Erythropoietin attenuates oxidative stress and apoptosis in Schwann cells isolated from streptozotocin-induced diabetic rats.

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Research Paper

Journal of Pharmacy And Pharmacology

Erythropoietin attenuates oxidative stress and apoptosis in Schwann cells isolated from streptozotocin-induced diabetic rats Ting Yua, Lei Lib, Yanwen Bic, Zhen Liua, Huaxiang Liud and Zhenzhong Lia a Department of Anatomy, Shandong University School of Medicine, Departments of cCardiosurgery and dRheumatology, Shandong University Qilu Hospital and bDepartment of Diagnosis, Jining Medical University, Shandong, China

Keywords apoptosis; diabetic neuropathy; erythropoietin; oxidative stress; Schwann cell Correspondence Zhenzhong Li, Department of Anatomy, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong Province 250012, China. E-mail: [email protected] Received January 2, 2014 Accepted February 23, 2014 doi: 10.1111/jphp.12244

Abstract Objectives High glucose-evoked oxidative stress and apoptosis within Schwann cells (SCs) are mechanisms facilitating the procession of diabetic peripheral neuropathy (DPN). Although erythropoietin (EPO) was demonstrated to have neuroprotective effects in neurodegenerative diseases, the effects of EPO on glucose-evoked oxidative stress and apoptosis of SCs remain unknown. Methods Primary cultured SCs isolated from streptozotocin (STZ)-induced diabetic peripheral neuropathic rats and normal control rats were exposed to high or normal glucose condition with or without EPO incubation for 72 h. Cell viability, apoptotic rate, cellular reactive oxygen species (ROS) level, total glutathione (GSH) level, EPO mRNA and erythropoietin receptor (EPOR) mRNA levels were assayed. Key findings SCs from diabetic rats showed a lower cell viability and a higher apoptotic rate. High glucose culture condition elevated ROS level and diminished total GSH level of SCs. EPO improved cell viability and decreased cell apoptotic rate of SCs. EPO also elevated total GSH level and decreased intracellular ROS level. SCs from diabetic rats exhibited higher EPO mRNA and EPOR mRNA levels than SCs from normal control rats. Conclusions The data of this study offered fresh viewpoints for interpreting the pathogenesis of DPN and novel pharmacological principles implicit in the therapeutic effect of EPO.

Introduction Diabetic peripheral neuropathy (DPN) is the most ordinary, least recognised and most poorly understood longterm complication of diabetes, with severe chronic pain to degrade the life quality of diabetic patients.[1] Although complicated mechanisms underlie the pathogenic process of diabetic neuropathy, researches point out that glucoseevoked oxidative stress is the unitive ligament among diabetic complications.[2–4] Glucose-evoked oxidative stress presents because of an imbalance between the creation of oxygen free radical and the function of antioxidant systems.[5] The oxidation of elevated glucose within the peripheral nervous system stimulates the production of cellular reactive oxygen species (ROS), which are correlated with membrane lipid peroxidation, nitration of proteins, formation of poly(ADP-ribosyl)ated protein polymers and 1150

degradation of DNA.[6,7] Consistent with this view, the prevention of ROS may repair metabolic disturbances and block the progression of DPN.[5,8] Glutathione (GSH), famous as a nucleophilic scavenger and an enzymecatalysed antioxidant, can participate in the clearance of free radical species that cause lipid peroxidation, DNA strand breakdown and enhanced mutagenicity.[9] Schwann cells (SCs), which are known as the peripheral myelin-forming cells, play an important role in maintaining nerve integrity during development and after nerve injury.[1,10] SCs are highly vulnerable to hyperglycaemia because of their glucose absorption via insulin-independent glucose transporter.[11,12] They respond to hyperglycaemic toxicity by reinforcing the antioxidant defence systems.[5,13] High glucose-mediated apoptosis of SCs is correlated to the

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1150–1160

Ting Yu et al.

