Available online at www.sciencedirect.com
ScienceDirect Journal of Nutritional Biochemistry xx (2014) xxx – xxx
Proanthocyanidins protect against early diabetic peripheral neuropathy by modulating endoplasmic reticulum stress☆ Ye Ding, Xiaoqian Dai, Zhaofeng Zhang, Yanfei Jiang, Xiaotao Ma, Xiaxia Cai, Yong Li⁎ Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Beijing 100191, PR China
Received 18 July 2013; received in revised form 16 February 2014; accepted 6 March 2014
Abstract Diabetic peripheral neuropathy (DPN) is the most common and troublesome complication of type 2 diabetes mellitus (T2DM). Recent findings reveal an important role of endoplasmic reticulum (ER) stress in the development of DPN and identify a potential new therapeutic target. Schwann cells (SC), the myelinating cells in peripheral nervous system, are highly susceptible to ER homeostasis. Grape seed proanthocyanidins (GSPs) have been reported to improve DPN of type 1 diabetic rats and relieve ER stress in skeletal muscles and pancreas of T2DM. We investigated the potential role of ER stress in SC in regulating DPN of T2DM and assessed whether early intervention of GSPs would prevent DPN by modulating ER stress. The present study was performed in Sprague–Dawley rats made T2DM with low-dose streptozotocin and a high-carbohydrate/high-fat diet and in rat SC cultured in serum from type 2 diabetic rats. Diabetic rats showed a typical characteristic of T2DM and slowing of nerve conduction velocity (NCV) in sciatic/tibial nerves. The lesions of SC, Ca2+ overload and ER stress were present in sciatic nerves of diabetic rats, as well as in cell culture models. GSPs administration significantly decreased the low-density lipoprotein level and increased NCV in diabetic rats. GSPs or their metabolites also partially prevented cell injury, Ca2+ overload and ER stress in animal and cell culture models. Therefore, ER stress is implicated in peripheral neuropathy in animal and cell culture models of T2DM. Prophylactic GSPs treatment might have auxiliary preventive potential for DPN partially by alleviating ER stress. © 2014 Elsevier Inc. All rights reserved. Keywords: Calcium; Diabetes; Endoplasmic reticulum stress; Neuroprotection; Proanthocyanidins; Schwann cells
1. Introduction Diabetic peripheral neuropathy (DPN) is the most common and troublesome complication of diabetes, leading to the greatest disability and mortality [1]. As changing lifestyles lead to increased caloric consumption and reduced physical activity, DPN continues to increase in numbers and significance in countries where the prevalence of type 2 diabetes mellitus (T2DM) is rising [2]. Incidence and prevalence of DPN increase with age and increasing duration of diabetes [3]. Once nerve damage is done, it is almost totally irreversible [4]. Moreover, up to 50% of DPN may be asymptomatic, and patients are at risk of insensate injury to their feet [5]. Therefore, the early recognition and prevention of DPN is the best strategy and may result in a reduced incidence of ulceration and consequently amputation. The observation that the lesions of Schwann cells (SC) existed earlier than ongoing demyelination and axonal degeneration highlights the significance of SC in the pathogenesis of DPN [6]. As an extension of their plasma membrane, SC generate unique, lipid-rich multilamellar myelin sheaths to ensure successful conduction of the ☆ This work was supported by the research grants from National Natural Science Foundation of PR China (81072293). ⁎ Corresponding author. Tel./fax: +86-10-82801177. E-mail address: [email protected] (Y. Li).
http://dx.doi.org/10.1016/j.jnutbio.2014.03.007 0955-2863/© 2014 Elsevier Inc. All rights reserved.
action potential along axons and maintain the axonal integrity [7]. During the active phase of myelination, SC must synthesize tremendous amounts of myelin membrane proteins, cholesterol and membrane lipids through the secretory pathway [8]. Therefore, SC are highly sensitive to perturbations of the secretory pathway, particularly endoplasmic reticulum (ER) homeostasis [9]. Recent studies have demonstrated that ER stress is emerging as an important mechanism of metabolic diseases [10,11]. Based on diabetic models, the contribution of ER stress in target organs (pancreatic β-cells, liver, adipose tissue and skeletal muscle) to the pathogenesis of diabetes has been well established [12–16]. Within the context of diabetic complications, ER stress has also been demonstrated to be linked to retinopathy, cardiomyopathy, nephropathy, endothelial dysfunction and neuropathy in central nervous system [17–21]. Furthermore, recent in vivo studies have reported that ER stress plays a key role in the pathogenesis of peripheral neuropathy in prediabetic and type 1 diabetic animals [22,23]. However, much less attention has been focused on the effect of ER stress on lesions of SC in models of DPN. Indeed, it has become increasingly clear that ER stress in myelinating cells is an important feature of chemically induced and inherited neuropathies [9,24,25]. Given an in vitro study of palmitic acid and high-glucose-induced SC that supports the hypothesis linking ER stress in SC and DPN [26], the relevant mechanisms of ER stress on DPN need further elucidation.
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Natural plant components with minimal or no side effects are being pursued as alternatives to pharmaceutical interventions. Grape seed proanthocyanidins (GSPs), which are derived from grape seeds, refer to a group of proanthocyanidins mostly containing dimers, trimers and other oligomers of catechin and epicatechin and their gallic acid esters. A previous study confirmed that 6 months of 250 or 500 mg/kg body weight (BW) GSPs treatment was safe and did not cause any detrimental effects in vivo [27]. Previous studies based on chemically induced type 1 diabetic animals reported that GSPs exerted anti-diabetic property [28,29]. Our recent study also found that GSPs partially ameliorated hyperglycemia and insulin resistance in type 2 diabetic rats in part by alleviation of ER stress in skeletal muscles and pancreas [15,16]. Moreover, GSPs showed activities that improved type 1 diabetic complications, including cardiomyopathy, nephropathy, retinopathy and neuropathies in central and peripheral nervous systems [30–34]. However, the effect of GSPs on the peripheral nerves of T2DM has not been elaborated. In the present study, using Sprague–Dawley rats made T2DM with low-dose streptozotocin and a high-carbohydrate/high-fat diet, we aimed to investigate whether early intervention of GSPs would result in the prevention of early structural and functional abnormalities in sciatic nerves and to assess whether ER stress in sciatic nerves was the target of GSPs. Meanwhile, given the previous finding that only (+)catechin (C) and (−)-epicatechin (EC) were identified in both rat plasma and nervous system after oral administration of GSPs [35], we used rat SC cultured in serum from type 2 diabetic rats to establish the contribution of ER stress in lesions of SC and to clarify whether the pretreatment of C and EC could protect SC via alleviating ER stress. 2. Methods and materials 2.1. Reagents GSPs (Lot No: 1003007–24) were purchased from Jianfeng Natural Products Co. Ltd. (Tianjin, China). The proanthocyanidin content was 96.64% while analyzed using HPLC with gas chromatography/MS detection. They contained 6.10% C, 6.78% EC, 55.59% dimeric forms, 11.91% trimeric forms, 6.55% tetrameric forms and small amounts of other polymeric forms. Basal diet and the high-carbohydrate/high-fat diet (66% basal diet, 15% lard, 10% plantation white sugar, 3% yolk powder and 6% casein) were produced by Beijing Keao Xieli Co. Ltd. (Beijing, China). Streptozotocin, C, EC, methyl thiazolyl tetrazolium (MTT) and tunicamycin were from Sigma-Aldrich (St. Louis, MO, USA). Free Ca2+ assay kit was from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). The lactate dehydrogenase (LDH) release assay kit and Fluo3 AM were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). FITCconjugated Annexin V and propidium iodide (PI) were obtained as a kit from Roche Molecular Biochemicals (Indianapolis, IN, USA). The antibodies for glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), Caspase-12, JNK, phosphorylated JNK (phospho-JNK) and β-Actin were obtained from Santa Cruz Biotechnology (California, USA). 2.2. Animal treatments
