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

Abstract

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.

Heme oxygenase (HO)-1 is a stress-responsive enzyme that has anti-inflammatory, antioxidant, and cytoprotective functions. Expectations are high that the regulation and amplification of HO-1 by pharmacological approaches would lead to the discovery of novel drugs for the treatment of a variety of diseases.¹⁾ HO-1 has emerged as an anti-inflammatory therapeutic target.²⁾ In particular, targeting HO-1 in neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, has been reported.³⁾ As HO-1 expression is involved in neuropathological changes, its regulation is essential for the development of new therapeutic approaches.³⁾ However, such metalloporphyrins as cobalt protoporphyrin IX, which are prototypical inducers of HO-1 and commonly used in experimental cell culture and animal models, are not applicable to clinical interventions because of their high toxicity.²⁾ It is expected that the upregulation of HO-1 by currently available pharmacological agents, whose safety and pharmacokinetics have already been confirmed clinically, would be useful for the treatment of a variety of diseases.

The biochemical activities of heme degradation products and their metabolic derivatives contribute to the cytoprotective function of HO-1. HO-1 catalyzes the degradation of heme to produce ferrous iron, carbon monoxide, and biliverdin, the latter of which is subsequently converted into bilirubin. Carbon monoxide is involved in inflammation regulation.⁴⁾ Bilirubin and biliverdin, which can scavenge peroxyl radicals, are cytoprotective antioxidants.⁵⁾ However, studies have shown that HO-1 overexpression increases reactive oxygen species (ROS) levels, thereby inducing cell death.^6,7) Pro-oxidant ferrous iron easily reacts with molecular oxygen to produce superoxide, which may have deleterious effects. During the reaction, HO-1 potentially generates a significant amount of hydrogen peroxide (H₂O₂), which is a source of hydroxyl radical. Superoxide dismutase (SOD) and catalase are responsible for scavenging superoxide and H₂O₂, respectively, resulting in reduced ROS accumulation. In addition, cells overexpress ferritin to protect themselves from iron toxicity.⁸⁾ Therefore, the regulation and amplification of HO-1, SOD, and catalase by pharmacological approaches may be a new therapeutic strategy for some diseases.

Epalrestat (5-[(1Z,2E)-2-methyl-3-phenyl propenylidene]-4-oxo-2-thioxo-3-thiazolidine acetic acid; EPS) is an inhibitor of aldose reductase, a rate-limiting enzyme in the polyol pathway (Fig. 1). Under hyperglycemic conditions, EPS reduces intracellular sorbitol accumulation, which is implicated in the pathogenesis of diabetic complications.⁹⁾ Recently, we found that EPS increases the intracellular levels of glutathione (GSH), which is important for protection against oxidative injury, in rat Schwann cells.¹⁰⁾ Schwann cells, which are myelin-forming cells in the peripheral nervous system, play a key role in the pathology of inflammation and metabolic disorders.¹¹⁾ Schwann cells are susceptible to oxidative stress and the specific targets of oxidative injury.¹²⁾ In rat Schwann cells, EPS increases GSH levels through transcriptional regulation by stimulating nuclear factor erythroid 2-related factor 2 (Nrf2) activation.¹⁰⁾ Nrf2 is a key transcription factor that plays a central role in regulating antioxidant gene expression. HO-1 is a representative Nrf2 target gene product.¹³⁾ Nrf2 controls not only HO-1 gene but also the genes of other antioxidant proteins, including SOD, catalase, and ferritin.^8,14,15) EPS may be associated with the increase of antioxidant protein expression. In this study, we examined the effects of EPS on the expression of HO-1, SOD, and catalase in rat Schwann cells. In addition, we investigated HO-1 expression in the human neuroblastoma cell line SH-SY5Y.

Fig. 1. Chemical Structure of Epalrestat

MATERIALS AND METHODS

Cell Culture and Treatment with EPS

Rat Schwann cells and the human neuroblastoma cell line SH-SY5Y were purchased from Sumitomo Dainippon Pharma Co., Ltd. (Osaka, Japan). Cells were grown to 80–90% confluence in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), L-glutamine (4 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Then, the cells were passaged by trypsinization.

Before treating the cells with EPS (Wako Pure Chemical Industries, Ltd., Osaka, Japan), the culture medium was replaced with DMEM containing 2% FBS. EPS (10, 50, 100 µM) was subsequently added to the medium.

Knockdown of Nrf2 with Small Interfering RNA (siRNA)

Oligonucleotides directed against rat Nrf2 and control siRNA (Ambion, Austin, TX, U.S.A.) were transfected into Schwann cells using Lipofectamine RNAiMAX (Invitrogen, Eugene, OR, U.S.A.), according to the manufacturer’s protocol. Briefly, both Nrf2 siRNA and control siRNA were diluted with Opti-MEM medium and then, diluted Lipofectamine RNAiMAX was added. The transfection mixture was incubated at room temperature for 20 min. When cells reached 30–50% confluence, the culture medium was replaced with DMEM (without FBS) and the transfection mixture was added to each well. The final concentration of siRNA was 50 nM.

