Quercetin alleviates high glucose-induced Schwann cell damage by autophagy

Ling Qu, Xiaochun Liang, Bei Gu, Wei Liu

1 Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Peking Union Medical College, China Academy of Medical Sciences, Beijing, China

2 Cell Center, Institute of Basic Medical Science, Peking Union Medical College, China Academy of Medical Sciences, Beijing, China

Quercetin alleviates high glucose-induced Schwann cell damage by autophagy

Ling Qu1, Xiaochun Liang1, Bei Gu2, Wei Liu1

1 Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Peking Union Medical College, China Academy of Medical Sciences, Beijing, China

2 Cell Center, Institute of Basic Medical Science, Peking Union Medical College, China Academy of Medical Sciences, Beijing, China

Ling Qu, M.D., Department of

Traditional Chinese Medicine, Peking

Union Medical College Hospital, Peking

Union Medical College, China Academy of Medical Sciences, Beijing 100730, China, quling@pumch.cn.

Quercetin can reverse high glucose-induced inhibition of neural cell proliferation, and therefore may have a neuroprotective effect in diabetic peripheral neuropathy. It is dif fi cult to obtain primary Schwann cells and RSC96 cells could replace primary Schwann cells in studies of the role of autophagy in the mechanism underlying diabetic peripheral neuropathy. Here, we show that under high glucose conditions, there are fewer autophagosomes in immortalized rat RSC96 cells and primary rat Schwann cells than under control conditions, the proliferative activity of both cell types is signi fi cantly impaired, and the expression of Beclin-1 and LC3, the molecular markers for autophagy, is signi fi cantly lower. After intervention with quercetin, the autophagic and proliferative activity of both cell types is rescued. These results suggest that quercetin can alleviate high glucose-induced damage to Schwann cells by autophagy.

nerve regeneration; quercetin; diabetic peripheral neuropathy; high glucose; RSC96; primary Schwann cells; proliferation; ultrastructure; autophagy; Beclin-1; LC3; NSFC grant; neural regeneration

Funding: This study was supported by the National Natural Science Foundation of China, No. 30572438; the Beijing Science Nuture Foundation, No. 7132189; a grant from Science Foundation of Peking Union Medical College Hospital, No. 2013-098.

Qu L, Liang XC, Gu B, Liu W. Quercetin alleviates high glucose-induced Schwann cell damage by autophagy. Neural Regen Res. 2014;9(12)：1195-1203.

Introduction

Diabetic peripheral neuropathy (DPN) is one of the major chronic complications of diabetes (Tsapas et al., 2014). The pathogenesis of DPN has not been elucidated, and effective therapies for the condition are still lacking (Zychowska et al., 2013). Autophagy has recently been found to play important roles in the maintenance of islet β cell structure and function, improve insulin resistance, and delay the onset of complications of chronic diabetes such as diabetic nephropathy, retinopathy, cardiomyopathy and atherosclerosis (Marsh et al., 2007; Ebato et al., 2008; Fujimoto et al., 2009; Masini et al., 2009; Lo et al., 2010; Ost et al., 2010; Younce et al., 2010; Kitada et al., 2011; Mellor et al., 2011; Peng et al., 2011; Wu et al., 2011; Xie et al., 2011; He et al., 2012; Hu et al., 2012; Miranda et al., 2012; Quan et al., 2012). However, little is understood about the relationship between autophagy and DPN.

Schwann cells are specialized glial cells in the peripheral nervous system, playing important roles in maintaining neuronal structure and function and repairing damaged nerves. Schwann cell dysfunction is one of the important pathogenic mechanisms underlying diabetes-associated abnormalities in neural regeneration and repair (Kennedy et al., 2005). It is believed that hyperglycemia leads to increased aldose reductase activity and polyol metabolism in Schwann cells, and the resultant abnormal metabolites cause the organelle’s damage and morphological changes such as swelling and vacuolation (Mizisin et al., 1997; Kalichman et al., 1998; Murakawa et al., 2002). Furthermore, clearance of defective organelles is closely related to the autophagy process (Jung and Lee, 2010; Wang et al., 2011; Grimaldi et al., 2012). Autophagy is an important physiological process and a major defensive mechanism of the body under stresses such as nutrient or energy deprivation (Gonzalez et al., 2011). Autophagy can remove the damaged organelles, but also provide the materials for cell survival under stressful conditions. The Beclin1 gene (also called Becn1), is homologous to yeast Atg6/Vps30 and is involved in the initiation of autophagosome formation. The expression of Beclin-1 protein is positively correlated to the occurrence of autophagy, and is therefore used to monitor and determine autophagy dynamics when combined with other biochemical parameters (Yamahara et al., 2013). LC3 is the analog of the Atg8 gene product in yeast. LC3 precursor (ProLC3) was fi rst processed to form soluble LC3-I, and then activated by Atg7 and modi fi ed by Atg3 to create the membrane binding form LC3-II. LC3-II is located on pre-autophagosomes and autophagosomes, and its abundance is positively proportional to the number of autophagicvacuoles. Therefore, it serves as a molecular marker for autophagy (Klionsky et al., 2012).