centrifugation (3500 rpm for 10 min at 4°C) and used for analysis of insulin and lipids. The level of insulin was determined by means of radioimmune assay. The levels of triglyceride (TG), cholesterol (CHO), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were determined by automatic biochemistry analyzer. 2.4. Measurement of nerve conduction velocity (NCV) Sixteen weeks after induction of diabetes, NCV was detected through a Pclab-UE biomedical signal acquisition and processing system (Beijing Microsignalstar Technology Development Co. Ltd., China). After being deeply anesthetized with 10% chloral hydrate, rats were killed by cervical dislocation. The left sciatic/tibial nerve of each rat was rapidly dissected with a glass needle and placed in an insulated box. Nerve conduction was made and recorded as previously reported [34]. The sciatic nerves were then dissected, frozen in liquid nitrogen and stored at −80°C for Western blot. 2.5. Pathological observation and measurement of free Ca2+ in sciatic nerves Following the NCV measurement, the right sciatic nerve of each rat was also removed. Half of nerves in each group were immediately frozen in liquid nitrogen and stored separately at −80°C for the measurement of the concentration of free Ca2+, which was assayed according to the manufacturer's protocol and expressed as nmol/ mg protein. The other half nerves were used for electron microscopic analysis. A small portion of the same region from each group was immersed overnight in 2.5% glutaraldehyde (pH 7.4). Following embedding in Epon 812, microsections were cut and stained on the copper grid with 4% uranyl acetate and Reynold's lead citrate. The ultrastructure was then checked from magnification 4000× to magnification 12,000×. 2.6. Cell culture and treatments RSC96 SC were obtained from the American Type Culture Collection and cultured as previously described [36]. When grown upon reaching 70% confluence, cells were preincubated in serum-free medium for 4 h prior to each experiment. To induce cell injury, cells were challenged with DMEM containing 10% serum from diabetic rats (DRS). Cells treated with 10% serum from healthy rats (HRS) were used as controls. Ten healthy male Sprague–Dawley rats and 20 diabetic rats, which were induced by the same method as mentioned above, were used for serum extracts. The serum of rats was collected as previously reported [37]. In this set of experiments, cells were pretreated with different doses of C/EC mixtures (5, 10 and 20 μmol/L, respectively) for 3 h prior to 10% serum stimulation and continuously treated with 10% serum and different doses of C/EC mixtures. The classical ER stress activator tunicamycin (5 μg/ml) was added during the last 12 h of treatment (Fig. 1b). 2.7. Evaluation of cell injury Cell viability was evaluated by MTT assay. Cells were washed twice with PBS and cultured in phenolsulfonphthalein-free DMEM with MTT (1 g/L) at 37°C for 4 h. Blue formazan crystals were then resolved with DMSO. Absorbance was measured at 595 nm by a microplate reader using DMSO as the blank. Cell lysis was determined by LDH release assay. After the culture supernatant was collected, 1% Triton X-100 was added to cells to release all LDH. LDH in both culture supernatant and cell lysate were quantified by measuring the absorbance at 490 nm using a microplate reader. Assay was performed according to the manufacturer's instructions. The percent LDH release was calculated as follows: LDH release (%)=(LDH in culture supernatant/LDH in culture supernatant+LDH in cell lysate)×100. Cell apoptosis/necrosis was measured by Annexin V/PI double staining. Prepared cells (1×106) were washed with PBS and resuspended in binding buffer and then incubated for 10 min at room temperature in the dark with a FITC-conjugated, antiAnnexin V antibody and PI. Flow cytometric analysis was performed within 30 min of staining. The extent of apoptosis/necrosis observed was reported as a percentage using the formula: apoptotic/necrotic fraction (%)=[(Annexin V+PI−events)+(Annexin V +PI+events)]/[(Annexin V+PI−events)+(Annexin V+PI+events)+(Annexin V−PI −events)]×100.
All animal care and experimental procedures followed the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 85–23, revised 1996) and the protocols approved by the Institutional Animal Care and Use Committee of Peking University. Fifty male Sprague–Dawley rats (180–200 g), obtained from Animal Service of Health Science Center (Peking University), were housed 2 per cage and randomly divided into 4 groups. Group 1 (n=12, vehicle-treated normal ones) and group 2 (n=12, 250 mg/kg BW GSPs-treated normal ones) were both fed the basal diet; group 3 (n=13, vehicle-treated diabetic ones) and group 4 (n=13, 250 mg/kg BW GSPs-treated diabetic ones) were induced by 8 weeks of the high-carbohydrate/highfat diet and 2 injections of 25 mg/kg BW streptozotocin as described previously [15,16]. During the experimental period, the vehicle-treated normal and diabetic groups were given water, and the other two groups were given GSPs by stomach tube. After induction of diabetes, all groups were allowed free access to a basal diet (Fig. 1a). BW and food intake were measured once a week.
After digestion with trypsin, cells were harvested by centrifugation. For loading with Fluo-3, cells were incubated for 1 h at 37°C with Fluo-3 AM (5 μmol/L) in the dark. Then, cells were rinsed twice in Ca2+- and Mg2+-free PBS and analyzed with a FACScan flow cytometer. Excitation was from an argon laser at 488 nm. Emission at 530 nm (bandwidth 30 nm) was measured on a linear scale. Raw data for free Ca2+ content were expressed as mean fluorescence intensity.
2.3. Measurement of blood parameters
2.9. Western blot
Blood samples were collected at the end of 1 week and 16 weeks after induction of diabetes. The 6-h fasted rats were deeply anesthetized with 10% chloral hydrate. Blood samples were then collected from snipped tails by tail milking. Plasma glucose was immediately determined using a rapid glucometer. Serum was separated by
Forty micrograms of protein extracts from sciatic nerves or SC was separated by PAGE and immunoblotting was performed with antibodies for GRP78, CHOP, Caspase12, phospho-JNK, JNK and β-Actin. Relative protein quantification was done with Quantity One software.
2.8. Measurement of cytoplasmic free Ca2+
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Fig. 1. Schematic representation of the experimental procedures. The present study was performed in Sprague–Dawley rats made diabetic with low-dose streptozotocin and a highcarbohydrate/high-fat diet (a) and in rat SC cultured in serum from type 2 diabetic rats (b). C, (+)-catechin; EC, (−)-epicatechin.
2.10. Statistical analysis
3.3. Electron microscopic analysis of sciatic nerves
Values of Pb.05 were considered statistically significant. Results are presented as means±S.E.M. All data were statistically analyzed by one-way analysis of variance, followed by LSD (equal variances assumed) or Dunnett's T3 (equal variances not assumed) for multiple comparisons.
As depicted in Fig. 2, normal nucleus, mitochondria and ER in SC and normal structure and morphology of myelin sheath with moderate homogenous electron density were seen in GSPs treated and untreated normal control rats. However, sciatic nerves showed evidence of damage in diabetic control rats, including swelling and dilatation of mitochondria and ER in SC and deranged myelin sheath with lighter electron density. The protective effect of GSPs on diabetic rats was evident with moderate increases in normal mitochondria and ER. Regretfully, treatment of diabetic rats with GSPs only improved morphology of myelin sheath to a certain extent, and myelin sheath was still slightly splitting under electron microscope.
3. Results 3.1. Physical and biochemical characteristics in normal and diabetic rats As shown in Table 1, there were no differences in food consumption, plasma glucose, serum insulin, TG, CHO, LDL and HDL between normal and GSPs control groups throughout the study period, whereas the level of BW was significantly lower in GSPs control rats than that in normal control ones at the end of the study. As expected, a high-carbohydrate/high-fat diet for 8 weeks accompanying by low-dose streptozotocin twice injection significantly increased food consumption, plasma glucose, serum insulin, TG, CHO and LDL in diabetic rats when compared with normal control rats. At the end of the study, diabetic rats still showed a typical characteristic of T2DM as emaciation, polyphagia, hyperglycemia, hyperinsulinemia and blood lipid disorder. However, the GSPs treatment slightly improved the raised diabetic parameters, among which the difference in the level of LDL was statistically significant when compared with diabetic control ones.
3.4. The concentration of free Ca2+ and ER stress markers in sciatic nerves As shown in Table 1 and Fig. 3, the concentration of free Ca2+ and ER stress markers (GRP78, CHOP, phospho-JNK, total JNK and cleaved Caspase-12) were all expressed at low levels in both normal and GSPs control groups. However, these stress markers were all significantly elevated in sciatic nerves of diabetic controls when compared with normal controls. The GSPs treatment slightly reduced the concentration of free Ca2+ and ER stress markers, among which the expression of CHOP was statistically decreased when compared with diabetic control group.