Measurement of Protein

HO-1, SOD, and catalase protein levels were analyzed by Western blotting. The cells were treated with EPS, washed with Dulbecco’s phosphate buffered saline (DPBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Pierce, Rockford, IL, U.S.A.) containing protease inhibitors. The lysate was centrifuged at 10000×g for 15 min and 15 µg of protein in the supernatant was resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with the following primary antibodies: anti-rabbit HO-1 polyclonal antibody (Abcam, Cambridge, U.K.), anti-rabbit SOD1 polyclonal antibody (Abcam), and anti-mouse catalase monoclonal antibody (Sigma-Aldrich, St. Louis, MO, U.S.A.). Following primary antibody incubation, the membrane was incubated with horseradish-peroxidase (HRP)-conjugated secondary antibodies. Chemiluminescence was detected with an ECL Plus Western blot detection kit (GE Healthcare, Buckinghamshire, U.K.). Band intensities were quantified using ImageJ software.

For Western blot analysis of Nrf2, nuclear and cytosolic protein extracts were prepared using an NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Rockford, IL, U.S.A.) according to the manufacturer’s instructions. Nuclear and cytosolic proteins (20 µg) were resolved by 12% SDS-PAGE. Then, the proteins were transferred to a PVDF membrane where they were stained with primary antibody, anti-rabbit Nrf2 monoclonal antibody (Abcam), and HRP conjugated secondary antibody.

In addition, we measured HO-1, SOD, and catalase protein levels by fluorescence microscopy studies. Briefly, cells treated with EPS were fixed with 4% p-formaldehyde. HO-1, SOD, and catalase proteins were detected by reacting with primary antibodies for HO-1, SOD1, and catalase and Alexa Fluor^® 488 conjugated secondary antibodies (Cell Signaling Technology, Beverly, MA, U.S.A.). Following incubation with the antibodies, the cells were washed with DPBS and analyzed by confocal laser scanning microscopy (Carl Zeiss LSM700, Jena, Germany).

Measurement of mRNA

Quantitative RT-PCR analysis was carried out to measure mRNA levels. Total RNA from the treated cells was extracted with RNAspin Mini (GE Healthcare) according to the manufacturer’s protocol. mRNAs were reverse-transcribed into cDNA with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, U.S.A.). Quantitative RT-PCR was performed with a 7500 Fast Real-Time PCR System (Applied Biosystems). Primers for rat HO-1 (Rn01536933_m1), rat SOD1 (Rn01477289_m1), rat catalase (Rn00560930_m1), rat ferritin (Rn00820640_g1), rat Nrf2 (Rn00477784_m1), human HO-1 (Hs01110250_m1), and GAPDH (Rn01775763_g1 or Hs02758991_g1) were purchased from Applied Biosystems. mRNA levels were acquired from the value of the threshold cycle (C_t) of HO-1, SOD1, catalase, ferritin, and Nrf2 normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative mRNA levels were compared and expressed as percentage of control levels. Data are representative of three experiments.

Determination of Intracellular ROS and Free Iron

5-(6)-Carboxy-2′,7′-dichlorofluorescein diacetate (Sigma-Aldrich) and RhoNox-1 (Goryo Chemical, Inc., Sapporo, Japan) were used to estimate intracellular ROS and free ferrous iron levels, respectively. The cells were treated with EPS and incubated in medium containing 5-(6)-carboxy-2′,7′-dichlorofluorescein diacetate (10 µM) or RhoNox-1 (5 µM) for 20 min or 1 h, respectively. After the cells were washed with DPBS, changes in intracellular ROS and free iron levels were visualized as green or red fluorescence by confocal laser scanning microscopy.

Other Procedures

Cell viability was assessed by using CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS assay) from Promega (Madison, WI, U.S.A.), as described previously.¹⁰⁾ Protein concentrations were determined using the Bradford method with bovine serum albumin as the standard.

Statistical Analysis

All experiments were performed independently at least three times. Data were combined and expressed as the mean±standard deviation (S.D.). Statistical significance was determined using one-way ANOVA with Bonferroni post-hoc tests. A p value of <0.05 was considered to be significant.

RESULTS

Effect of EPS on HO-1 Levels in Schwann Cells

HO-1 is known as a typical antioxidant protein regulated by Nrf2 activation.¹³⁾ In our previous work, we demonstrated that the activation of Nrf2 was observed after treatment of Schwann cells with EPS.¹⁰⁾ In this study, we first examined the effect of EPS on HO-1 expression in Schwann cells (Fig. 2). Schwann cells were treated with EPS at 10, 50, and 100 µM for 24 h. Then, HO-1 protein levels in the EPS-treated Schwann cells were estimated by fluorescence microscopy studies (Fig. 2A) and Western blot analysis (Fig. 2B). Both fluorescence microscopy studies and Western blot analysis demonstrated a dose-dependent increase in HO-1 protein levels in the EPS-treated Schwann cells. Treatment with 10 µM EPS did not induce a significant increase in HO-1 protein levels, whereas treatment with 50 and 100 µM EPS markedly increased HO-1 protein levels. In the treatment with 50 and 100 µM EPS, the increases were 3.9- and 13.7-fold by Western blot analysis, respectively, relative to control. Figure 2C shows that EPS at 50 and 100 µM caused a dramatic increase in HO-1 mRNA levels. In Schwann cells treated with 10 µM EPS, no significant change was observed in HO-1 mRNA levels. These results indicate that EPS increases HO-1 levels in Schwann cells through transcription regulation.

