (PKR)-like ER kinase (PERK) and C/EBP homology protein (CHOP) and downregulation of Bcl-2 induced by high glucose. Moreover, Rb1 inhibited both the elevation of intracellular reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential induced by high glucose. In addition, the high glucose-induced cell apoptosis, activation of ER stress, ROS accumulation and mitochondrial dysfunction can also be attenuated by the inhibitor of ER stress 4-phenylbutyric acid (4-PBA) and anti-oxidant N-acetylcysteine(NAC). In conclusion, these results suggest that Rb1 may protect neurons against high glucose-induced cell injury through inhibiting CHOP signaling pathway as well as oxidative stress and mitochondrial dysfunction.

Olive (Olea europaea L.) leaf extract attenuates early diabetic neuropathic pain through prevention of high glucose-induced apoptosis: In vitro and in vivo studies

Since the leaves of olive have been recommended in the literature as a remedy for the treatment of diabetes and they also contain antioxidant agents, we decided to investigate the possible effects of olive leaf extract (OLE) on in vitro and in vivo models of diabetic pain neuropathy. The high glucose-induced cell damage in naive and NGF-treated Pheochromocytoma (PC12) cells and streptozotocin-induced diabetic rats were used. Tail-flick test was used to access nociceptive threshold. Cell viability was determined by MTT assay. Biochemical markers of neural apoptosis were evaluated using immunoblotting. We found that elevation of glucose (4 times of normal) sequentially increases functional cell damage and caspase-3 activation in NGF-treated PC12 cells. Incubation of cells with OLE (200, 400 and 600 μg/ml) decreased cell damage. Furthermore, the diabetic rats developed neuropathic pain which was evident from decreased tail-flick latency (thermal hyperalgesia). Activated caspase 3 and Bax/Bcl2 ratio were significantly increased in spinal cord of diabetic animals. OLE treatment (300 and 500 mg/kg per day) ameliorated hyperalgesia, inhibited caspase 3 activation and decreased Bax/Bcl2 ratio. Furthermore, OLE exhibited potent DPPH free radical scavenging capacity. The results suggest that olive leaf extract inhibits high glucose-induced neural damage and suppresses diabetes-induced thermal hyperalgesia. The mechanisms of these effects may be due, at least in part, to reduce neuronal apoptosis and suggest therapeutic potential of olive leaf extract in attenuation of diabetic neuropathic pain.