3.2. NCV of sciatic/tibial nerves
3.5. Cell injury in RSC96 SC
NCV of sciatic/tibial nerves in GSPs control group was similar to that in normal control rats. However, NCV was significantly reduced in diabetic control rats when compared with normal control rats. Interestingly, administration of GSPs to diabetic rats significantly increased the level of NCV (Table 1).
As indicated in Fig. 4, cell viability, the extents of LDH release and apoptosis/necrosis fraction in cells treated with HRS and metabolites of GSPs were similar to that in cells only treated with HRS. However, in contrast with the control group, exposure to DRS significantly decreased cell viability and increased cell lysis and cell apoptosis/
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Table 1 BW, food intake, plasma glucose, serum insulin, TG, CHO, LDL, HDL, NCV and free Ca2+ content of sciatic nerves in normal and diabetic rats Parameter
Normal rats Vehicle
At the end of 1 week after induction of diabetes BW (g) 470.13±11.30 Food intake (g/day) 19.48±0.61 Plasma glucose (mg/dl) 81.90±4.99 Serum insulin (mIU/L) 21.27±0.79 TG (mmol/L) 1.37±0.14 CHO (mmol/L) 1.87±0.11 LDL (mmol/L) 0.35±0.03 HDL (mmol/L) 1.64±0.12 At the end of 16 weeks after induction of diabetes BW (g) 595.38±8.08 Food intake (g/day) 22.45±1.62 Plasma glucose (mg/dl) 103.5±3.97 Serum insulin (mIU/L) 19.71±2.67 TG (mmol/L) 1.20±0.09 CHO (mmol/L) 2.23±0.14 LDL (mmol/L) 0.42±0.04 HDL (mmol/L) 1.80±0.11 NCV (m/s) 48.26±1.19 Free Ca2+ (nmol/mg pro) 3.20±0.33
Diabetic rats GSPs
Vehicle
GSPs
461.13±15.81 18.85±0.59 84.29±4.23 21.88±1.02 1.32±0.14 1.88±0.09 0.32±0.04 1.60±0.12
453.88±20.46 30.32±0.89 ⁎⁎ 284.40±12.37 ⁎⁎ 29.02±2.14 ⁎⁎ 3.51±0.31 ⁎⁎ 2.70±0.17 ⁎⁎ 0.91±0.07 ⁎⁎
445.75±17.03 29.45±1.39 ⁎⁎ 283.05±15.14 ⁎⁎ 28.77±1.29 ⁎⁎ 3.53±0.36 ⁎⁎ 2.72±0.21 ⁎⁎ 0.89±0.07 ⁎⁎
553.50±10.91 ⁎ 20.85±1.81 95.63±3.97 21.03±2.01 1.18±0.10 2.13±0.15 0.35±0.04 1.77±0.08 46.25±1.04 3.12±0.35
415.00±20.03 ⁎⁎ 38.93±1.15 ⁎⁎ 381.15±21.08 ⁎⁎ 34.27±3.62 ⁎⁎ 2.42±0.17 ⁎⁎ 3.85±0.35 ⁎ 1.41±0.13 ⁎⁎ 2.41±0.25 ⁎ 39.48±1.02 ⁎⁎ 5.77±0.49 ⁎
1.81±0.14
1.76±0.11 443.13±11.82 35.92±1.38 331.65±19.76 32.32±4.44 2.19±0.17 3.37±0.25 0.92±0.09 # 2.55±0.16 43.46±1.86 # 5.17±0.31
Data were means±S.E.M. of 8 rats of each group. ⁎ Pb.05 vs. vehicle-treated normal rats. ⁎⁎ Pb.01 vs. vehicle-treated normal rats. # Pb.05 vs. vehicle-treated diabetic rats.
Fig. 2. Pathological observations on sciatic nerves of normal and diabetic rats. Scale bar=1 μm. Ax, axon; C, control rats; D, diabetic rats; MC, mitochondria; MNF, myelinated nerve fiber; N, nucleus.
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Fig. 3. Effect of GSPs on ER stress in sciatic nerves of normal and diabetic rats. An aliquot of protein extracts was subject to Western blot analysis using the 78-kDa glucose-regulated protein (GRP78), CHOP, Caspase-12, JNK and phospho-JNK antibodies. β-Actin protein levels were used as a control (a). A ratio between specific protein and β-Actin (or JNK) was calculated as means±S.E.M. of 3 determinations from 3 individual rats in each group (b–e). ⁎Pb.05 and ⁎⁎Pb.01 vs. vehicle-treated control rats, #Pb.05 and ##Pb.01 vs. vehicle-treated diabetic rats. C, control rats; D, diabetic rats.
necrosis. Interestingly, C and EC pretreatment partially ameliorated cell injury in DRS-treated cells, among which the differences between cells treated with DRS, C (10 μmol/L) and EC (10 μmol/L) and cells only treated with DRS were statistically significant.
3.6. Ca2+ overload and ER stress markers in RSC96 SC The mean fluorescence intensity of cytoplasmic free Ca2+ and the low levels of ER stress markers in cells that were treated with HRS were not significantly different with or without metabolites of GSPs
(Figs. 4 and 5). However, in response to DRS, the free Ca2+ content and ER stress markers (GRP78, CHOP, phospho-JNK and cleaved Caspase-12) were all significantly elevated when compared with control group. The mixture of C and EC blocked the increase in cytoplasmic free Ca2+ content in DRS-treated cells. The inhibitory rates of 2.5, 5 and 10 μmol/L of the mixture reached 5.40%, 8.77% and 10.98%, respectively. Furthermore, The pretreatment with the mixture (at the dose of 5 and 10 μmol/L, respectively) for 3 h prior to DRS stimulation also clearly alleviated ER stress. The expressions of GRP78 and phospho-JNK were statistically decreased when compared with cells only treated with DRS. Moreover, the activation of ER stress
Fig. 4. Comparison of cell injury (a–c) and Ca2+ overload (d) in RSC96 SC. Cell injury was evaluated by MTT assay (a), LDH release assay (b) and Annexin V/PI double staining (c). For MTT and LDH release analysis, data were means±S.E.M. from 10 experiments. The other data were means±S.E.M. from 8 experiments. ⁎Pb.05 and ⁎⁎Pb.01 vs. only HRS-treated cells, # Pb.05 and ##Pb.01 vs. only DRS-treated cells. C, (+)-catechin; EC, (−)-epicatechin.
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Fig. 5. Effects of (+)-catechin (C) and (−)-epicatechin (EC) on ER stress in RSC96 SC. An aliquot of protein extracts was subject to Western blot analysis using the 78-kDa glucoseregulated protein (GRP78), CHOP, Caspase-12, JNK and phospho-JNK antibodies. β-Actin protein levels were used as a control (a). A ratio between specific protein and β-Actin (or JNK) was calculated as means±S.E.M. of 3 independent experiments (b–e). ⁎Pb.05 and ⁎⁎Pb.01 vs. only HRS-treated cells, #Pb.05 and ##Pb.01 vs. only DRS-treated cells.