Fig. 2. Effect of EPS on HO-1 in Schwann Cells

Schwann cells were treated with EPS at the indicated concentrations for 24 h. HO-1 protein levels were estimated by fluorescence microscopy studies (A) and by Western blot analysis (B), and HO-1 mRNA levels were quantified by using real-time RT-PCR (C). Pictures shown are representative of three independent experiments. Scale bar in A indicates 20 µm. Values in B and C are the mean±S.D. of three experiments. * Significant difference from the value of control (p<0.05). (D) Schwann cells were transfected with control siRNA (siControl) or Nrf2 siRNA (siNrf2) and were treated or not treated with 50 µM EPS for 24 h. Subsequently, HO-1 mRNA levels were measured. Values are the mean±S.D. of three experiments. * Significant difference from the value of non-treated siControl (p<0.05). ^# Significant difference from the value of siControl treated with EPS (p<0.05).

Next, we examined whether Nrf2 levels could alter the increases in HO-1 levels in cells treated with EPS, by means of Nrf2 knockdown in Schwann cells. Schwann cells were transfected with control siRNA (siControl) or Nrf2 siRNA (siNrf2). Nrf2 mRNA levels in the cells transfected with Nrf2 siRNA were reduced by approximately 90% relative to those in control siRNA transfected cells (data not shown). As shown in Fig. 2D, the increase in HO-1 mRNA levels after treatment with 50 µM EPS was completely inhibited by the knockdown of Nrf2 by siRNA. These results indicate that EPS induces HO-1 upregulation via Nrf2-mediated signaling in Schwann cells.

Effect of EPS on Intracellular Free Iron and Ferritin Levels in Schwann Cells

HO-1 catalyzes the degradation of heme to produce ferrous iron, which easily reacts with molecular oxygen to produce superoxide that is subsequently converted into H₂O₂. It has been demonstrated that the overexpression of HO-1 promotes ROS formation and cell death.^6,7) As shown in Fig. 2, EPS at 50 and 100 µM caused a dramatic increase in HO-1 levels, implying that the excess free iron released by HO-1 may accelerate ROS production in the intracellular milieu. It was reported that the treatment of Schwann cells with EPS at 10 and 50 µM for 24 h had no effect on cell viability; slightly lowered cell viability was observed at the EPS concentration of 100 µM and loss of viability was noted in only 4% of the EPS-treated cells compared to control.¹⁰⁾ When Schwann cells were treated with EPS at 10, 50, and 100 µM for 4 h, there were little or no changes in intracellular ROS levels as measured by fluorescence microscopy with the fluorescent probe 5-(6)-carboxy-2′,7′-dichlorofluorescein diacetate (Fig. 3A).

Fig. 3. Effect of EPS on Intracellular ROS, Free Iron, and Ferritin Levels in Schwann Cells

Schwann cells were treated with EPS at the indicated concentrations for 4 h (A) or 24 h (B–D). Intracellular ROS (A) and ferrous iron (B) levels were estimated by fluorescence microscopy using 5-(6)-carboxy-2′,7′-dichlorofluorescein diacetate and RhoNox-1, respectively. Each panel shows the typical fluorescence intensity from three independent experiments. Bright-field images are shown in the upper part of A. Scale bar, 20 µm. (C) Ferritin mRNA levels were quantified by using real-time RT-PCR. Values in C are the mean±S.D. of three experiments. * Significant difference from the value of control (p<0.05). (D) Schwann cells were transfected with control siRNA (siControl) or Nrf2 siRNA (siNrf2) and were treated or not treated with 50 µM EPS for 24 h. Subsequently, ferritin mRNA levels were measured. Values are the mean±S.D. of three experiments. * Significant difference from the value of non-treated siControl (p<0.05). ^# Significant difference from the value of siControl treated with EPS (p<0.05).

Then, we further examined the effect of EPS on free iron levels. Figure 3B shows the intracellular free ferrous iron levels estimated by fluorescence microscopy studies using the novel turn-on fluorescent probe RhoNox-1 for the selective detection of labile ferrous iron through the formation of a red fluorescent product.¹⁶⁾ EPS did not induce the intracellular accumulation of free ferrous iron; in fact, a dose-dependent decrease in red fluorescence was observed in Schwann cells treated with EPS.

Iron is sequestered by ferritin, an intracellular iron repository protein that is co-induced with HO-1.¹⁷⁾ Figure 3C demonstrates that EPS at 50 and 100 µM upregulated ferritin mRNA levels. The treatment with 10 µM EPS did not cause a significant increase in ferritin mRNA levels. Nrf2 knockdown by siRNA suppressed the increase in ferritin mRNA levels after EPS treatment (Fig. 3D). It seems that EPS acts to store free iron in the non-toxic form by increasing ferritin via Nrf2 during HO-1 induction.