pathogenesis of DPN.[14] The SCs may be a crucial target for therapeutic strategies against DPN.[13] Erythropoietin (EPO) and its receptor (EPOR) that were initially recognised for their hematopoietic effects were demonstrated to have neuroprotective effects in neurodegenerative diseases recently.[15] Non-erythropoietic effects of EPO consist of the preservation of neural integrity[16,17]and the defence from or the enhanced restoration after neuronal damage.[18–20] Recent study has confirmed that targeting dorsal root ganglion with EPO gene transfer can interfere the development of DPN.[21] However, the effects of EPO on glucose-evoked oxidative stress and apoptosis of SCs remain unknown. In the current study, primary-cultured SCs isolated from streptozotocin (STZ)-induced diabetic peripheral neuropathic rats and normal control rats were exposed to high or normal glucose condition with or without EPO incubation for 72 h. Cell viability, apoptotic rate, cellular ROS level, total GSH level, EPO mRNA and EPOR mRNA levels were assayed to investigate whether and how EPO participates in the protection of SCs from glucose-evoked oxidative stress and apoptosis.

Materials and Methods Preparations of diabetic neuropathic pain animal model All animals involved in the current study were male Wistar rats (from the Experimental Animal Center at Shandong University of China) with body weight range from 200– 250 g. All animals were cared for in accordance with the National Institutes of Health guide for the care and use of laboratory animals (revised 1996). All experimental procedures were reviewed by and had prior approval by the Ethical Committee for Animal Experimentation of the Shandong University. All efforts were made to minimise animal suffering, to reduce the number of animal used and to utilise alternatives to in-vivo techniques. The rats were induced diabetes by a single intravenous injection (i.v.) of STZ (Sigma, St Louis, MO, USA; 55 mg/kg freshly dissolved in 0.1 mol/l citric acid buffer, pH 4.5). Age-matched rats injected with the same volume of the vehicle solution were used as control group. The diabetic peripheral neuropathic rats were defined as a blood glucose level >16 mmol/l from 3 days to 8 weeks after STZ injection and with evident neuropathic hyperalgesia (mechanical threshold ≤ 5.0 g and thermal threshold ≤ 10 s) at 8 weeks after STZ administration.

Evaluation of mechanical and thermal hyperalgesia The mechanical and thermal hyperalgesia behavioural tests were finished by the investigators who were blinded to the

Erythropoietin on Schwann cells

animal groups. The thresholds to mechanical and thermal stimuli were detected alternatively between the left and right hind paws with Von Frey filaments (BME-403, Chinese Academy of Medical Sciences Institute of Biomedical Engineering, Tianjin, China) and a plantar analgesia tester (BME-400C, Chinese Academy of Medical Sciences Institute of Biomedical Engineering). Each rat underwent training sessions before behavioural tests. Mechanical hyperalgesia was judged by the 50% withdrawal threshold to the Von Frey filaments stimulation. Each rat for mechanical stimulation test was acclimated individually for 15 min in a clear plastic cage with a wire mesh bottom that allowed full access to the rat hind paw. A series of standard Von Frey filaments were used serially to stimulate the plantar intermediate region of the rat hind paw. The rats were given a pressure that was just adequate to bend the filament for 5 s. A positive response was considered as a brisk withdrawal of the testing hind paw. The 50% threshold was calculated according to the sequence of positive and negative scores as previous described.[22] The withdrawal threshold to a noxious radiant heat stimulation was used to evaluate the thermal hyperalgesia. Each rat for thermal hyperalgesia assessment was acclimated to the environment for 15 min and then was placed on a thin glass platform maintained at 30°C. A radiant heat source with constant intensity was used to stimulate the plantar intermediate region of the rat hind paw. The withdrawal latency was determined from the beginning of thermal stimulation until a brisk withdrawal of the testing hind paw. A cut-off of 30 s was used to prevent hind paw from tissue injury.