Quercetin is widely present in traditional Chinese medicine and food, and is known to have antioxidant, anticancer, anti-inflammatory, antidiabetic, antihypertensive, neuroprotective and immunoregulatory effects (Olaleye et al., 2013; Bądziul et al., 2014; Liao and Lin, 2014; Milackova et al., 2014). Recently, it was reported that quercetin increased the proliferation of neurons or glial cells that were inhibited by high concentrations of glucose, and reduced oxidative stress-mediated damage (Shi et al., 2013; Wu et al., 2014). Quercetin was also implicated in the mechanism underlying the reduction of apoptosis through autophagy induction (Wang et al., 2011; Kim et al., 2013). Whether or not quercetin protects Schwann cells through autophagy pathways remains unclear.

Most cultured Schwann cells are primary cultures of peripheral nerve tissue from normal experimental animals. Primary cultured cells exhibit more accurate biological characteristics and fewer differences in proliferation and cytokine expression than cells from other sources (Qu et al., 2008). However, it is difficult to obtain primary Schwann cells because of the complex techniques involved, low cell yield, poor cell activity and high costs (Pan et al., 2005; Fu et al., 2012). RSC96 cells are an immortalized cell line derived from the long-term culture of rat primary Schwann cells. Although proteome differences have been found between primary Schwann cells and RSC96 cells that highlight several differentially expressed proteins with potential biological signi fi cance (Ji et al., 2012), RSC96 cells have been used in many studies of peripheral nerve injury and regeneration (Hai et al., 2002; Chang et al., 2011; Yin et al., 2012; Gui et al., 2013; Huang et al., 2014). However, whether RSC96 cells can be used as a substitute for primary rat Schwann cells in the study of autophagy is unclear. To the best of our knowledge, the differences in autophagy between primary Schwann cells and RSC96 cells have not been investigated under high glucose conditions.

In the present study, we observed the effects of quercetin on the morphology, proliferation and autophagy activity of primary Schwann cells and RSC96 cells cultured in high concentrations of glucose, with the aim of providing information for future studies on the relationship between autophagy and DPN and the search for effective therapies for DPN.

Materials and Methods

Animals

Male Sprague-Dawley rats, aged 3 days, were purchased from the Institute of Laboratory Animals of the Chinese Medical Science Academy, Beijing, China (license No. SCXK (Jing) 2005-0013). The study was approved by the Animal Ethics Committee of Peking Union Medical College Hospital in China.

Cells

RSC96 cells were purchased from the Cell Bank, Chinese Academy of Sciences, Shanghai, China.

Preparation of quercetin

Quercetin (C15H10O7, molecular weight 302.23) was purchased from Sigma, St. Louis, MO, USA. Stock solution of quercetin was dissolved in deionized water at a concentration of 25 μmol/L and fi ltered through a 0.22 μm fi lter before use.

Primary culture of Schwann cells

Rats were sacrificed under anesthesia and bilateral sciatic nerves dissected out and subjected to enzymatic digestion. Dulbecco’s modi fi ed Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) was supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 2 mmol/L L-glutamate, 1 mmol/L sodium pyruvate and 12.5 mmol/L hydroxyethyl piperazine ethanesulfonic acid (Sigma). Schwann cells were puri fi ed by differential speed adherence after 5 days and treated with G-418 (Sigma; 100 μg/mL) 7 days later.

Culture of RSC96 cells

RSC96 cells were revived. The medium was the same as that used for primary culture of Schwann cells and was changed once every 2 or 3 days.