Effect of EPS on SOD and Catalase Levels in Schwann Cells

Nrf2 controls not only HO-1 gene but also the genes of many cytoprotective enzymes, including SOD and catalase.^14,15) We examined whether EPS could alter SOD and catalase levels. SOD1 and catalase protein levels in Schwann cells treated with EPS were estimated by fluorescence microscopy studies and Western blot analysis (Fig. 4). As shown in Figs. 4A and B, slight increases in SOD1 and catalase protein levels were observed at EPS concentrations of 50 and 100 µM, as measured by fluorescence microscopy studies. The increases in SOD1 protein levels after treatment with 50 and 100 µM EPS were 1.8- and 2.2-fold, respectively, relative to control, as determined by Western blot analysis. (Fig. 4C). At those EPS concentrations, the increases in catalase protein levels were 1.4- and 1.8-fold, respectively (Fig. 4D). EPS at 50 and 100 µM increased both SOD1 and catalase mRNA levels (Figs. 5A, B). In addition, Nrf2 knockdown by siRNA suppressed the increases in SOD1 and catalase mRNA levels after treatment with 50 µM EPS (Figs. 5C, D). The results suggest that EPS can induce SOD1 and catalase upregulation via Nrf2. However, the ability of EPS to increase SOD1 and catalase levels is suppressed compared with its ability to increase HO-1 levels.

Fig. 4. Effect of EPS on SOD1 and Catalase Protein Levels in Schwann Cells

Schwann cells were treated with EPS at the indicated concentrations for 24 h. SOD1 and catalase protein levels were estimated by fluorescence microscopy studies (A: SOD1, B: catalase) and by Western blot analysis (C: SOD1, D: catalase). Pictures shown are representative of three independent experiments. Scale bar in A and B indicates 20 µm. Values in C and D are the mean±S.D. of three experiments. * Significant difference from the value of control (p<0.05).

Fig. 5. Effect of EPS on SOD1 and Catalase mRNA Levels in Schwann Cells

Schwann cells were treated with EPS at the indicated concentrations for 24 h. SOD1 (A) and catalase (B) mRNA levels were estimated by real-time RT-PCR assay. Values in A and B are the mean±S.D. of three experiments. * Significant difference from the value of control (p<0.05). In C and D, Schwann cells were transfected with control siRNA (siControl) or Nrf2 siRNA (siNrf2) and were treated or not treated with 50 µM EPS for 24 h. Subsequently, SOD1 (C) and catalase (D) mRNA levels were measured. Values are the mean±S.D. of three experiments. *Significant difference from the value of non-treated siControl (p<0.05). ^# Significant difference from the value of siControl treated with EPS (p<0.05).

Effect of EPS on HO-1 Levels and Nrf2 in SH-SY5Y Cells

It is known that HO-1 induction may play an important role in Parkinson’s disease, a degenerative disorder of the nervous system.¹⁸⁾ We examined the effects of EPS on HO-1 expression using the human neuroblastoma cell line SH-SY5Y, which is a model of neuronal cells. SH-SY5Y cells were treated with EPS at 10, 50, and 100 µM for 24 h. EPS at those concentrations had no effect on cell viability (data not shown). As shown in Figs. 6A and B, treatment with EPS at 50 and 100 µM caused an increase in HO-1 levels even in SH-SY5Y cells. At those EPS concentrations, the increases were 3.2- and 17.1-fold, respectively, relative to control, as determined by Western blot analysis. A concomitant increase in HO-1 mRNA level was observed (Fig. 6C).

Fig. 6. Effect of EPS on HO-1 and Nrf2 in SH-SY5Y Cells

SH-SY5Y cells were treated with EPS at the indicated concentrations. After a 24-h incubation, HO-1 protein levels were estimated by fluorescence microscopy studies (A) and by Western blot analysis (B). Pictures shown are representative of three independent experiments. Scale bar in A indicates 20 µm. (C) HO-1 mRNA levels were estimated by real-time RT-PCR assay. In D and E, SH-SY5Y cells were treated with EPS for 4 h. Subsequently, Nrf2 protein expression in nuclear and cytosolic extracts of SH-SY5Y cells were assayed by Western blotting. Lamin B1 and β-actin were used as nuclear and cytosolic extract control, respectively. Values are the mean±S.D. of three experiments. * Significant difference from the value of control (p<0.05).

Figure 6D demonstrates that EPS induced an increase in nuclear Nrf2 protein level. The increase was 6.6- and 11.1-fold by treatment with 50 and 100 µM EPS, respectively. EPS at 10 µM did not significantly increase the nuclear Nrf2 protein level. The results in Fig. 6D were similar to those in Figs. 6A–C; Nrf2 activation and HO-1 upregulation were observed under the same conditions for 50 and 100 µM EPS treatment. As can be seen from Fig. 6E, EPS at the concentrations used had no influence on cytosolic Nrf2 protein level. The results indicate that EPS increases HO-1 levels by stimulating the Nrf2 pathway in not only Schwann cells but also SH-SY5Y cells.

DISCUSSION

EPS (Ono Pharmaceuticals, Osaka, Japan) is the only aldose reductase inhibitor currently available for the treatment of diabetic neuropathy. EPS is easily absorbed by neural tissue and inhibits aldose reductase with minimum adverse effects.¹⁹⁾ The usual dosage of EPS for oral use is 50 mg three times a day. The plasma EPS concentration of 3.9 µg/mL (12 µM) was measured 1 h after a single oral dose of 50 mg.²⁰⁾ Using rat Schwann cells and human SH-SY5Y cells, we demonstrated for the first time that EPS at near-plasma concentration increases HO-1 expression in the component cells of the nervous system.