Isolation of Schwann cells and culture preparations After 8 weeks of STZ or vehicle solution injection, the animals were anesthetised with 3% sodium pentobarbital (1 ml/kg body weight, intraperitoneal injection). The bilateral sciatic nerves (between sciatic notch and the ankle) were isolated from each animal. Upon the epineurium removal, the sciatic nerve fragments were minced into small pieces and incubated with 0.5% trypsin (Sigma) and 1% collagenase (type IA, Invitrogen Corporation, Camarillo, CA, USA) at 37°C for 1 h to be isolated as dispersed cells. After being resuspended in DMEM/F-12 (Gibco, Grand Island, NY, USA) media, which contained 10% fetal bovine serum, the dissociated SCs were plated at 0.5 × 105 cells/well in a volume of 0.1 ml for 96-well clusters or at 1.5 × 105 cells/well in a volume of 0.5 ml for 24-well clusters precoated with poly-L-lysine (0.1 mg/ml; Sigma). For high glucose challenge or EPO incubation, the additional glucose (30 mmol/l) or EPO (Peprotech, Rocky Hill, NJ, USA; 10 U/ ml) was added into the culture media. According to the


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experimental design, the cultured SCs were divided into six groups: (1) diabetes + normal glucose; (2) diabetes + normal glucose + EPO; (3) diabetes + high glucose; (4) diabetes + high glucose + EPO; (5) normal + high glucose; and (6) normal control + normal glucose group. All these culture preparations were incubated in a 5% CO2 incubator for 72 h at 37°C.

1 h at 20°C and permeabilised for 2 min on ice with 0.1% Triton X-100 dissolved in 0.1% sodium citrate. The TUNEL reaction mixture was applied and incubated with the cells for 1 h at 37°C in the darkness. After being washed with 0.01 mol/l PBS, the samples were placed on glass slides with a DAPI-containing anti-fade mounting medium. The apoptotic rate of SCs was expressed as the proportion of TUNEL-positive (red) with DAPI-positive (blue) nuclei.

Double fluorescence labelling At the end of culture, the cells in the 24-well clusters were rinsed quickly in 0.01 mol/l phosphate-buffered saline (PBS; pH 7.4) to clear the rest culture media attached on the cell surface. After being fixed in 4% paraformaldehyde for 40 min, the cells were permeabilised with 3% Triton X-100 (Sigma, St Louis, MO, USA) for 1 h. Non-specific sites were blocked by incubating the cells with 10% normal goat serum for 1 h at room temperature. After that, the cells were incubated with mouse monoclonal S-100 antibody (1 : 1000; Abcam, Cambridge, MA, USA) overnight at 4°C and then with the goat antimouse secondary antibody conjugated to fluorescein isothiocyanate (1 : 200) for 1 h in the darkness. After being washed with 0.01 mol/l PBS, the cells were covered with a 4′,6-diamidino-2-phenylindole (DAPI)-containing antifade mounting medium (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The purity of the SCs was expressed as the proportion of S-100-positive (green) cells with DAPI-positive (blue) nuclei.

Evaluation of cell viability At the end of the culture, the cell viability was assessed by the water-soluble tetrazolium salt-1 (WST-1 (containing 4-(3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1, 3-benzene disulfonate)) assay with a WST-1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, Beijing, China). Briefly, 10 μl of the WST-1-reagent was applied to the 96-well clusters for 4 h at 37°C. The absorbance of the cells in different experimental groups (Aexperimental) was normalised to that of the cells in control group. Abackground was the absorbance of the culture medium plus WST-1 without cells. The viability of the SCs was calculated by the following formula:

Cell visibility = ( Aexperimental − Abackground ) ( Acontrol − Abackground ) × 100%

Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay for cell apoptosis The apoptotic cells were labelled by TUNEL assay using the situ cell-death detection kit (Roche, Mannheim, Germany). The air-dried cells were fixed in 4% paraformadelyde for 1152

Measurement of total glutathione level At the end of the culture, the level of total GSH in SCs was assessed by GSH reductase quantification method with a Total GSH Assay Kit (Beyotime). The SCs were collected and were lysed in the protein removal reagent S. The lysate was centrifuged at 10 000g for 10 min and the supernatant was used for the total GSH assay according to the manufacturer’s protocol. The total GSH level was expressed as nmol/mg protein.