by pharmacological approach was also observed. Tunicamycin (5 μg/ ml), the classical ER stress activator, reversed the inhibition of ER stress induced by C/EC mixtures in DRS-treated cells, among which the difference in the expression of phospho-JNK was statistically significant when compared with DRS-treated cells with C (10 μmol/L) and EC (10 μmol/L) treatment. Interestingly, unlike in vivo study as mentioned above, the expression of total JNK remained mainly unchanged among these groups in vitro. 4. Discussion The findings reported herein support the pathogenetic role of SC in DPN and the lesions of SC in animal and cell culture models of T2DM are partially associated with ER stress. Furthermore, the incomplete but significant prevention of neuropathy and cell injury by GSPs or their metabolites suggests that GSPs might have auxiliary therapeutic potential for DPN partially by alleviating Ca2+ overload and ER stress in SC. In the present study, the animal model induced by a highcarbohydrate/high-fat diet for 8 weeks accompanying by low-dose streptozotocin twice injection is an excellent model of T2DM because it develops polyphagia, hyperglycemia, hyperinsulinemia and blood lipid disorder throughout the study period. In T2DM, reduction NCV is one of the earliest neuropathic abnormalities [38]. Diabetic rats exhibiting the characteristic nerve conduction slowing in sciatic/tibial nerves 16 weeks after induction of diabetes comprise a suitable model for exploration of new pathogenetic mechanisms of DPN. Researchers commonly comment that axon and Schwann cell deficits appear to proceed independently of each other [7]. In accordance with this comment, ultrastructural lesions were found in myelin sheath and SC, but no lesion was observed in axons in the present study. Although some in vivo studies have revealed diabetes-induced typical apoptotic nuclear and cytoplasmic changes in SC of streptozotocin-diabetic animals, many investigators have resisted the concept of apoptotic processes [39–41]. Indeed, little consistent evidence remains for morphological changes in peripheral nerves of diabetic models,
which most likely results from multiple factors, including animal species and age, small sample size, changes in dosage and injection techniques of streptozotocin, differences in time points of observation and duration of hyperglycemia. In the present study, we only found swelling and dilatation of mitochondria and ER, but without apparent chromatic agglutination in SC 16 weeks after induction of diabetes. In order to explore the detailed pathogenetic role of SC in DPN, we studied in vitro using Schwann cell line RSC96, which was used to study peripheral nerve injury previously [42,43]. Exposure of SC to 10% serum from type 2 diabetic rats significantly decreased cell viability and increased cell death. The findings are in agreement with those in DRG neurons cultured in 15% serum from a type 2 diabetic patient with neuropathy [44]. Previous studies mainly observed SC under hyperglycemic and/or hyperlipidemic conditions. Hyperglycemic (150 mmol/L for 24 h) defined media showed nuclear blebbing of apoptotic cells in SC [40]. The percentage of apoptotic cells was also increased after incubation with palmitate (0.05–0.50 mmol/L) for 48 h [45]. However, the results that hyperglycemia (60 mmol/L for 4 days) does not cause death of SC also existed [46]. Based on recent findings that chronic hyperglycemia alone (50 mmol/L for 24–48 h) did not induce cell death but its presence strongly magnified the effects of lipotoxic injury in SC [26], we hypothesize that some factors might present in the serum of diabetics (e.g., lipid overload and autoimmune immunoglobulin), in addition to hyperglycemia per se, and result in an override of the cell defensive response and contribute to cell death. In the past, the effect of GSPs on DPN was only studied in streptozotocin-induced type 1 diabetic rats. It was shown that 24 weeks of GSPs (250 mg/kg BW) treatment prevented against structural and functional abnormalities in peripheral nerves, including increasing NCV, decreasing the severe segmental demyelination and improving impaired SC in sciatic nerves [34]. In the present study, oral supplementation with the same dosage of GSPs for 24 weeks prevented nerve conduction slowing and structural abnormalities of SC (mainly swelling and dilatation of mitochondria and ER) despite
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only partial preservation of myelin sheath in peripheral nerves of type 2 diabetic rats. Moreover, the treatment of metabolites of GSPs achieved a partial but significant prevention of cell injury in SC cultured in serum from type 2 diabetic rats. These data suggest that although peripheral nerves might not have maintained completely normal function, prophylactic GSPs treatment may be efficacious in delaying the development of DPN in T2DM. We also investigated the effects of GSPs or their metabolites on normal peripheral nerves or SC. It was shown that GSPs or their metabolites in normal sciatic nerves or SC were safe under the conditions investigated in this study. Considerable evidence suggests that impaired cellular Ca2+ homeostasis, largely due to dysfunction of the intracellular Ca2+ stores such as ER and mitochondria, is an early molecular marker linked to the onset of DPN [47]. Our results are basically in agreement with this point. Elevated free Ca2+ content in sciatic nerves was existed 16 weeks after induction of diabetes. Previous in vitro studies showed that palmitate in the presence of high glucose resulted in an early and more robust release of Ca2+ into the cytosol from the ER in SC [26]. Moreover, Ca2+ influx was enhanced by the exposure of DRG neurons to 10% serum from diabetic BB/W rats for 18–24 h or 15% serum from a type 2 diabetic patient with neuropathy for 24 h [44,48]. In a similar manner, we showed that cytoplasmic free Ca2+ content was significantly elevated in DRS-treated SC. Although the precise mechanisms how cellular Ca2+ homeostasis is altered by diabetes and how the alteration contributes to the development of DPN remain to be elucidated, amelioration of abnormal Ca2+ homeostasis can be a therapeutic target in DPN. For example, a previous study found that reducing excessive cytosolic Ca2+ levels induced by palmitate in the presence of high glucose using the calcium chelator inhibited the cell death process in SC [26]. Our results indicated that metabolites of GSPs decreased cytoplasmic free Ca2+ content in DRS-treated SC with an apparent dose dependency, which may be one of its mechanisms of cytoprotective action. Unfortunately, GSPs treatment only partially reduced Ca2+ overload in sciatic nerves, suggesting that the dosage used in the present study may not have been sufficient to achieve a complete prevention and studies are still needed to address the action of GSPs in this regard. A number of cell stress conditions, such as hyperglycemia, dyslipidemia and abnormal cellular Ca2+ homeostasis in T2DM, will disrupt ER homeostasis and subsequently lead to the accumulation of unfolded or misfolded proteins in the ER lumen [10]. This activates an adaptive unfolded protein response (UPR), which aims to reconcile the imbalance. One of the most described mechanisms of UPR activation is the competition model, in which the ER chaperone protein GRP78 plays an essential role in the activation of different ER stress transducers [10]. If the imbalance persists, the UPR can ultimately lead to cell death, which is mediated in part by increased expression of CHOP and activities of JNK and Caspase-12 [11]. Based on Zucker rats, high fat diet-fed mice and streptozotocin-induced type 1 diabetic rats, ER stress manifest in up-regulation of the expressions of GRP78 and CHOP was identified in sciatic nerves [22,23]. Like in vivo studies, previous in vitro studies under hyperglycemic and/or hyperlipidemic conditions mainly showed that the expressions of ER stress genes and proteins (GRP78 and CHOP) were altered [26,45]. Intriguingly, in addition to the expressions of GRP78 and CHOP, we also found that the activities of JNK and Caspase-12 were all significantly elevated in both sciatic nerves and DRS-treated SC. These significant findings further confirm the important role of ER stress in DPN. So far, little information is available describing the effect of beneficial components of plants on ER stress in DPN. To our knowledge, the only one of phytochemicals, which was demonstrated to improve peripheral neuropathy by alleviating ER stress in a inherited neuropathic model, was curcumin [49]. In the present study, elevated ER stress markers (especially the expression of CHOP and the
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activity of Caspase-12) in peripheral nerves of type 2 diabetic rats were partially reversed by GSPs treatment. This is consistent with the results of our previous study, which indicated that the same dosage of GSPs mainly regulated the same ER stress markers in skeletal muscles of type 2 diabetic rats [15]. Indeed, inhibition of ER stress by chemicals or gene knockout technology was demonstrated to alleviate peripheral nerve dysfunction in prediabetic and type 1 diabetic animals [22,23]. Importantly, the results of our in vitro study further showed that metabolites of GSPs markedly inhibited elevated ER stress markers (especially the expression of GRP78 and the activities of JNK and Caspase-12) in rat SC cultured in serum from type 2 diabetic rats, and this activity was disrupted by ER stress activator. Although not measured in detail in the present study, these series of events would confirm that GSPs might have auxiliary preventive potential for DPN partially by alleviating ER stress. The present study has some unanswered questions that should be included in further studies. First, the results of ER stress markers in vivo were not equal to that in vitro under both GSPs (or metabolites of GSPs) treated and untreated diabetic models. Second, previous studies indicated that CHOP activity in SC was distinct from other cell types and it only promoted demyelination in the absence of cell death [24]. However, our data and other in vitro studies based on hyperglycemic and/or hyperlipidemic conditions demonstrated an increase in apoptotic cells and expression of significant level of CHOP in SC [26,45]. Third, it was in dispute as to whether the short-term exposure of SC to 10% serum from type 2 diabetic rats in culture could adequately simulate the condition of human diabetic complications. Finally, the present experiment was preventive, and there was incomplete protection of nerve function by GSPs, it would be of practical interest to ascertain whether GSPs could reverse established neuropathic changes.
Acknowledgments The authors would like to thank Lei Bao, Yujie Li, Feng Zhang, Lulu Jing and Jiaojiao Gu for their assistance in the experiment.