Grouping

Six groups were designated as follows: primary Schwann cell cultures treated with DMEM (PC group), DMEM + 125 mmol/L glucose (PG group), or DMEM + 125 mmol/L glucose + 25 μmol/L quercetin (PQ group); RSC96 cells were treated with DMEM (RC group), DMEM + 125 mmol/L glucose (RG group), or DMEM + 125 mmol/L glucose + 25 μmol/L quercetin (RQ group).

Immuno fl uorescence identi fi cation of Schwann cells

Cells were seeded and incubated under each condition for 24 hours before being treated with acetone for 15 minutes at 4°C and 0.1% Triton-X 100 for 10 minutes at 4°C. Thereafter, they were treated with mouse anti-S-100 antibody (1:50 dilution; Boster, Wuhan, China) at 4°C overnight (PBS as negative control). Following PBS washes, the cells were incubated with FITC-conjugated Af fi nipure goat anti-mouse IgG (Beijing Xiya Jinqiao Biotechnology Co., Ltd., Beijing, China) at 37°C for 20 minutes and stained with fresh DAPI for 10 minutes. The fl uorescent intensity of different groups of cells was analyzed using an Olympus FluoView FV 1000 (Olympus Corporation, Tokyo, Japan; excitation 364 nm, emission 488 nm for DAPI; excitation 488 nm, emission 525 nm for FITC).

Ultrastructure of Schwann cells

Primary Schwann cells and RSC96 cells were fi xed in 2.5% glutaraldehyde, treated with 1% osmium tetroxide and embedded in Epon812 (Shell Chemical, Houston, TX, USA), and dehydrated with ethanol and acetone. Ultrathin sections (60-80 nm) were made and stained with uranyl acetate and lead citrate (Alfa Aesar (China), Shanghai, China). Transmission electron microscopy (JEM1010, JEOL (Beijing), Beijing, China) was used to observe the ultrastructure of Schwann cells.

Figure 1 Identi fi cation and morphology of primary Schwann cells and RSC96 cells.

Figure 2 Effect of high glucose concentration on the ultrastructure of primary Schwann cells and RSC96 cells..

Determination of Schwann cell proliferation and viability by MTT assay

MTT (Sigma) was used to evaluate the proliferation of both types of Schwann cells after seeding. 200 μL of 10% MTT was added to each well for 2-4 hours at 37°C and mixed with 200 μL of dimethyl sulfoxide for 30-60 minutes. Absorbance values were measured at 570 nm by multi-mode readers (BioTek Instruments Inc, Vermont, USA). Five wellsfrom each group were selected for detection. The test was performed twice.

Figure 3 Effect of quercetin on the proliferative ability of primary Schwann cells and RSC96 cells.

Determination of Beclin-1 and LC3 expression by immuno fl uorescence staining

Schwann cells were seeded at 2 × 105/mL on poly-l-lysine-coated coverslips. When they reached 70% con fl uence, the cells were starved overnight and observed in duplicate under the three conditions described above (control, or high glucose with or without quercetin). The cells were treated with 5% bovine serum albumin, followed by primary antibody (dilution 1:100; rabbit anti-Beclin-1 antibody and rabbit anti-LC3 A/B antibody; Abcam, Cambridge, UK) at 4°C overnight. PBS was used in place of primary antibody as the negative control.

Following PBS washes, the cells were incubated with secondary antibody (TRITC-conjugated Affinipure goat anti-rabbit IgG; Beijing Xiya Jinqiao Biotechnology Co., Ltd.) at 37°C for 20 minutes, and stained with fresh DAPI for 10 minutes. The fl uorescent intensity of different groups of cells was analyzed using an Olympus FluoView FV 1000 (Olympus Corporation; excitation 364 nm, emission 488 nm for DAPI; excitation 547 nm, emission 620 nm for TRITC). Average absorbance values of each cell were measured by FV10-ASW 3.0 analysis software (Olympus Corporation). Five fi elds of view were randomly chosen from each group for analysis. The experiment was performed twice.

Statistical analysis

Statistical analysis was performed using SPSS 13.0 software (SPSS, Chicago, IL, USA). One-sample Kolmogorov-Smirnov Z test was used to determine whether the data fit the normal distribution before analysis. Normally-distributed data were expressed as mean ± standard deviation. The inter-group differences were compared by one-way analysis of variance and the least signi fi cant difference test. A level of P＜ 0.05 was considered statistically signi fi cant.