Measurement of intracellular reactive oxygen species level For measuring the level of intracellular ROS, the SCs after treatments were incubated with 10 μmol/l cellpermeable oxidation-sensitive fluorescent probe: 2′,7′dichlorodihydrofluorescein diacetate (Sigma). After being incubated at 37°C in the darkness for 20 min, the cells were rinsed in 0.01 mol/l PBS and the unloaded probe was removed. The cells were imaged at a wavelength of 485 nm. The exposure duration was set at 150 ms and the fluorescent densities were measured with Image-Pro Plus 5.1 (Media Cybernetics Inc, Rockville, MD, USA).

Real-time PCR analysis of EPO mRNA and EPOR mRNA expression in SCs At the end of the culture, the mRNA levels of EPO and EPOR in SCs were analysed by real-time PCR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control. Total RNA from SCs was abstracted using TRIzol (TakaRa Biotechnology, Dalian, China). cDNA was synthesised with a cDNA synthesis kit (Thermo Scientific Molecular Biology, Lithuania, EU). The primer sequences for EPO, EPOR and GAPDH were as follows: EPO 5′-TTA CCG TCC CAG ATA CCA AAG T-3′ (forward) and 5′-AAG CAG TGA AGT GAG GCT ACG −3′ (reverse). EPOR-TCT CAT TCT CGT CCT CAT CTC A −3′ (forward) and 5′-GAC CCT CAA ACT CAT TCT CTG G-3′ (reverse). GAPDH 5′-GGC ACA GTC AAG GCT GAG AAT G −3′ (forward) and 5′-ATG GTG GTG AAG ACG CCA GTA −3′ (reverse). Real-time RT-PCR was performed by the eppendorf Realplex PCR system (Eppendorf,


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Figure 1 The mechanical and thermal thresholds of diabetic and nondiabetic rats. (a) The mechanical threshold of diabetic rats decreased significantly 3 weeks after STZ injection. (b) The thermal threshold of diabetic rats decreased significantly 4 weeks after STZ injection. Bar graphs with error bars represent mean ± SEM (n = 10). *P < 0.05 vs control at the same time point.

Hamburg, Germany). The comparative cycle of threshold fluorescence (Ct) method was used and the relative transcript amount of the target gene was normalised to that of GAPDH using the 2-ΔΔCt method.

There was no change of mechanical and thermal thresholds in age-related control rats during the 8-week experimental time (Figure 1).

Purity of Schwann cells Statistical analysis All experiments were performed in triplicate for each condition as one experiment. Ten experiments (n = 10) were finished for behavioural tests. Five experiments (n = 5) were finished for other analysis. All the data were presented as mean ± SEM. The statistical analysis was performed with GraphPad Software (GraphPad Software Inc, San Diego, CA, USA). Kruskal–Wallis test was used to compare the differences. Individual differences between treatments were identified using Dunn’s post hoc test. A P value < 0.05 was defined to be significant for analysis of all results.

Results The mechanical and thermal thresholds of rats The responses of rats to Von Frey filaments and noxious radiant heat stimulations were tested weekly to determine mechanical and thermal sensitivity. The mechanical threshold of diabetic rats decreased significantly as early as 3 weeks after STZ injection. The worsened decrease of the mechanical threshold of diabetic rats was observed from 5 weeks after STZ treatment and persisted throughout the study. Four weeks after the induction of diabetes, there was significant decrease in thermal threshold of diabetic rats. The severe thermal hyperalgesia was observed from 6 weeks after STZ treatment and persisted throughout the study.