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ScienceDirect Journal of Nutritional Biochemistry xx (2014) xxx – xxx
Proanthocyanidins protect against early diabetic peripheral neuropathy by modulating endoplasmic reticulum stress☆ Ye Ding, Xiaoqian Dai, Zhaofeng Zhang, Yanfei Jiang, Xiaotao Ma, Xiaxia Cai, Yong Li⁎ Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Beijing 100191, PR China
Received 18 July 2013; received in revised form 16 February 2014; accepted 6 March 2014
Abstract Diabetic peripheral neuropathy (DPN) is the most common and troublesome complication of type 2 diabetes mellitus (T2DM). Recent findings reveal an important role of endoplasmic reticulum (ER) stress in the development of DPN and identify a potential new therapeutic target. Schwann cells (SC), the myelinating cells in peripheral nervous system, are highly susceptible to ER homeostasis. Grape seed proanthocyanidins (GSPs) have been reported to improve DPN of type 1 diabetic rats and relieve ER stress in skeletal muscles and pancreas of T2DM. We investigated the potential role of ER stress in SC in regulating DPN of T2DM and assessed whether early intervention of GSPs would prevent DPN by modulating ER stress. The present study was performed in Sprague–Dawley rats made T2DM with low-dose streptozotocin and a high-carbohydrate/high-fat diet and in rat SC cultured in serum from type 2 diabetic rats. Diabetic rats showed a typical characteristic of T2DM and slowing of nerve conduction velocity (NCV) in sciatic/tibial nerves. The lesions of SC, Ca2+ overload and ER stress were present in sciatic nerves of diabetic rats, as well as in cell culture models. GSPs administration significantly decreased the low-density lipoprotein level and increased NCV in diabetic rats. GSPs or their metabolites also partially prevented cell injury, Ca2+ overload and ER stress in animal and cell culture models. Therefore, ER stress is implicated in peripheral neuropathy in animal and cell culture models of T2DM. Prophylactic GSPs treatment might have auxiliary preventive potential for DPN partially by alleviating ER stress. © 2014 Elsevier Inc. All rights reserved. Keywords: Calcium; Diabetes; Endoplasmic reticulum stress; Neuroprotection; Proanthocyanidins; Schwann cells
1. Introduction Diabetic peripheral neuropathy (DPN) is the most common and troublesome complication of diabetes, leading to the greatest disability and mortality [1]. As changing lifestyles lead to increased caloric consumption and reduced physical activity, DPN continues to increase in numbers and significance in countries where the prevalence of type 2 diabetes mellitus (T2DM) is rising [2]. Incidence and prevalence of DPN increase with age and increasing duration of diabetes [3]. Once nerve damage is done, it is almost totally irreversible [4]. Moreover, up to 50% of DPN may be asymptomatic, and patients are at risk of insensate injury to their feet [5]. Therefore, the early recognition and prevention of DPN is the best strategy and may result in a reduced incidence of ulceration and consequently amputation. The observation that the lesions of Schwann cells (SC) existed earlier than ongoing demyelination and axonal degeneration highlights the significance of SC in the pathogenesis of DPN [6]. As an extension of their plasma membrane, SC generate unique, lipid-rich multilamellar myelin sheaths to ensure successful conduction of the ☆ This work was supported by the research grants from National Natural Science Foundation of PR China (81072293). ⁎ Corresponding author. Tel./fax: +86-10-82801177. E-mail address: [email protected] (Y. Li).
http://dx.doi.org/10.1016/j.jnutbio.2014.03.007 0955-2863/© 2014 Elsevier Inc. All rights reserved.
action potential along axons and maintain the axonal integrity [7]. During the active phase of myelination, SC must synthesize tremendous amounts of myelin membrane proteins, cholesterol and membrane lipids through the secretory pathway [8]. Therefore, SC are highly sensitive to perturbations of the secretory pathway, particularly endoplasmic reticulum (ER) homeostasis [9]. Recent studies have demonstrated that ER stress is emerging as an important mechanism of metabolic diseases [10,11]. Based on diabetic models, the contribution of ER stress in target organs (pancreatic β-cells, liver, adipose tissue and skeletal muscle) to the pathogenesis of diabetes has been well established [12–16]. Within the context of diabetic complications, ER stress has also been demonstrated to be linked to retinopathy, cardiomyopathy, nephropathy, endothelial dysfunction and neuropathy in central nervous system [17–21]. Furthermore, recent in vivo studies have reported that ER stress plays a key role in the pathogenesis of peripheral neuropathy in prediabetic and type 1 diabetic animals [22,23]. However, much less attention has been focused on the effect of ER stress on lesions of SC in models of DPN. Indeed, it has become increasingly clear that ER stress in myelinating cells is an important feature of chemically induced and inherited neuropathies [9,24,25]. Given an in vitro study of palmitic acid and high-glucose-induced SC that supports the hypothesis linking ER stress in SC and DPN [26], the relevant mechanisms of ER stress on DPN need further elucidation.
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Natural plant components with minimal or no side effects are being pursued as alternatives to pharmaceutical interventions. Grape seed proanthocyanidins (GSPs), which are derived from grape seeds, refer to a group of proanthocyanidins mostly containing dimers, trimers and other oligomers of catechin and epicatechin and their gallic acid esters. A previous study confirmed that 6 months of 250 or 500 mg/kg body weight (BW) GSPs treatment was safe and did not cause any detrimental effects in vivo [27]. Previous studies based on chemically induced type 1 diabetic animals reported that GSPs exerted anti-diabetic property [28,29]. Our recent study also found that GSPs partially ameliorated hyperglycemia and insulin resistance in type 2 diabetic rats in part by alleviation of ER stress in skeletal muscles and pancreas [15,16]. Moreover, GSPs showed activities that improved type 1 diabetic complications, including cardiomyopathy, nephropathy, retinopathy and neuropathies in central and peripheral nervous systems [30–34]. However, the effect of GSPs on the peripheral nerves of T2DM has not been elaborated. In the present study, using Sprague–Dawley rats made T2DM with low-dose streptozotocin and a high-carbohydrate/high-fat diet, we aimed to investigate whether early intervention of GSPs would result in the prevention of early structural and functional abnormalities in sciatic nerves and to assess whether ER stress in sciatic nerves was the target of GSPs. Meanwhile, given the previous finding that only (+)catechin (C) and (−)-epicatechin (EC) were identified in both rat plasma and nervous system after oral administration of GSPs [35], we used rat SC cultured in serum from type 2 diabetic rats to establish the contribution of ER stress in lesions of SC and to clarify whether the pretreatment of C and EC could protect SC via alleviating ER stress. 2. Methods and materials 2.1. Reagents GSPs (Lot No: 1003007–24) were purchased from Jianfeng Natural Products Co. Ltd. (Tianjin, China). The proanthocyanidin content was 96.64% while analyzed using HPLC with gas chromatography/MS detection. They contained 6.10% C, 6.78% EC, 55.59% dimeric forms, 11.91% trimeric forms, 6.55% tetrameric forms and small amounts of other polymeric forms. Basal diet and the high-carbohydrate/high-fat diet (66% basal diet, 15% lard, 10% plantation white sugar, 3% yolk powder and 6% casein) were produced by Beijing Keao Xieli Co. Ltd. (Beijing, China). Streptozotocin, C, EC, methyl thiazolyl tetrazolium (MTT) and tunicamycin were from Sigma-Aldrich (St. Louis, MO, USA). Free Ca2+ assay kit was from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). The lactate dehydrogenase (LDH) release assay kit and Fluo3 AM were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). FITCconjugated Annexin V and propidium iodide (PI) were obtained as a kit from Roche Molecular Biochemicals (Indianapolis, IN, USA). The antibodies for glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), Caspase-12, JNK, phosphorylated JNK (phospho-JNK) and β-Actin were obtained from Santa Cruz Biotechnology (California, USA). 2.2. Animal treatments
