Epalrestat suppresses cadmium-induced cytotoxicity through Nrf2 in endothelial cells.

Cadmium (Cd) is an industrial and environmental pollutant that targets the vascular endothelium. The vascular system is critically affected by Cd toxicity. Recent studies have indicated an association between Cd and vascular diseases, although the mechanisms of Cd implications in vascular diseases are not clear. The purpose of the present study was to determine whether epalrestat (EPS), which is used for the treatment of diabetic neuropathy, protects against Cd-induced cytotoxicity in bovine aortic endothelial cells (BAECs). In the present study, the effects of EPS at near-plasma concentration were examined on Cd-induced cytotoxicity in BAECs. Cd-induced cytotoxicity was suppressed by pretreatment with EPS. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that serves a role in regulating the expression of glutamate cysteine ligase, the rate-limiting enzyme in glutathione (GSH) synthesis. In a previous study, EPS was demonstrated to increase GSH levels in BAECs in association with the Nrf2 pathway. In the present study, EPS increased GSH levels in BAECs exposed to Cd. The protective ability of EPS against the Cd-induced cytotoxicity disappeared following Nrf2 small interfering RNA transfection. In addition, EPS affected the intracellular levels of Cd, Cd transporter ZIP8 and metallothionein. To the best of our knowledge, the current study demonstrated, for the first time, that EPS suppresses Cd-induced cytotoxicity in BAECs. The upregulation of GSH may be associated with the suppression of Cd-induced cytotoxicity by EPS. From these findings, it may be proposed that the regulation of GSH, ZIP8 and metallothionein by EPS is a promising therapeutic approach to prevent Cd-induced toxicity.

[Protective Effect of Epalrestat against Oxidative Stress-induced Cytotoxicity].

Epalrestat (EPS), approved in Japan, is currently the only aldose reductase inhibitor that is available for the treatment of diabetic neuropathy. Recently, we found that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH) in rat Schwann cells. GSH, the most abundant non-protein thiol antioxidant in cells, is important for protection against oxidative stress. Oxidative stress is associated with the development and progression of many pathological conditions, such as atherosclerosis, diabetes, and neurodegeneration. In this study, we tested the hypothesis that EPS enhances resistance to oxidative stress, by using rat Schwann cells. To determine whether EPS protects Schwann cells from oxidative stress, we performed experiments by using radical generators, drugs, and heavy metals as the source of oxidative stress. EPS reduced the cytotoxicity induced by 2,2-azobis-[2-(2-imidazolin-2-yl) propane] dihydrochloride, 6-hydroxydopamine, cisplatin, palmitate, cadmium chloride, and manganese (II) sulfate, indicating that EPS plays a role in protecting cells from oxidative stress. We suggest that EPS has the potential to prevent the development and progression of disorders caused by oxidative stress.

Epalrestat Upregulates Heme Oxygenase-1, Superoxide Dismutase, and Catalase in Cells of the Nervous System.

Heme oxygenase (HO)-1 has potent antioxidant and anti-inflammatory functions. Recent studies have shown that the upregulation of HO-1 is beneficial to counteract neuroinflammation, making HO-1 a new therapeutic target for neurological diseases. We have reported that epalrestat (EPS), which is currently used for the treatment of diabetic neuropathy, increases HO-1 levels through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) in bovine aortic endothelial cells. In this study, we tested the hypothesis that EPS upregulates HO-1 via Nrf2 activation in the component cells of the nervous system, by using rat Schwann cells and human SH-SY5Y cells. Treatment of Schwann cells with EPS at near-plasma concentration led to a dramatic increase in HO-1 levels. Nrf2 knockdown by small interfering RNA (siRNA) suppressed the EPS-induced HO-1 expression. EPS did not promote the intracellular accumulation of free ferrous ion and reactive oxygen species, by increasing ferritin via Nrf2 during HO-1 induction. Moreover, EPS stimulated the expression of superoxide dismutase 1 and catalase, which also are Nrf2 target gene products. It also markedly increased HO-1 levels in SH-SY5Y cells through the activation of Nrf2. We demonstrated for the first time that EPS upregulates HO-1, superoxide dismutase, and catalase by activating Nrf2. We suggest that EPS has the potential to prevent several neurological diseases.

Glycolaldehyde induces endoplasmic reticulum stress and apoptosis in Schwann cells.