S-100 is a well-established marker for SCs.[23,24] In the current experiment, the SCs were derived from the sciatic nerve that excluded the other S-100-expressing cells. At 72-h post-culture, the purity of SCs in this culture system was expressed as the proportion of S-100-expressing cells with DAPI-stained nuclei. The immunocytochemical staining results demonstrated that the rate of SCs was 95.2 ± 3.4% (Figure 2).

Cell viability of Schwann cells The viability and metabolic activity of SCs were measured by the WST-1 assay. The dehydrogenases of viable cells could reduce WST-1 stable tetrazolium salts to watersoluble, orange formazan dyes.[25] Therefore, the absorbance reflects the number of metabolically active cells under different culture conditions. Compared with SCs from normal control rats, the viability of SCs from diabetic rats significantly decreased. High (35.6 mmol/l) or normal (5.6 mmol/l) glucose culture conditions had no significant influence on cell viability, regardless of whether the SCs were from diabetic or nondiabetic rats. EPO (10 U/ml) significantly improved SCs viability from diabetic rats in normal and high glucose culture conditions (Figure 3).

Apoptotic rate of Schwann cells DNA cleavage, a notable marker of apoptosis, may yield double-stranded and single-stranded DNA breaks (nicks).


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Figure 2 Double fluorescence labeling of S-100 and DAPI. (a) S-100-labelled SCs. (b) The DAPI-labelled nuclei of all cells in culture. (c) Overlay of A and B, the arrow shows an S-100 negative fibroblast. Scale bar = 50 μM. The percentage of SCs was 95.2 ± 3.4%.

increased. High glucose (35.6 mmol/l) or normal glucose (5.6 mmol/l) culture condition had no significant influence on the apoptotic rate, regardless of whether the SCs were from diabetic or nondiabetic rats. EPO (10 U/ml) significantly reduced the apoptotic rate of SCs from diabetic rats in normal and high glucose culture conditions (Figure 4).

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Figure 3 Assessment of cell viability of SCs with WST-1 assay. Compared with SCs from normal control rats, the cell viability of SCs from diabetic rats significantly decreased. High glucose (35.6 mmol/l) or normal glucose (5.6 mmol/l) culture condition had no significant influence on cell viability, regardless of whether the SCs were from diabetic or non-diabetic rats. EPO (10 U/ml) significantly improved SCs viability from diabetic rats in normal and high glucose culture conditions. Bar graphs with error bars represent mean ± SEM (n = 5). *P < 0.05 vs normal control + normal glucose. #P < 0.05 vs diabetes + normal glucose. $P < 0.05 vs diabetes + high glucose.

TUNEL staining detects both types of DNA breaks based on the enzymatic labelling of free 3′ DNA ends. The apoptotic SCs were visualised with TUNEL and were counterstained with DAPI. Compared with SCs from normal control rats, the apoptotic rate of SCs from diabetic rats significantly 1154

As an important intracellular antioxidant, the total GSH level is a putative indicator of cellular oxidative stress.[26] Compared with SCs from normal control rats, the total GSH level of SCs from diabetic rats significantly decreased. High glucose (35.6 mmol/l) culture condition further reduced total GSH level compared with normal glucose (5.6 mmol/l) culture condition, regardless of whether the SCs were from diabetic or nondiabetic rats. EPO (10 U/ml) significantly increased the total GSH level in SCs from diabetic rats in normal and high glucose culture conditions (Figure 5).

Level of intracellular reactive oxygen species in Schwann cells The intracellular ROS level was expressed as the fluorescent density in SCs. Compared with SCs from normal control rats, the fluorescent density in SCs from diabetic rats significantly increased. High glucose (35.6 mmol/l) culture condition further increased fluorescent density in SCs compared with normal glucose (5.6 mmol/l) culture condition, regardless of whether the SCs were from diabetic or nondiabetic rats. EPO (10 U/ml) significantly decreased the intracellular ROS level of SCs from diabetic rats in normal and high glucose culture conditions (Figure 6).