centrifugation (3500 rpm for 10 min at 4°C) and used for analysis of insulin and lipids. The level of insulin was determined by means of radioimmune assay. The levels of triglyceride (TG), cholesterol (CHO), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were determined by automatic biochemistry analyzer. 2.4. Measurement of nerve conduction velocity (NCV) Sixteen weeks after induction of diabetes, NCV was detected through a Pclab-UE biomedical signal acquisition and processing system (Beijing Microsignalstar Technology Development Co. Ltd., China). After being deeply anesthetized with 10% chloral hydrate, rats were killed by cervical dislocation. The left sciatic/tibial nerve of each rat was rapidly dissected with a glass needle and placed in an insulated box. Nerve conduction was made and recorded as previously reported [34]. The sciatic nerves were then dissected, frozen in liquid nitrogen and stored at −80°C for Western blot. 2.5. Pathological observation and measurement of free Ca2+ in sciatic nerves Following the NCV measurement, the right sciatic nerve of each rat was also removed. Half of nerves in each group were immediately frozen in liquid nitrogen and stored separately at −80°C for the measurement of the concentration of free Ca2+, which was assayed according to the manufacturer's protocol and expressed as nmol/ mg protein. The other half nerves were used for electron microscopic analysis. A small portion of the same region from each group was immersed overnight in 2.5% glutaraldehyde (pH 7.4). Following embedding in Epon 812, microsections were cut and stained on the copper grid with 4% uranyl acetate and Reynold's lead citrate. The ultrastructure was then checked from magnification 4000× to magnification 12,000×. 2.6. Cell culture and treatments RSC96 SC were obtained from the American Type Culture Collection and cultured as previously described [36]. When grown upon reaching 70% confluence, cells were preincubated in serum-free medium for 4 h prior to each experiment. To induce cell injury, cells were challenged with DMEM containing 10% serum from diabetic rats (DRS). Cells treated with 10% serum from healthy rats (HRS) were used as controls. Ten healthy male Sprague–Dawley rats and 20 diabetic rats, which were induced by the same method as mentioned above, were used for serum extracts. The serum of rats was collected as previously reported [37]. In this set of experiments, cells were pretreated with different doses of C/EC mixtures (5, 10 and 20 μmol/L, respectively) for 3 h prior to 10% serum stimulation and continuously treated with 10% serum and different doses of C/EC mixtures. The classical ER stress activator tunicamycin (5 μg/ml) was added during the last 12 h of treatment (Fig. 1b). 2.7. Evaluation of cell injury Cell viability was evaluated by MTT assay. Cells were washed twice with PBS and cultured in phenolsulfonphthalein-free DMEM with MTT (1 g/L) at 37°C for 4 h. Blue formazan crystals were then resolved with DMSO. Absorbance was measured at 595 nm by a microplate reader using DMSO as the blank. Cell lysis was determined by LDH release assay. After the culture supernatant was collected, 1% Triton X-100 was added to cells to release all LDH. LDH in both culture supernatant and cell lysate were quantified by measuring the absorbance at 490 nm using a microplate reader. Assay was performed according to the manufacturer's instructions. The percent LDH release was calculated as follows: LDH release (%)=(LDH in culture supernatant/LDH in culture supernatant+LDH in cell lysate)×100. Cell apoptosis/necrosis was measured by Annexin V/PI double staining. Prepared cells (1×106) were washed with PBS and resuspended in binding buffer and then incubated for 10 min at room temperature in the dark with a FITC-conjugated, antiAnnexin V antibody and PI. Flow cytometric analysis was performed within 30 min of staining. The extent of apoptosis/necrosis observed was reported as a percentage using the formula: apoptotic/necrotic fraction (%)=[(Annexin V+PI−events)+(Annexin V +PI+events)]/[(Annexin V+PI−events)+(Annexin V+PI+events)+(Annexin V−PI −events)]×100.
All animal care and experimental procedures followed the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 85–23, revised 1996) and the protocols approved by the Institutional Animal Care and Use Committee of Peking University. Fifty male Sprague–Dawley rats (180–200 g), obtained from Animal Service of Health Science Center (Peking University), were housed 2 per cage and randomly divided into 4 groups. Group 1 (n=12, vehicle-treated normal ones) and group 2 (n=12, 250 mg/kg BW GSPs-treated normal ones) were both fed the basal diet; group 3 (n=13, vehicle-treated diabetic ones) and group 4 (n=13, 250 mg/kg BW GSPs-treated diabetic ones) were induced by 8 weeks of the high-carbohydrate/highfat diet and 2 injections of 25 mg/kg BW streptozotocin as described previously [15,16]. During the experimental period, the vehicle-treated normal and diabetic groups were given water, and the other two groups were given GSPs by stomach tube. After induction of diabetes, all groups were allowed free access to a basal diet (Fig. 1a). BW and food intake were measured once a week.
After digestion with trypsin, cells were harvested by centrifugation. For loading with Fluo-3, cells were incubated for 1 h at 37°C with Fluo-3 AM (5 μmol/L) in the dark. Then, cells were rinsed twice in Ca2+- and Mg2+-free PBS and analyzed with a FACScan flow cytometer. Excitation was from an argon laser at 488 nm. Emission at 530 nm (bandwidth 30 nm) was measured on a linear scale. Raw data for free Ca2+ content were expressed as mean fluorescence intensity.
2.3. Measurement of blood parameters
2.9. Western blot
Blood samples were collected at the end of 1 week and 16 weeks after induction of diabetes. The 6-h fasted rats were deeply anesthetized with 10% chloral hydrate. Blood samples were then collected from snipped tails by tail milking. Plasma glucose was immediately determined using a rapid glucometer. Serum was separated by
Forty micrograms of protein extracts from sciatic nerves or SC was separated by PAGE and immunoblotting was performed with antibodies for GRP78, CHOP, Caspase12, phospho-JNK, JNK and β-Actin. Relative protein quantification was done with Quantity One software.
2.8. Measurement of cytoplasmic free Ca2+
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Fig. 1. Schematic representation of the experimental procedures. The present study was performed in Sprague–Dawley rats made diabetic with low-dose streptozotocin and a highcarbohydrate/high-fat diet (a) and in rat SC cultured in serum from type 2 diabetic rats (b). C, (+)-catechin; EC, (−)-epicatechin.
2.10. Statistical analysis
3.3. Electron microscopic analysis of sciatic nerves
Values of Pb.05 were considered statistically significant. Results are presented as means±S.E.M. All data were statistically analyzed by one-way analysis of variance, followed by LSD (equal variances assumed) or Dunnett's T3 (equal variances not assumed) for multiple comparisons.
As depicted in Fig. 2, normal nucleus, mitochondria and ER in SC and normal structure and morphology of myelin sheath with moderate homogenous electron density were seen in GSPs treated and untreated normal control rats. However, sciatic nerves showed evidence of damage in diabetic control rats, including swelling and dilatation of mitochondria and ER in SC and deranged myelin sheath with lighter electron density. The protective effect of GSPs on diabetic rats was evident with moderate increases in normal mitochondria and ER. Regretfully, treatment of diabetic rats with GSPs only improved morphology of myelin sheath to a certain extent, and myelin sheath was still slightly splitting under electron microscope.
3. Results 3.1. Physical and biochemical characteristics in normal and diabetic rats As shown in Table 1, there were no differences in food consumption, plasma glucose, serum insulin, TG, CHO, LDL and HDL between normal and GSPs control groups throughout the study period, whereas the level of BW was significantly lower in GSPs control rats than that in normal control ones at the end of the study. As expected, a high-carbohydrate/high-fat diet for 8 weeks accompanying by low-dose streptozotocin twice injection significantly increased food consumption, plasma glucose, serum insulin, TG, CHO and LDL in diabetic rats when compared with normal control rats. At the end of the study, diabetic rats still showed a typical characteristic of T2DM as emaciation, polyphagia, hyperglycemia, hyperinsulinemia and blood lipid disorder. However, the GSPs treatment slightly improved the raised diabetic parameters, among which the difference in the level of LDL was statistically significant when compared with diabetic control ones.
3.4. The concentration of free Ca2+ and ER stress markers in sciatic nerves As shown in Table 1 and Fig. 3, the concentration of free Ca2+ and ER stress markers (GRP78, CHOP, phospho-JNK, total JNK and cleaved Caspase-12) were all expressed at low levels in both normal and GSPs control groups. However, these stress markers were all significantly elevated in sciatic nerves of diabetic controls when compared with normal controls. The GSPs treatment slightly reduced the concentration of free Ca2+ and ER stress markers, among which the expression of CHOP was statistically decreased when compared with diabetic control group.