Nrf2 is a critical transcription factor for protecting cells from oxidative injury. In our previous reports, we have described that EPS activates Nrf2 in rat Schwann cells and bovine aortic endothelial cells.^10,21) Schwann cells, the myelin forming cells in the peripheral nervous system, are crucial for the proper function and maintenance of peripheral nerves.¹¹⁾ Schwann cells are susceptible to oxidative stress because of their large population of mitochondria.¹²⁾ It is reported that immortalized human fetal Schwann cells show marked susceptibility to oxidative stress induced by H₂O₂.²²⁾ Our present study revealed that treatment of Schwann cells with EPS increased HO-1 levels (Fig. 2). The knockdown of Nrf2, which regulates HO-1 expression, completely suppressed the increase in HO-1 levels after EPS treatment. Taken together, the results offer solid evidence that EPS increases HO-1 levels by activating Nrf2 in Schwann cells. Moreover, EPS stimulated the expression of superoxide dismutase 1 and catalase (Fig. 4).

Genes coding for cellular antioxidant enzymes, such as HO-1, contain a promoter-enhancer sequence known as the antioxidant response element (ARE).¹⁴⁾ Nrf2 is usually retained in the cytoplasm as an inactive complex with its cytosolic repressor, Kelch-like ECH associated protein 1 (Keap1).^8,23,24) When the Keap1–Nrf2 complex is dissociated by some form of cellular stimuli, Nrf2 is translocated into the nucleus where it binds to ARE. EPS has an α,β-unsaturated ketone moiety in its structure. α,β-Unsaturated ketones act as inducers of ARE genes by oxidizing sulfhydryl groups in Keap1, which leads to the dissociation and nuclear translocation of Nrf2.²⁵⁾ Therefore, the EPS-stimulated Nrf2 activation is most likely achieved by altering Keap1 structure. Unlike EPS, other aldose reductase inhibitors, such as sorbinil and alrestatin,^26,27) failed to increase HO-1 levels (data not shown). This result indicates that sorbinil and alrestatin, which do not have an α,β-unsaturated aldehyde or ketone, have no influence on the Nrf2 pathway. Aldose reductase is known as a target gene of Nrf2.^28,29) However, it seems unlikely that aldose reductase is induced by EPS because our previous study indicated that the reductase activity in Schwann cells was decreased by 30–50% after treatment with 10–100 µM EPS.¹⁰⁾ In addition, these findings suggest that the inhibition of aldose reductase does not contribute to the ability of EPS to increase HO-1 levels by activating Nrf2. Our study of bovine aortic endothelial cells revealed that LY294002, an inhibitor of phosphatidylinositol 3-kinase,³⁰⁾ abolished the EPS-stimulated GSH synthesis, suggesting that the kinase is associated with Nrf2 activation induced by EPS.²¹⁾ In contrast, in the case of Schwann cells, it was unlikely that the action of EPS was associated with the kinase because LY294002 had no influence on EPS-stimulated GSH synthesis (data not shown). On the other hand, Nrf2 was degraded by the ubiquitin-proteasome system and proteasome inhibitors enhanced HO-1 mRNA and protein accumulation.³¹⁾ EPS had no effect on proteasome activity in Schwann cells (data not shown). In addition, EPS-induced HO-1 upregulation was observed in the presence of clasto-lactacystin-β-lactone, a proteasome-specific inhibitor³²⁾ (data not shown). We suggest that ubiquitin-proteasome is not involved in the mechanism underlying HO-1 induction by EPS. At present, it remains unclear how EPS activates the Nrf2–Keap1 pathway in Schwann cells; its clarification will require further studies.

HO-1 is induced in response to oxidative stress and protects cells from oxidative injury.³³⁾ The HO-1-mediated protection is related to the degradation of its substrate, heme.³⁴⁾ Non-protein-bound free heme is highly toxic and may cause oxidative stress.²⁾ It has been shown that free heme has proinflammatory properties.²⁾ The by-products of the HO-1 reaction also contribute to the protective response. For example, CO exerts cytoprotective effects through its potent anti-inflammatory, anti-apoptotic, and vasodilatory properties.^4,18) Bile pigments biliverdin and bilirubin are peroxy radical scavengers.⁵⁾ However, some studies suggested that HO-1 induction might not always be beneficial and the release of redox-active iron from heme might enhance oxidative stress.^35,36) The circulating redox-active iron is capable of promoting the generation of ROS, such as superoxide and H₂O₂, which is a source of hydroxyl radical. A recent study suggested that the increased expression of HO-1, which is mostly localized on the endoplasmic reticulum, resulted in significant translocation to the mitochondria and induced oxidative stress by disrupting mitochondrial function.⁷⁾ Several studies also showed that HO-1 overexpression promoted mitochondrial sequestration of non-transferrin iron and induced macroautophagy that contributed to pathological iron deposition and bioenergetic failure in age-related neurodegenerative disorders.^7,37,38) In the present study, EPS dramatically increased HO-1 levels. This implies that the increased HO-1 expression by EPS may lead to excessive free iron accumulation and subsequently ROS production in the intracellular milieu. In addition, because mild oxidative stress activates the Nrf2 pathway,³⁹⁾ it is assumed that EPS has the potential to induce oxidative stress. However, our results indicated that EPS failed to elevate ROS production (Fig. 3). EPS did not accumulate intracellular free iron; in fact, a decrease in free iron levels was observed in Schwann cells treated with EPS, probably by upregulating ferritin expression. These results suggest that EPS increases HO-1 levels without producing unintended negative effects on Schwann cells. Conveniently, the increased expression of SOD and catalase may contribute to the ability to preserve cell viability (Fig. 4). In order to identify the relationship between HO-1 induction and ROS levels, we examined whether ferritin levels could alter the increase in ROS levels in cells treated with EPS, by performing ferritin knockdown in Schwann cells. Nevertheless, EPS had a negligible effect on ROS levels in cells transfected with ferritin siRNA (data not shown). Because EPS induced a dramatic increase in GSH levels¹⁰⁾ in the same manner as that observed in HO-1 and SOD/catalase levels, it appeared that EPS failed to elevate ROS production even in ferritin-knockdown cells. It has been demonstrated that HO-1 protects cells from damage induced by oxidative stress in diverse cell types.⁴⁰⁾ In addition, HO-1 exerts cytoprotective effects by preventing apoptosis.⁴⁰⁾ These findings suggest that HO-1 upregulation by EPS may prevent the development and progression of disorders caused by oxidative stress.