Results

Identi fi cation and morphology of primary Schwann cells and RSC96 cells

Primary Schwann cells were long and thin, spindle-shaped or bipolar-like, with bright edges. Their nuclei were oval or spindle-shaped and contained little nucleoplasm. The cells appeared interconnected and fasciculated (Figure 1A). RSC96 cells were also oval, spindle-shaped or bipolar-like, but smaller than primary Schwann cells. Their nuclei were oval and full (Figure 1B). Both primary Schwann cells and RSC96 cells were stained green by the marker S-100 (Figure 1C, D).

Effect of high concentrations of glucose on the ultrastructure of primary Schwann cells and RSC96 cells

In the PC group (Figure 2A, B), there were a few microvilli on the surface of the primary Schwann cells. Nuclei were ovoid and located to one side of the cells. Mitochondria, autophagosomes and other organelles were identifiable. In the PG group (Figure 2C, D), Schwann cells were of different sizes. Some had lobulated nuclei. Mitochondria and numerous lysosomes were seen in the cell matrix. Vacuolar structures were seen, but not autophagosomes. In the RC group (Figure 2E, F), cells and their nuclei were ovoid and possessed distinct nucleoli and uniform chromatin. Cellular organelles including mitochondria, autophagosomes and autolysosomes were visible. In the RG group (Figure 2G, H), most nucleoli were distinct, and chromatin was less uniform. Mitochondria were swollen with an increased number of vacuoles and autophagosomes, and autolysosomes were less visible, compared with the other groups.

Effect of quercetin on the viability of primary Schwann cells and RSC96 cells

MTT assay showed that at 72 hours, proliferative ability of cells in the PG and RG groups was significantly lower than that in the PC and RC groups, respectively (P ＜ 0.05). However, in the PQ and RQ groups, proliferative ability was significantly greater than that in the PG and RG groups, respectively (P ＜ 0.05), and not signi fi cantly different from their respective controls (P ＞ 0.05;Figure 3).

Effect of quercetin on Beclin-1 expression in primary Schwann cells and RSC96 cells

Immuno fl uorescent analysis showed that in the PC and RC groups, Beclin-1 expression was patchy in distribution. In the PG and RG groups, Beclin-1 expression was noticeably weaker than in the control groups, whereas the signal in the PQ and RQ groups was stronger than that in the high glucose groups (Figure 4A).

Figure 4 Effect of quercetin on Beclin-1 expression in primary Schwann cells and RSC96 cells.

Semi-quantitative analysis (Figure 4B) revealed that expression levels of Beclin-1 in the PG and RG groups were significantly lower than those in the PC and RC groups (P ＜ 0.01), while the expression of Beclin-1 in the PQ and RQ groups was signi fi cantly higher than in the PG and RG groups, respectively (P ＜ 0.01), with no difference detected between the control and quercetin-treated groups in either cell type (P ＞ 0.05). The results indicate that Beclin-1 expression is similar in the two kinds of Schwann cells cultured with high glucose and quercetin.

Expression levels of Beclin-1 in the RC group were signi ficantly greater than in the PC group (P ＜ 0.01;Figure 4B), but no differences in Beclin-1 expression were observed between the two cell types under test conditions (RG or RQ vs.PG or PQ, respectively).

Figure 5 Effect of quercetin on LC3 expression in primary Schwann cells and RSC96 cells.

Effect of quercetin on LC3 expression in primary Schwann cells and RSC96 cells

Immuno fl uorescent analysis revealed that in the PC and RC groups, LC3 expression was patchy in distribution. In the PG and RG groups, LC3 expression was signi fi cantly weaker than in the corresponding control groups, while the signal in the PQ and RQ groups was signi fi cantly stronger than in the respective high glucose groups (Figure 5A).

Semi-quantitative analysis of LC3 expression (Figure 5B) in the same cell types under different conditions showed thatthe expression level of LC3 in the PG group was signi fi cantly lower than that in the PC and PQ groups (P ＜ 0.05), with no signi fi cant difference between the PC and PQ groups (P＞ 0.05). The expression level of LC3 in the RG group was signi fi cantly lower than that in the RC group (P ＜ 0.01), and LC3 expression in the RQ group was significantly higher than that in the RG group (P ＜ 0.01), but there was no significant difference between LC3 expression in the RQ and RC groups (P ＞ 0.05). LC3 expression levels were not signi ficantly different between cell types under any condition (P ＞0.05;Figure 5B).