3.2. NCV of sciatic/tibial nerves
3.5. Cell injury in RSC96 SC
NCV of sciatic/tibial nerves in GSPs control group was similar to that in normal control rats. However, NCV was significantly reduced in diabetic control rats when compared with normal control rats. Interestingly, administration of GSPs to diabetic rats significantly increased the level of NCV (Table 1).
As indicated in Fig. 4, cell viability, the extents of LDH release and apoptosis/necrosis fraction in cells treated with HRS and metabolites of GSPs were similar to that in cells only treated with HRS. However, in contrast with the control group, exposure to DRS significantly decreased cell viability and increased cell lysis and cell apoptosis/
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Table 1 BW, food intake, plasma glucose, serum insulin, TG, CHO, LDL, HDL, NCV and free Ca2+ content of sciatic nerves in normal and diabetic rats Parameter
Normal rats Vehicle
At the end of 1 week after induction of diabetes BW (g) 470.13±11.30 Food intake (g/day) 19.48±0.61 Plasma glucose (mg/dl) 81.90±4.99 Serum insulin (mIU/L) 21.27±0.79 TG (mmol/L) 1.37±0.14 CHO (mmol/L) 1.87±0.11 LDL (mmol/L) 0.35±0.03 HDL (mmol/L) 1.64±0.12 At the end of 16 weeks after induction of diabetes BW (g) 595.38±8.08 Food intake (g/day) 22.45±1.62 Plasma glucose (mg/dl) 103.5±3.97 Serum insulin (mIU/L) 19.71±2.67 TG (mmol/L) 1.20±0.09 CHO (mmol/L) 2.23±0.14 LDL (mmol/L) 0.42±0.04 HDL (mmol/L) 1.80±0.11 NCV (m/s) 48.26±1.19 Free Ca2+ (nmol/mg pro) 3.20±0.33
Diabetic rats GSPs
Vehicle
GSPs
461.13±15.81 18.85±0.59 84.29±4.23 21.88±1.02 1.32±0.14 1.88±0.09 0.32±0.04 1.60±0.12
453.88±20.46 30.32±0.89 ⁎⁎ 284.40±12.37 ⁎⁎ 29.02±2.14 ⁎⁎ 3.51±0.31 ⁎⁎ 2.70±0.17 ⁎⁎ 0.91±0.07 ⁎⁎
445.75±17.03 29.45±1.39 ⁎⁎ 283.05±15.14 ⁎⁎ 28.77±1.29 ⁎⁎ 3.53±0.36 ⁎⁎ 2.72±0.21 ⁎⁎ 0.89±0.07 ⁎⁎
553.50±10.91 ⁎ 20.85±1.81 95.63±3.97 21.03±2.01 1.18±0.10 2.13±0.15 0.35±0.04 1.77±0.08 46.25±1.04 3.12±0.35
415.00±20.03 ⁎⁎ 38.93±1.15 ⁎⁎ 381.15±21.08 ⁎⁎ 34.27±3.62 ⁎⁎ 2.42±0.17 ⁎⁎ 3.85±0.35 ⁎ 1.41±0.13 ⁎⁎ 2.41±0.25 ⁎ 39.48±1.02 ⁎⁎ 5.77±0.49 ⁎
1.81±0.14
1.76±0.11 443.13±11.82 35.92±1.38 331.65±19.76 32.32±4.44 2.19±0.17 3.37±0.25 0.92±0.09 # 2.55±0.16 43.46±1.86 # 5.17±0.31
Data were means±S.E.M. of 8 rats of each group. ⁎ Pb.05 vs. vehicle-treated normal rats. ⁎⁎ Pb.01 vs. vehicle-treated normal rats. # Pb.05 vs. vehicle-treated diabetic rats.
Fig. 2. Pathological observations on sciatic nerves of normal and diabetic rats. Scale bar=1 μm. Ax, axon; C, control rats; D, diabetic rats; MC, mitochondria; MNF, myelinated nerve fiber; N, nucleus.
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Fig. 3. Effect of GSPs on ER stress in sciatic nerves of normal and diabetic rats. An aliquot of protein extracts was subject to Western blot analysis using the 78-kDa glucose-regulated protein (GRP78), CHOP, Caspase-12, JNK and phospho-JNK antibodies. β-Actin protein levels were used as a control (a). A ratio between specific protein and β-Actin (or JNK) was calculated as means±S.E.M. of 3 determinations from 3 individual rats in each group (b–e). ⁎Pb.05 and ⁎⁎Pb.01 vs. vehicle-treated control rats, #Pb.05 and ##Pb.01 vs. vehicle-treated diabetic rats. C, control rats; D, diabetic rats.
necrosis. Interestingly, C and EC pretreatment partially ameliorated cell injury in DRS-treated cells, among which the differences between cells treated with DRS, C (10 μmol/L) and EC (10 μmol/L) and cells only treated with DRS were statistically significant.
3.6. Ca2+ overload and ER stress markers in RSC96 SC The mean fluorescence intensity of cytoplasmic free Ca2+ and the low levels of ER stress markers in cells that were treated with HRS were not significantly different with or without metabolites of GSPs
(Figs. 4 and 5). However, in response to DRS, the free Ca2+ content and ER stress markers (GRP78, CHOP, phospho-JNK and cleaved Caspase-12) were all significantly elevated when compared with control group. The mixture of C and EC blocked the increase in cytoplasmic free Ca2+ content in DRS-treated cells. The inhibitory rates of 2.5, 5 and 10 μmol/L of the mixture reached 5.40%, 8.77% and 10.98%, respectively. Furthermore, The pretreatment with the mixture (at the dose of 5 and 10 μmol/L, respectively) for 3 h prior to DRS stimulation also clearly alleviated ER stress. The expressions of GRP78 and phospho-JNK were statistically decreased when compared with cells only treated with DRS. Moreover, the activation of ER stress
Fig. 4. Comparison of cell injury (a–c) and Ca2+ overload (d) in RSC96 SC. Cell injury was evaluated by MTT assay (a), LDH release assay (b) and Annexin V/PI double staining (c). For MTT and LDH release analysis, data were means±S.E.M. from 10 experiments. The other data were means±S.E.M. from 8 experiments. ⁎Pb.05 and ⁎⁎Pb.01 vs. only HRS-treated cells, # Pb.05 and ##Pb.01 vs. only DRS-treated cells. C, (+)-catechin; EC, (−)-epicatechin.
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Fig. 5. Effects of (+)-catechin (C) and (−)-epicatechin (EC) on ER stress in RSC96 SC. An aliquot of protein extracts was subject to Western blot analysis using the 78-kDa glucoseregulated protein (GRP78), CHOP, Caspase-12, JNK and phospho-JNK antibodies. β-Actin protein levels were used as a control (a). A ratio between specific protein and β-Actin (or JNK) was calculated as means±S.E.M. of 3 independent experiments (b–e). ⁎Pb.05 and ⁎⁎Pb.01 vs. only HRS-treated cells, #Pb.05 and ##Pb.01 vs. only DRS-treated cells.