One study has described that HO-1 is a target for neuroprotection and neuroinflammation in neurodegenerative diseases.³⁾ Several phytochemicals, such as resveratrol, curcumin, flavonoids, and carnosol, can upregulate HO-1 expression via the Nrf2 pathway.⁴¹⁾ Studies on the regulation and amplification of HO-1 by pharmacological approaches may lead to the discovery of novel drugs for the treatment of a variety of diseases.¹⁾ Meanwhile, it is necessary to examine currently available pharmacological agents for their ability to induce HO-1 with minimum adverse effects. In drug reprofiling strategies, the actions of drugs in clinical use, whose safety and pharmacokinetics in humans have already been confirmed, are examined comprehensively at the molecular level and the results are used for the development of new medicines.⁴²⁾ One example is statins, which have initially been introduced to prevent arteriosclerosis due to their cholesterol-lowering effects, but have been recognized to exert anti-inflammatory effects via HO-1 induction.^43,44) Moreover, 5-aminosalicylic acid, one of the standard therapies for inflammatory bowel disease, has been shown to exhibit protective anti-inflammatory effects at least in part through the upregulation of HO-1 in an animal model of colitis.⁴⁵⁾ A recent study has indicated that the protective effect of metformin, an antihyperglycemic agent used in type 2 diabetes, on oxidative stress is in part dependent on the induction of HO-1.⁴⁶⁾ In this study, we showed that EPS induced HO-1 expression in Schwann cells, suggesting the beneficial effect of EPS. Analysis of SOD1 and catalase revealed that EPS induced their antioxidant protein expression via the Nrf2-mediated pathway. Moreover, EPS upregulated HO-1 expression in not only Schwann cells but also SH-SY5Y cells. A very recent study using SH-SY5Y cells has revealed that sulfuretin, one of the major flavonoid glycosides, induces Nrf2-dependent HO-1 expression and has preventive and/or therapeutic potential for the management of Alzheimer’s disease.⁴⁷⁾ In addition, berberine, a well-known alkaloid, protects SH-SY5Y cells from cell death induced by Parkinson’s disease related neurotoxin 6-hydroxydopamine through the induction of HO-1.⁴⁸⁾ From the findings of the present study, we expect that the pharmacological actions of EPS would lead to breakthroughs in drug discovery and development. EPS might be useful as a therapeutic agent for the treatment of Alzheimer’s disease and Parkinson’s disease.

In summary, we demonstrated that EPS, approved in Japan for use in treating subjective neuropathy symptoms, upregulates HO-1 expression in Schwann cells and SH-SY5Y cells in association with the Nrf2 pathway. The upregulation of HO-1 is considered to contribute to enhancing neuroprotection. Our findings have led us to propose that targeting the upregulation of HO-1, ferritin, and SOD/catalase by EPS is a promising therapeutic approach in nervous system diseases.