Discussion

Schwann cell dysfunction caused by diabetes mellitus leads to reduced regeneration and repair capability in peripheral nerves (Eckersley, 2002; Kennedy et al., 2005). It is believed that demyelination of nerve fi bers may be caused by metabolic disorders in Schwann cells. In vivo studies revealed myelin breakdown, lamella disorganization, demyelination, and swollen Schwann cell mitochondria in the sciatic nerve tissues of diabetic rats (Chopra et al., 1969; Bestetti et al., 1981; Zemp et al., 1981; Mizisin and Powell, 1997; Kalichman et al., 1998; Murakawa et al., 2002; Shi et al., 2013). The damage to cell ultrastructure seen in the presence of high concentrations of glucose correlates with that observed with cellular metabolic disorders (Kalichman et al., 1998; Ishibashi et al., 2002; Liu et al., 2013; Prasath and Subramanian, 2013). Energy metabolism disorders induce autophagy pathways to degrade self-phospholipids and cytoplasmic proteins to maintain energy supply and anabolism (Uchiyama et al., 2008; Yamahara et al., 2013; Fierabracci, 2014; Mizukami et al., 2014).

In the present study, we found that double-membrane autophagosomes 300-900 nm in diameter (average 500 nm) were seen in both RSC96 cells and primary Schwann cells in the basic medium. However, in the presence of high concentrations of glucose, these two kinds of Schwann cells were irregular in shape and had swollen organelles and nuclei, and many vacuoles. Most of the vacuoles were 1,000 nm in diameter and did not have the double membrane structure, differing from autophagosomes described in the literature (Marsh et al., 2007; Ebato et al., 2008; Masini et al., 2009; Fujimoto et al., 2009; Klionsky et al., 2012). Furthermore, we also found that protein expression of Beclin-1 and LC3, used to monitor autophagy (Klionsky et al., 2012; Yamahara et al., 2013), was downregulated under high glucose conditions. Together, our results show that high concentration glucose suppresses autophagy in Schwann cells.

We also found that quercetin not only protected the proliferative activity of RSC96 cells and primary Schwann cells from high glucose-mediated damage, but also upregulated protein expression levels of Beclin-1 and LC3. Quercetin is widely used in traditional Chinese medicine and food and can reduce the inhibition of nerve cell proliferation caused by high concentrations of glucose. Reductions in oxidative stress-mediated damage and apoptosis have also been shown by autophagy induction (Wang et al., 2011; Kim et al., 2013; Shi et al., 2013; Wu et al., 2014). On the basis of these results, we postulated that quercetin alleviates the impairment of proliferation induced by high glucose, and that this might correlate with autophagy in Schwann cells. We therefore compared the morphology of RSC96 cells and primary rat Schwann cells, and the effects of quercetin on the proliferative and autophagy ability in these two cell types when cultured in high glucose medium. We found that the morphology of both kinds of Schwann cell was similar under optical and transmission electron microscopes, and stained positive for the marker S-100. We also found that RSC96 cells and primary cultured rat sciatic nerve Schwann cells are shaped differently according to protein expression. Furthermore, changes in ultrastructure, proliferative activity and autophagy were similar between cell types in high glucose and after quercetin treatment. These results indicate that RSC96 cells could be used as a substitute for primary rat Schwann cells in studies of autophagy.

In summary, the present study reports that quercetin increases proliferation and upregulates autophagy in two different cell models in the presence of high concentrations of glucose. RSC96 cells and primary rat Schwann cells were similar both in morphology and in their responses to high glucose and quercetin. The use of RSC96 cells would avoid problems frequently arising in primary culture, such as a long cycle, complicated technique and limited cell yield. RSC96 cells could replace primary Schwann cells in studies of the role of autophagy in the mechanism underlying DPN and related drug screening models. In future studies, we will address the regulation of autophagy under high glucose conditions and evaluate the differences in biological activity between RSC96 cells and primary Schwann cells in the fi eld of autophagy.

Acknowledgments:We would like to thank Professor Zhang H and Wang XW from Cell Center, Institute of Basic Medical Science, Peking Union Medical College, China Academy of Medical Sciences, and Liu X from the Department of Confocal Laser Microscopy, Institute of Basic Medical Science, China, for their help in this study.

Author contributions:Qu L designed and performed the majority of the experiments, obtained and analyzed experimental data and wrote the manuscript. Liang XC revised the manuscript. Gu B and Liu W contributed to the preparation of the cell models. All authors approved the final version of this manuscript.Con fl icts of interest:None declared.