The expression of mRNAs for EPO and EPOR in SCs The EPO mRNA and EPOR mRNA levels in SCs were determined by real-time PCR. High glucose (35.6 mmol/l)


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Figure 4 TUNEL assay for apoptosis of SCs and DNA breaks. (a) diabetes + normal glucose. (b) diabetes + normal glucose + EPO. (c) diabetes + high glucose. (d) diabetes + high glucose + EPO. (e) normal + high glucose. (f) normal control + normal glucose. (A2-F2) The highlight of the box in (A1-F1). (g) The quantitative analysis of the proportion of TUNEL-positive (red) with DAPI-positive (blue) nuclei in different experimental conditions. Scale bar = 50 μM. Bar graphs with error bars represent mean ± SEM (n = 5). *P < 0.05 vs normal control + normal glucose. #P < 0.05 vs diabetes + normal glucose. $P < 0.05 vs diabetes + high glucose.


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Figure 5 Total GSH levels of SCs in different experimental conditions. Compared with SCs from normal control rats, the total GSH level of SCs from diabetic rats significantly decreased. High glucose (35.6 mmol/l) culture condition further reduced total GSH level compared with normal glucose (5.6 mmol/l) culture condition, regardless of whether the SCs were from diabetic or nondiabetic rats. EPO (10 U/ml) significantly increased total GSH level of SCs from diabetic rats in normal and high glucose culture conditions. Bar graphs with error bars represent mean ± SEM (n = 5). *P < 0.05 vs normal control + normal glucose. #P < 0.05 vs diabetes + normal glucose. $P < 0.05 vs diabetes + high glucose.

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culture condition increased the level of EPO mRNA, but not EPOR mRNA in SCs from normal control rats. SCs from diabetic rats exhibited higher EPO mRNA and EPOR mRNA levels than normal control rats, regardless of whether the SCs were cultured in high or normal glucose condition. EPO (10 U/ml) treatment seemed to have no significant influence on EPO mRNA and EPOR mRNA expression in SCs from diabetic rats in normal and high glucose culture conditions (Figure 7).

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Figure 6 Intracellular ROS levels of SCs in different experimental conditions. (a) diabetes + normal glucose. (b) diabetes + normal glucose + EPO. (c) diabetes + high glucose. (d) diabetes + high glucose + EPO. (e) normal + high glucose. (f) normal control + normal glucose. (g) The quantitative analysis of fluorescent density in SCs. Scale bar = 50 μM. Bar graphs with error bars represent mean ± SEM (n = 5). *P < 0.05 vs normal control + normal glucose. #P < 0.05 vs diabetes + normal glucose. $P < 0.05 vs diabetes + high glucose.

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Figure 7 The expression of EPO mRNA and EPOR mRNA in SCs in different experimental conditions. The amount of mRNA was detected by realtime PCR and expressed as the ratio of mRNA to GAPDH. (a) EPO mRNA levels in different groups. (b) EPOR mRNA levels in different groups. Bar graphs with error bars represent mean ± SEM (n = 5). *P < 0.05 vs normal control + normal glucose.

Discussion It is well known that hyperglycaemia plays a significant role in the pathogenic process of diabetic complications, but the mechanisms underlying the development and progression of DPN are not well understood. DPN predominantly occurs in a distal symmetrical pattern, with progressive reduced intraepidermal nerve fibre density[26,27] over the duration of diabetes, and a proximal-distal graded loss of myelinated fibre density in the peripheral nerves.[28] Currently, there is no effective therapeutic regimen available for DPN beyond tight blood glucose control which seems difficult to maintain. Exploring the pathogenic process of DPN and pursuing new pharmacological targets are particularly important for new effective therapeutic strategies. In this study, we found that both mechanical and thermal thresholds of diabetic rats significantly decreased, reflecting hyperalgesia symptoms and SC dysfunction in association with diabetes. The cell viability and apoptotic rate of SCs from diabetic and normal rats did not change in high glucose culture condition, but SCs from diabetic rats showed a lower cell viability and a higher apoptotic rate compared with SCs from normal rats. Exposure of SCs from diabetic and non-diabetic rats to high glucose culture condition triggered elevated cellular ROS level and this oxidative stress may be evoked or aggravated by impaired antioxidant defences, as demonstrated by the decreased level of total GSH in SCs. EPO improved cell viability and inhibited