Acknowledgment

This study was supported in part by an Education and Research Grant from Hokkaido Pharmaceutical University School of Pharmacy.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Motterlini R, Foresti R. Heme oxygenase-1 as a target for drug discovery. Antioxid. Redox Signal., 20, 1810–1826 (2014).
• 2) Paine A, Eiz-Vesper B, Blasczyk R, Immenschuh S. Signaling to heme oxygenase-1 and its anti-inflammatory therapeutic potential. Biochem. Pharmacol., 80, 1895–1903 (2010).
• 3) Jazwa A, Cuadrado A. Targeting heme oxygenase-1 for neuroprotection and neuroinflammation in neurodegenerative diseases. Curr. Drug Targets, 11, 1517–1531 (2010).
• 4) Ryter SW, Choi AM. Heme oxygenase-1/carbon monoxide: from metabolism to molecular therapy. Am. J. Respir. Cell Mol. Biol., 41, 251–260 (2009).
• 5) Kapitulnik J. Bilirubin: an endogenous product of heme degradation with both cytotoxic and cytoprotective properties. Mol. Pharmacol., 66, 773–779 (2004).
• 6) Kim DH, Kim JH, Kim EH, Na HK, Cha YN, Chung JH, Surh YJ. 15-Deoxy-Δ^12,14-prostaglandin J₂ upregulates the expression of heme oxygenase-1 and subsequently matrix metalloproteinase-1 in human breast cancer cells: possible roles of iron and ROS. Carcinogenesis, 30, 645–654 (2009).
• 7) Bansal S, Biswas G, Avadhani NG. Mitochondria-targeted heme oxygenase-1 induces oxidative stress and mitochondrial dysfunction in macrophages, kidney fibroblasts and in chronic alcohol hepatotoxicity. Redox Biol., 2, 273–283 (2014).
• 8) Kalyanaraman B. Teaching the basics of redox biology to medical and graduate students: oxidants, antioxidants and disease mechanisms. Redox Biol., 1, 244–257 (2013).
• 9) Steele JW, Faulds D, Goa KL. Epalrestat: a review of its pharmacology, and therapeutic potential in late-onset complications of diabetes mellitus. Drugs Aging, 3, 532–555 (1993).
• 10) Sato K, Yama K, Murao Y, Tatsunami R, Tampo Y. Epalrestat increases intracellular glutathione levels in Schwann cells through transcription regulation. Redox Biol., 2, 15–21 (2013).
• 11) Lehmann HC, Höke A. Schwann cells as a therapeutic target for peripheral neuropathies. CNS Neurol. Disord. Drug Targets, 9, 801–806 (2010).
• 12) Eckersley L, Ansselin AD, Tomlinson DR. Effects of experimental diabetes on axonal and Schwann cell changes in sciatic nerve isografts. Brain Res. Mol. Brain Res., 92, 128–137 (2001).
• 13) Qi H, Chen B, Le XC, Rong J. Concomitant induction of heme oxygenase-1 attenuates the cytotoxicity of arsenic species from lumbricus extract in human liver HepG2 cells. Chem. Biodivers., 9, 739–754 (2012).
• 14) Vincent AM, Kato K, McLean LL, Soules ME, Feldman EL. Sensory neurons and Schwann cells respond to oxidative stress by increasing antioxidant defense mechanisms. Antioxid. Redox Signal., 11, 425–438 (2009).
• 15) Kim HJ, Vaziri ND. Contribution of impaired Nrf2–Keap1 pathway to oxidative stress and inflammation in chronic renal failure. Am. J. Physiol. Renal Physiol., 298, F662–F671 (2010).
• 16) Hirayama T, Okuda K, Nagasawa H. A highly selective turn-on fluorescent probe for iron(II) to visualize labile iron in living cells. Chem. Sci., 4, 1250–1256 (2013).
• 17) Agarwal A, Bolisetty S. Adaptive responses to tissue injury: role of heme oxygenase-1. Trans. Am. Clin. Climatol. Assoc., 124, 111–122 (2013).
• 18) Hung SY, Liou HC, Kang KH, Wu RM, Wen CC, Fu WM. Overexpression of heme oxygenase-1 protects dopaminergic neurons against 1-methyl-4-phenylpyridinium-induced neurotoxicity. Mol. Pharmacol., 74, 1564–1575 (2008).
• 19) Hotta N, Akanuma Y, Kawamori R, Matsuoka K, Oka Y, Shichiri M, Toyota T, Nakashima M, Yoshimura I, Sakamoto N, Shigeta Y. Long-term clinical effects of epalrestat, an aldose reductase inhibitor, on diabetic peripheral neuropathy: the 3-year, multicenter, comparative aldose reductase inhibitor-diabetes complications trial. Diabetes Care, 29, 1538–1544 (2006).
• 20) Ono Pharmaceutical Co., Ltd., Kinedak (epalrestat) Package Insert, Osaka, Japan (2009).
• 21) Yama K, Sato K, Abe N, Murao Y, Tatsunami R, Tampo Y. Epalrestat increases glutathione, thioredoxin, and heme oxygenase-1 by stimulating Nrf2 pathway in endothelial cells. Redox Biol., 4, 87–96 (2015).
• 22) Lehmann HC, Chen W, Mi R, Wang S, Liu Y, Rao M, Höke A. Human Schwann cells retain essential phenotype characteristics after immortalization. Stem Cells Dev., 21, 423–431 (2012).
• 23) Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1–Nrf2–ARE pathway. Annu. Rev. Pharmacol. Toxicol., 47, 89–116 (2007).
• 24) Kansanen E, Kuosmanen SM, Leinonen H, Levonen AL. The Keap1–Nrf2 pathway: Mechanisms of activation and dysregulation in cancer. Redox Biol., 1, 45–49 (2013).