by pharmacological approach was also observed. Tunicamycin (5 μg/ ml), the classical ER stress activator, reversed the inhibition of ER stress induced by C/EC mixtures in DRS-treated cells, among which the difference in the expression of phospho-JNK was statistically significant when compared with DRS-treated cells with C (10 μmol/L) and EC (10 μmol/L) treatment. Interestingly, unlike in vivo study as mentioned above, the expression of total JNK remained mainly unchanged among these groups in vitro. 4. Discussion The findings reported herein support the pathogenetic role of SC in DPN and the lesions of SC in animal and cell culture models of T2DM are partially associated with ER stress. Furthermore, the incomplete but significant prevention of neuropathy and cell injury by GSPs or their metabolites suggests that GSPs might have auxiliary therapeutic potential for DPN partially by alleviating Ca2+ overload and ER stress in SC. In the present study, the animal model induced by a highcarbohydrate/high-fat diet for 8 weeks accompanying by low-dose streptozotocin twice injection is an excellent model of T2DM because it develops polyphagia, hyperglycemia, hyperinsulinemia and blood lipid disorder throughout the study period. In T2DM, reduction NCV is one of the earliest neuropathic abnormalities [38]. Diabetic rats exhibiting the characteristic nerve conduction slowing in sciatic/tibial nerves 16 weeks after induction of diabetes comprise a suitable model for exploration of new pathogenetic mechanisms of DPN. Researchers commonly comment that axon and Schwann cell deficits appear to proceed independently of each other [7]. In accordance with this comment, ultrastructural lesions were found in myelin sheath and SC, but no lesion was observed in axons in the present study. Although some in vivo studies have revealed diabetes-induced typical apoptotic nuclear and cytoplasmic changes in SC of streptozotocin-diabetic animals, many investigators have resisted the concept of apoptotic processes [39–41]. Indeed, little consistent evidence remains for morphological changes in peripheral nerves of diabetic models,
which most likely results from multiple factors, including animal species and age, small sample size, changes in dosage and injection techniques of streptozotocin, differences in time points of observation and duration of hyperglycemia. In the present study, we only found swelling and dilatation of mitochondria and ER, but without apparent chromatic agglutination in SC 16 weeks after induction of diabetes. In order to explore the detailed pathogenetic role of SC in DPN, we studied in vitro using Schwann cell line RSC96, which was used to study peripheral nerve injury previously [42,43]. Exposure of SC to 10% serum from type 2 diabetic rats significantly decreased cell viability and increased cell death. The findings are in agreement with those in DRG neurons cultured in 15% serum from a type 2 diabetic patient with neuropathy [44]. Previous studies mainly observed SC under hyperglycemic and/or hyperlipidemic conditions. Hyperglycemic (150 mmol/L for 24 h) defined media showed nuclear blebbing of apoptotic cells in SC [40]. The percentage of apoptotic cells was also increased after incubation with palmitate (0.05–0.50 mmol/L) for 48 h [45]. However, the results that hyperglycemia (60 mmol/L for 4 days) does not cause death of SC also existed [46]. Based on recent findings that chronic hyperglycemia alone (50 mmol/L for 24–48 h) did not induce cell death but its presence strongly magnified the effects of lipotoxic injury in SC [26], we hypothesize that some factors might present in the serum of diabetics (e.g., lipid overload and autoimmune immunoglobulin), in addition to hyperglycemia per se, and result in an override of the cell defensive response and contribute to cell death. In the past, the effect of GSPs on DPN was only studied in streptozotocin-induced type 1 diabetic rats. It was shown that 24 weeks of GSPs (250 mg/kg BW) treatment prevented against structural and functional abnormalities in peripheral nerves, including increasing NCV, decreasing the severe segmental demyelination and improving impaired SC in sciatic nerves [34]. In the present study, oral supplementation with the same dosage of GSPs for 24 weeks prevented nerve conduction slowing and structural abnormalities of SC (mainly swelling and dilatation of mitochondria and ER) despite
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only partial preservation of myelin sheath in peripheral nerves of type 2 diabetic rats. Moreover, the treatment of metabolites of GSPs achieved a partial but significant prevention of cell injury in SC cultured in serum from type 2 diabetic rats. These data suggest that although peripheral nerves might not have maintained completely normal function, prophylactic GSPs treatment may be efficacious in delaying the development of DPN in T2DM. We also investigated the effects of GSPs or their metabolites on normal peripheral nerves or SC. It was shown that GSPs or their metabolites in normal sciatic nerves or SC were safe under the conditions investigated in this study. Considerable evidence suggests that impaired cellular Ca2+ homeostasis, largely due to dysfunction of the intracellular Ca2+ stores such as ER and mitochondria, is an early molecular marker linked to the onset of DPN [47]. Our results are basically in agreement with this point. Elevated free Ca2+ content in sciatic nerves was existed 16 weeks after induction of diabetes. Previous in vitro studies showed that palmitate in the presence of high glucose resulted in an early and more robust release of Ca2+ into the cytosol from the ER in SC [26]. Moreover, Ca2+ influx was enhanced by the exposure of DRG neurons to 10% serum from diabetic BB/W rats for 18–24 h or 15% serum from a type 2 diabetic patient with neuropathy for 24 h [44,48]. In a similar manner, we showed that cytoplasmic free Ca2+ content was significantly elevated in DRS-treated SC. Although the precise mechanisms how cellular Ca2+ homeostasis is altered by diabetes and how the alteration contributes to the development of DPN remain to be elucidated, amelioration of abnormal Ca2+ homeostasis can be a therapeutic target in DPN. For example, a previous study found that reducing excessive cytosolic Ca2+ levels induced by palmitate in the presence of high glucose using the calcium chelator inhibited the cell death process in SC [26]. Our results indicated that metabolites of GSPs decreased cytoplasmic free Ca2+ content in DRS-treated SC with an apparent dose dependency, which may be one of its mechanisms of cytoprotective action. Unfortunately, GSPs treatment only partially reduced Ca2+ overload in sciatic nerves, suggesting that the dosage used in the present study may not have been sufficient to achieve a complete prevention and studies are still needed to address the action of GSPs in this regard. A number of cell stress conditions, such as hyperglycemia, dyslipidemia and abnormal cellular Ca2+ homeostasis in T2DM, will disrupt ER homeostasis and subsequently lead to the accumulation of unfolded or misfolded proteins in the ER lumen [10]. This activates an adaptive unfolded protein response (UPR), which aims to reconcile the imbalance. One of the most described mechanisms of UPR activation is the competition model, in which the ER chaperone protein GRP78 plays an essential role in the activation of different ER stress transducers [10]. If the imbalance persists, the UPR can ultimately lead to cell death, which is mediated in part by increased expression of CHOP and activities of JNK and Caspase-12 [11]. Based on Zucker rats, high fat diet-fed mice and streptozotocin-induced type 1 diabetic rats, ER stress manifest in up-regulation of the expressions of GRP78 and CHOP was identified in sciatic nerves [22,23]. Like in vivo studies, previous in vitro studies under hyperglycemic and/or hyperlipidemic conditions mainly showed that the expressions of ER stress genes and proteins (GRP78 and CHOP) were altered [26,45]. Intriguingly, in addition to the expressions of GRP78 and CHOP, we also found that the activities of JNK and Caspase-12 were all significantly elevated in both sciatic nerves and DRS-treated SC. These significant findings further confirm the important role of ER stress in DPN. So far, little information is available describing the effect of beneficial components of plants on ER stress in DPN. To our knowledge, the only one of phytochemicals, which was demonstrated to improve peripheral neuropathy by alleviating ER stress in a inherited neuropathic model, was curcumin [49]. In the present study, elevated ER stress markers (especially the expression of CHOP and the
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activity of Caspase-12) in peripheral nerves of type 2 diabetic rats were partially reversed by GSPs treatment. This is consistent with the results of our previous study, which indicated that the same dosage of GSPs mainly regulated the same ER stress markers in skeletal muscles of type 2 diabetic rats [15]. Indeed, inhibition of ER stress by chemicals or gene knockout technology was demonstrated to alleviate peripheral nerve dysfunction in prediabetic and type 1 diabetic animals [22,23]. Importantly, the results of our in vitro study further showed that metabolites of GSPs markedly inhibited elevated ER stress markers (especially the expression of GRP78 and the activities of JNK and Caspase-12) in rat SC cultured in serum from type 2 diabetic rats, and this activity was disrupted by ER stress activator. Although not measured in detail in the present study, these series of events would confirm that GSPs might have auxiliary preventive potential for DPN partially by alleviating ER stress. The present study has some unanswered questions that should be included in further studies. First, the results of ER stress markers in vivo were not equal to that in vitro under both GSPs (or metabolites of GSPs) treated and untreated diabetic models. Second, previous studies indicated that CHOP activity in SC was distinct from other cell types and it only promoted demyelination in the absence of cell death [24]. However, our data and other in vitro studies based on hyperglycemic and/or hyperlipidemic conditions demonstrated an increase in apoptotic cells and expression of significant level of CHOP in SC [26,45]. Third, it was in dispute as to whether the short-term exposure of SC to 10% serum from type 2 diabetic rats in culture could adequately simulate the condition of human diabetic complications. Finally, the present experiment was preventive, and there was incomplete protection of nerve function by GSPs, it would be of practical interest to ascertain whether GSPs could reverse established neuropathic changes.
Acknowledgments The authors would like to thank Lei Bao, Yujie Li, Feng Zhang, Lulu Jing and Jiaojiao Gu for their assistance in the experiment.
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