Bądziul D, Jakubowicz-Gil J, Paduch R, Głowniak K, Gawron A (2014) Combined treatment with quercetin and imperatorin as a potent strategy for killing HeLa and Hep-2 cells. Mol Cell Biochem. Mar 30. [Epub ahead of print]

Bestetti G, Rossi GL, Zemp C (1981) Changes in peripheral nerves of rats four months a■er induction of streptozotocin diabetes. A qualitative and quantitative study. Acta Neuropathol 54:129-134.

Chang CY, Lee YH, Jiang-Shieh YF, Chien HF, Pai MH, Chen HM, Fong TH, Wu CH (2011) Novel distribution of cluster of di ff erentiation 200 adhesion molecule in glial cells of the peripheral nervous system of rats and its modulation after nerve injury. Neuroscience 183:32-46.

Chopra JS, Hurwitz LJ, Montgomery DA (1969) The pathogenesis of sural nerve changes in diabetes mellitus. Brain 92:391-418.

Ebato C, Uchida T, Arakawa M, Komatsu M, Ueno T, Komiya K, Azuma K, Hirose T, Tanaka K, Kominami E, Kawamori R, Fujitani Y, Watada H (2008) Autophagy is important in islet homeostasis and compensatory increase of beta cell mass in response to high-fat diet. Cell Metab 8:325-332.

Eckersley L (2002) Role of the Schwann cell in diabetic neuropathy. Int Rev Neurobiol 50:293-321.

Ferreira PE, Lopes CR, Alves AM, Alves ÉP, Linden DR, Zanoni JN, Buttow NC (2013) Diabetic neuropathy: an evaluation of the use of quercetin in the cecum of rats. World J Gastroenterol 19:6416-6426.

Fierabracci A (2014) ■e putative role of proteolytic pathways in the pathogenesis of Type 1 diabetes mellitus: ■e ‘autophagy’ hypothesis. Med Hypotheses 82:553-557.

Fu HY, Wang RY (2012) Progress on the in vitro culture and puri fi cation of Schwann cells. Hainan Yixue 23:118-121.

Fujimoto K, Hanson PT, Tran H, Ford EL, Han Z, Johnson JD, Schmidt RE, Green KG, Wice BM, Polonsky KS (2009) Autophagy regulates pancreatic beta cell death in response to Pdx1 de fi ciency and nutrient deprivation. J Biol Chem 284:27664-27673.

Gonzalez CD, Lee MS, Marchetti P, Pietropaolo M, Towns R, Vaccaro MI, Watada H, Wiley JW (2011) ■e emerging role of autophagy in the pathophysiology of diabetes mellitus. Autophagy 7:2-11.

Grimaldi C, Chiarini F, Tabellini G, Ricci F, Tazzari PL, Battistelli M, Falcieri E, Bortul R, Melchionda F, Iacobucci I, Pagliaro P, Martinelli G, Pession A, Barata JT, McCubrey JA, Martelli AM (2012) AMP-dependent kinase / mammalian target of rapamycin complex 1 signaling in T-cell acute lymphoblastic leukemia: therapeutic implications. Leukemia 26:91-100.

Gui T, Wang Y, Zhang L, Wang W, Zhu H, Ding W (2013) Krüppel-like factor 6 rendered rat Schwann cell more sensitive to apoptosis via upregulating FAS expression. PLoS One 8:e82449.

Hai M, Muja N, DeVries GH, Quarles RH, Patel PI (2002) Comparative analysis of Schwann cell lines as model systems for myelin gene transcription studies, J Neurosci Res 69:497-508.

He C, Bassik MC, Moresi V, Sun K, Wei Y, Zou Z, An Z, Loh J, Fisher J, Sun Q, Korsmeyer S, Packer M, May HI, Hill JA, Virgin HW, Gilpin C, Xiao G, Bassel-Duby R, Scherer PE, Levine B (2012) Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis. Nature 481:511-515.

Hu P, Lai D, Lu P, Gao J, He H (2012) ERK and Akt signaling pathways are involved in advanced glycation end product-induced autophagy in rat vascular smooth muscle cells. Int J Mol Med 29:613-618.