• 25) Magesh S, Chen Y, Hu L. Small molecule modulators of Keap1–Nrf2–ARE pathway as potential preventive and therapeutic agents. Med. Res. Rev., 32, 687–726 (2012).
• 26) Beyer-Mears A, Cruz E. Reversal of diabetic cataract by sorbinil, an aldose reductase inhibitor. Diabetes, 34, 15–21 (1985).
• 27) Gabbay KH, Spack N, Loo S, Hirsch HJ, Ackil AA. Aldose reductase inhibition: studies with alrestatin. Metabolism, 28 (Suppl 1), 471–476 (1979).
• 28) Nishinaka T, Yabe-Nishimura C. Transcription factor Nrf2 regulates promoter activity of mouse aldose reductase (AKR1B3) gene. J. Pharmacol. Sci., 97, 43–51 (2005).
• 29) Kang ES, Woo IS, Kim HJ, Eun SY, Paek KS, Kim HJ, Chang KC, Lee JH, Lee HT, Kim JH, Nishinaka T, Yabe-Nishimura C, Seo HG. Up-regulation of aldose reductase expression mediated by phosphatidylinositol 3-kinase/Akt and Nrf2 is involved in the protective effect of curcumin against oxidative damage. Free Radic. Biol. Med., 43, 535–545 (2007).
• 30) Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem., 269, 5241–5248 (1994).
• 31) Stewart D, Killeen E, Naquin R, Alam S, Alam J. Degradation of transcription factor Nrf2 via the ubiquitin–proteasome pathway and stabilization by cadmium. J. Biol. Chem., 278, 2396–2402 (2003).
• 32) Fenteany G, Standaert RF, Lane WS, Choi S, Corey EJ, Schreiber SL. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science, 268, 726–731 (1995).
• 33) Okita Y, Kamoshida A, Suzuki H, Itoh K, Motohashi H, Igarashi K, Yamamoto M, Ogami T, Koinuma D, Kato M. Transforming growth factor-β induces transcription factors MafK and Bach1 to suppress expression of the heme oxygenase-1 gene. J. Biol. Chem., 288, 20658–20667 (2013).
• 34) Agarwal A, Bolisetty S. Adaptive responses to tissue injury: role of heme oxygenase-1. Trans. Am. Clin. Climatol. Assoc., 124, 111–122 (2013).
• 35) Gutteridge JM. Fate of oxygen free radicals in extracellular fluids. Biochem. Soc. Trans., 10, 72–73 (1982).
• 36) Dennery PA, Spitz DR, Yang G, Tatarov A, Lee CS, Shegog ML, Poss KD. Oxygen toxicity and iron accumulation in the lungs of mice lacking heme oxygenase-2. J. Clin. Invest., 101, 1001–1011 (1998).
• 37) Schipper HM, Song W, Zukor H, Hascalovici JR, Zeligman D. Heme oxygenase-1 and neurodegeneration: expanding frontiers of engagement. J. Neurochem., 110, 469–485 (2009).
• 38) Schipper HM. Heme oxygenase-1 in Alzheimer’s disease: a tribute to Moussa Youdim. J. Neural Transm., 118, 381–387 (2011).
• 39) Bell KF, Al-Mubarak B, Fowler JH, Baxter PS, Gupta K, Tsujita T, Chowdhry S, Patani R, Chandran S, Horsburgh K, Hayes JD, Hardingham GE. Mild oxidative stress activates Nrf2 in astrocytes, which contributes to neuroprotective ischemic preconditioning. Proc. Natl. Acad. Sci. U.S.A., 108, E1–E2, author reply, E3–E4 (2011).
• 40) Lee SE, Yang H, Son GW, Park HR, Park CS, Jin YH, Park YS. Eriodictyol protects endothelial cells against oxidative stress-induced cell death through modulating ERK/Nrf2/ARE-dependent heme oxygenase-1 expression. Int. J. Mol. Sci., 16, 14526–14539 (2015).
• 41) Pae HO, Kim EC, Chung HT. Integrative survival response evoked by heme oxygenase-1 and heme metabolites. J. Clin. Biochem. Nutr., 42, 197–203 (2008).
• 42) Mizushima T. Drug discovery and development focusing on existing medicines: drug re-profiling strategy. J. Biochem., 149, 499–505 (2011).
• 43) Lee TS, Chang CC, Zhu Y, Shyy JY. Simvastatin induces heme oxygenase-1: a novel mechanism of vessel protection. Circulation, 110, 1296–1302 (2004).
• 44) Grosser N, Hemmerle A, Berndt G, Erdmann K, Hinkelmann U, Schürger S, Wijayanti N, Immenschuh S, Schröder H. The antioxidant defense protein heme oxygenase 1 is a novel target for statins in endothelial cells. Free Radic. Biol. Med., 37, 2064–2071 (2004).
• 45) Horváth K, Varga C, Berkó A, Pósa A, László F, Whittle BJ. The involvement of heme oxygenase-1 activity in the therapeutic actions of 5-aminosalicylic acid in rat colitis. Eur. J. Pharmacol., 581, 315–323 (2008).
• 46) Conde de la Rosa L, Vrenken TE, Buist-Homan M, Faber KN, Moshage H. Metformin protects primary rat hepatocytes against oxidative stress-induced apoptosis. Pharmacol. Res. Perspect., 3, e00125 (2015).
• 47) Kwon SH, Ma SX, Hwang JY, Lee SY, Jang CG. Involvement of the Nrf2/HO-1 signaling pathway in sulfuretin-induced protection against amyloid beta25–35 neurotoxicity. Neuroscience, 304, 14–28 (2015).
• 48) Bae J, Lee D, Kim YK, Gil M, Lee JY, Lee KJ. Berberine protects 6-hydroxydopamine-induced human dopaminergic neuronal cell death through the induction of heme oxygenase-1. Mol. Cells, 35, 151–157 (2013).