apoptosis of SCs isolated from diabetic rats in normal and high glucose culture conditions. EPO also attenuated oxidative stress by elevating the total GSH level of SCs and decreasing the intracellular ROS level. Moreover, SCs from diabetic rats exhibited higher EPO mRNA and EPOR mRNA levels than normal control rats, regardless of whether the SCs were grown in high or normal glucose culture condition. EPO treatment seemed to have no significant influence on EPO mRNA and EPOR mRNA levels in SCs in normal or high glucose culture condition. DPN is characterised by progressive, length-dependent loss of peripheral nerve axons in a stocking and glove (distal to proximal) pattern,[29] inducing pain, decreased sensation, and finally complete loss of sensation. In agreement with previous observations,[30,31] our data indicated that both mechanical and thermal thresholds significantly reduced four weeks after the induction of diabetes. The mechanical and thermal hyperalgesia persisted throughout the study, reflecting hyperalgesia symptoms and SCs dysfunction in diabetic condition. Because long-term DPN caused severe segmental demyelination and SCs death, the diabetic rats were executed at the relatively early stage of DPN to isolate enough SCs for cell culture. That is why we did not observe the decreased and finally complete loss of sensation. The role of SCs in DPN is confirmed by the proof of segmental demyelination (one special alteration in DPN), the occurrence of onion-bulb structures (an indicator of excessive SCs proliferation), and diminished neurotrophic support


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for axons.[10] Consistent with previous observations that diabetic demyelination is mainly due to the impaired function of SCs,[32] we found SCs dysfunction in the diabetic condition. SCs from diabetic rats showed a lower cell viability and a higher apoptotic rate compared with SCs from normal rats regardless of culture condition. Elevated blood glucose is thought to be origination of mechanisms leading to diabetic neuropathy and plays a pivotal role in this pathology. In-vitro studies with glucose concentrations of 30 ∼ 150 mmol/l showed that several markers of oxidative stress and apoptosis increased in cultured SCs from normal rats or mice.[15,33–35] In agreement with previous observations, our present work demonstrated that exposure of SCs from diabetic and nondiabetic rats to high glucose culture condition triggered elevated level of cellular ROS and this oxidative stress may be evoked or aggravated by impaired antioxidant defences, as demonstrated by the decreased level of total GSH in SCs. Inconsistent with previous studies, we found that cell viability and apoptotic rate of SCs from diabetic and normal rats did not change under high glucose concentration. The failure of high glucose to reduce cell viability and to activate apoptosis in SCs in vitro revealed the importance of the ‘STZ-induced diabetic phenotype’ of SCs, which we proposed accounted for the susceptibility of these SCs to glucose-induced metabolic stress. Whether such SCs had a metabolic memory related to previous high glucose-related stress is unclear.[36] However, these enhanced markers of oxidative stress and apoptosis in SCs isolated from STZ-induced diabetic rats matched well with in-vivo studies on experimentally diabetic rodents.[6,13,14] EPO has been demonstrated to exert neuroprotection in acrylamide and cisplatin toxic neuropathies,[37–39] HIV sensory neuropathy,[40] and experimental diabetic somatic neuropathy.[41,42] Some studies showed that EPO administration ameliorated autonomic and peripheral neuropathies in patients with moderate and advanced DPN.[42,43] However, the results of another study showed that 6 months of EPO administration in DPN patients managed with gabapentin could not improve nerve performance.[44] To investigate the underlying reason for this discrepancy and to explore whether and how EPO had cytoprotective effect against high glucose-induced cytotoxicity, SCs from rats with STZ-induced DPN were exposed to elevated or normal glucose condition. Our data indicated that EPO improved cell viability and decreased cell apoptosis in SCs isolated from diabetic rats in normal and high glucose culture conditions. EPO has been shown to regulate many constituents in the apoptotic cascade to avert cell death.[45] In the current study, EPO also attenuated oxidative stress by elevating the total GSH level of SCs and decreasing the intracellular ROS level. At the cellular level, EPO expression is regulated by oxygen tension rather than by the haemoglobin level.[46] Plasma EPO is often low in diabetic patients with or without anaemia,[47] and the failure to produce EPO in a 1158