Schwann cell injury is caused by diabetic neuropathy. The apoptosis of Schwann cells plays a pivotal role in diabetic nerve dysfunction. Glycolaldehyde is a precursor of advanced glycation end products that contribute to the pathogenesis of diabetic neuropathy. In this study, we examined whether glycolaldehyde induces endoplasmic reticulum (ER) stress and apoptosis in rat Schwann cells. Schwann cells treated with 500 µM glycolaldehyde showed morphological changes characteristic of apoptosis. Glycolaldehyde activated apoptotic signals, such as caspase-3 and caspase-8. Furthermore, it induced ER stress response involving RNA-dependent protein kinase-like ER kinase (PERK), inositol-requiring ER-to-nucleus signal kinase 1α (IRE1α), and eukaryotic initiation factor 2α (eIF2α). In addition, glycolaldehyde activated CCAAT/enhancer-binding homologous protein (CHOP), an ER stress response factor crucial to executing apoptosis. Knockdown of nuclear factor E2-related factor 2 (Nrf2), which is involved in the promotion of cell survival following ER stress, enhanced glycolaldehyde-induced cytotoxicity, indicating that Nrf2 plays a protective role in the cytotoxicity caused by glycolaldehyde. Taken together, these findings indicate that glycolaldehyde is capable of inducing apoptosis and ER stress in Schwann cells. The ER stress induced by glycolaldehyde may trigger the glycolaldehyde-induced apoptosis in Schwann cells. This study demonstrated for the first time that glycolaldehyde induced ER stress.

Epalrestat increases glutathione, thioredoxin, and heme oxygenase-1 by stimulating Nrf2 pathway in endothelial cells.

Epalrestat (EPS) is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Recently, we found that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH) in rat Schwann cells. GSH plays a crucial role in protecting endothelial cells from oxidative stress, thereby preventing vascular diseases. Here we show that EPS increases GSH levels in not only Schwann cells but also endothelial cells. Treatment of bovine aortic endothelial cells (BAECs), an in vitro model of the vascular endothelium, with EPS caused a dramatic increase in intracellular GSH levels. This was concomitant with the up-regulation of glutamate cysteine ligase, an enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Moreover, EPS stimulated the expression of thioredoxin and heme oxygenase-1, which have important redox regulatory functions in endothelial cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that regulates the expression of antioxidant genes. EPS increased nuclear Nrf2 levels in BAECs. Nrf2 knockdown by siRNA suppressed the EPS-induced glutamate cysteine ligase, thioredoxin-1, and heme oxygenase-1 expression. Interestingly, LY294002, an inhibitor of phosphatidylinositol 3-kinase, abolished the EPS-stimulated GSH synthesis, suggesting that the kinase is associated with Nrf2 activation induced by EPS. Furthermore, EPS reduced the cytotoxicity induced by H2O2 and tert-butylhydroperoxide, indicating that EPS plays a role in protecting cells from oxidative stress. Taken together, the results provide evidence that EPS exerts new beneficial effects on endothelial cells by increasing GSH, thioredoxin, and heme oxygenase-1 levels through the activation of Nrf2. We suggest that EPS has the potential to prevent several vascular diseases caused by oxidative stress.

Epalrestat increases intracellular glutathione levels in Schwann cells through transcription regulation.

Epalrestat (EPS), approved in Japan, is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Here we report that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH), which is important for protection against oxidative injury, through transcription regulation. Treatment of Schwann cells with EPS caused a dramatic increase in intracellular GSH levels. EPS increased the mRNA levels of γ-glutamylcysteine synthetase (γ-GCS), the enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that plays a central role in regulating the expression of γ-GCS. ELISA revealed that EPS increased nuclear Nrf2 levels. Knockdown of Nrf2 by siRNA suppressed the EPS-induced GSH biosynthesis. Furthermore, pretreatment with EPS reduced the cytotoxicity induced by H2O2, tert-butylhydroperoxide, 2,2'-azobis (2-amidinopropane) dihydrochloride, and menadione, indicating that EPS plays a role in protecting against oxidative stress. This is the first study to show that EPS induces GSH biosynthesis via the activation of Nrf2. We suggest that EPS has new beneficial properties that may prevent the development and progression of disorders caused by oxidative stress.