declining concentration of red blood cells suggests an impaired EPO response.[48] Another study manifested that some diabetic patients without advanced renal failure had an impaired EPO response to anaemia, which was related to autonomic neuropathy.[49] It is suggested that inflammatory cytokines take part in the impairment of EPO production under diabetic conditions.[50] The results of the current study showed that high glucose culture condition increased EPO mRNA level, but not EPOR mRNA level, in SCs from normal control rats. SCs from diabetic rats exhibited higher EPO mRNA and EPOR mRNA levels than normal control rats, regardless of whether the SCs were grown in high or normal glucose culture condition. EPO treatment seemed to have no significant influence on EPO mRNA or EPOR mRNA level in SCs from diabetic rats under normal and high glucose culture conditions. We inferred that during high glucose-induced free radical exposure, EPO and EPOR expression increased and maintained to attenuate the oxidative stress and to provide cellular protection against apoptotic cell death. EPO administration seemed to slightly decrease the EPO mRNA level and increase the EPOR mRNA level, although these changes were not significant due to the dose and action time of exogenous EPO.

Conclusions The data of this study indicated that EPO improved cell viability and decreased apoptosis of SCs isolated from diabetic rats in high and normal glucose culture conditions. EPO also attenuated oxidative stress by elevating total GSH level and by decreasing intracellular ROS level of SCs. SCs from diabetic rats exhibited higher EPO mRNA and EPOR mRNA levels than SCs from normal control rats, regardless of whether the SCs were grown in high or normal glucose culture condition. EPO treatment seemed to have no significant influence on the EPO mRNA or EPOR mRNA level in SCs in normal and high glucose culture conditions. These findings offer fresh viewpoints for interpreting the pathogenesis of DPN and novel pharmacological principles implicit in the therapeutic effect of EPO.

Declarations Conflict of interest The Author(s) declare(s) that they have no conflicts of interest to disclose.

Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81000517) and the Natural Science Foundation of Shandong Province of China (No. ZR2011HQ011).


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Fuzi attenuates diabetic neuropathy in rats and protects schwann cells from apoptosis induced by high glucose.

Rosuvastatin attenuates contrast-induced nephropathy through modulation of nitric oxide, inflammatory responses, oxidative stress and apoptosis in diabetic male rats.

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Attenuation of Oxidative Stress-Induced Cell Apoptosis in Schwann RSC96 Cells by Ocimum Gratissimum Aqueous Extract.

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Glycolaldehyde induces endoplasmic reticulum stress and apoptosis in Schwann cells.

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Cissus quadrangularis extract attenuates hyperglycaemia-mediated oxidative stress in streptozotocin-induced diabetic rats.

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Ginsenoside Rg1 ameliorates oxidative stress and myocardial apoptosis in streptozotocin-induced diabetic rats.

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Erythropoietin Modification Enhances the Protection of Mesenchymal Stem Cells on Diabetic Rat-Derived Schwann Cells: Implications for Diabetic Neuropathy.

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Yacon (Smallanthus sonchifolius) Leaf Extract Attenuates Hyperglycemia and Skeletal Muscle Oxidative Stress and Inflammation in Diabetic Rats.

Caffeic acid phenethyl ester attenuates ionize radiation-induced intestinal injury through modulation of oxidative stress, apoptosis and p38MAPK in rats.

Salvianolic acid B attenuates lipopolysaccharide-induced acute lung injury in rats through inhibition of apoptosis, oxidative stress and inflammation.