Huang CY, Kuo WW, Shibu MA, Hsueh MF, Chen YS, Tsai FJ, Yao CH, Lin CC, Pan LF, Ju DT (2014) Citrus medica var. sarcodactylis (Foshou) activates fi broblast growth factor-2 signaling to induce migration of RSC96 Schwann cells. Am J Chin Med 42:443-452.

Ishibashi Y, Sugimoto T, Ichikawa Y, Akatsuka A, Miyata T, Nangaku M, Tagawa H, Kurokawa K (2002) Glucose dialysate induces mitochondrial DNA damage in peritoneal mesothelial cells. Perit Dial Int 22:11-21.

Ji Y, Shen M, Wang X, Zhang S, Yu S, Chen G, Gu X, Ding F (2012) Comparative proteomic analysis of primary schwann cells and a spontaneously immortalized schwann cell line RSC 96: a comprehensive overview with a focus on cell adhesion and migration related proteins. J Proteome Res 11:3186-3198.

Jung HS, Lee MS (2010) Role of autophagy in diabetes and mitochondria. Ann N Y Acad Sci 1201:79-83.

Kalichman MW, Powell HC, Mizisin AP (1998) Reactive Degenerative Proliferative Schwann cell responses in experimental galactose and human diabetic neuropathy. Acta Neuropathol (Berl) 95:47-56.

Kennedy JM, Zochodne DW (2005) Impaired peripheral nerve regeneration in diabetes mellitus. J Peripher Nerv Syst 10:144-157.

Kim H, Moon JY, Ahn KS, Cho SK (2013) Quercetin induces mitochondrial mediated apoptosis and protective autophagy in human glioblastoma U373MG cells. Oxid Med Cell Longev 2013: Article ID 596496.

Kitada M, Takeda A, Nagai T, Ito H, Kanasaki K, Koya D (2011) Dietary restriction ameliorates diabetic nephropathy through anti-infl ammatory e ff ects and regulation of the autophagy via restoration of Sirt1 in diabetic Wistar fatty (fa/fa) rats: a model of type 2 diabetes. Exp Diabetes Res 2011:908185.

Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, Agholme L, Agnello M, Agostinis P, Aguirre-Ghiso JA, Ahn HJ, Ait-Mohamed O, Ait-Si-Ali S, Akematsu T, Akira S, Al-Younes HM, Al-Zeer MA, Albert ML, Albin RL, Alegre-Abarrategui J, et al. (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy. 8:445-544.

Liao YR, Lin JY. (2014) Quercetin, but not its metabolite quercetin-3-glucuronide, exerts prophylactic immunostimulatory activity and therapeutic antiinflammatory effects on lipopolysaccharide-treated mouse peritoneal macrophages ex vivo. J Agric Food Chem 62:2872-2880.

Liu J, Chen Z, Zhang Y, Zhang M, Zhu X, Fan Y, Shi S, Zen K, Liu Z (2013) Rhein protects pancreatic β-cells from dynamin-related protein-1-mediated mitochondrial fi ssion and cell apoptosis under hyperglycemia. Diabetes. 62:3927-3935.

Lo MC, Lu CI, Chen MH, Chen CD, Lee HM, Kao SH (2010) Glycoxidative stress-induced mitophagy modulates mitochondrial fates. Ann N Y Acad Sci 1201:1-7.

Marsh BJ, Soden C, Alarcón C, Wicksteed BL, Yaekura K, Costin AJ, Morgan GP, Rhodes CJ (2007) Regulated autophagy controls hormone content in secretory-de fi cient pancreatic endocrine beta-cells. Mol Endocrinol 21:2255-2269.

Masini M, Bugliani M, Lupi R, del Guerra S, Boggi U, Filipponi F, Marselli L, Masiello P, Marchetti P (2009) Autophagy in human type 2 diabetes pancreatic beta cells. Diabetologia 52:1083-1086.

Mellor KM, Bell JR, Young MJ, Ritchie RH, Delbridge LM (2011) Myocardial autophagy activation and suppressed survival signaling is associated with insulin resistance in fructose-fed mice. J Mol Cell Cardiol 50:1035-1043.

Milackova I, Prnova MS, Majekova M, Sotnikova R, Stasko M, Kovacikova L, Banerjee S, Veverka M, Stefek M (2014) 2-Chloro-1,4-naphthoquinone derivative of quercetin as an inhibitor of aldose reductase and anti-in fl ammatory agent. J Enzyme Inhib Med Chem [Epub ahead of print]

Miranda S, González-Rodríguez Á, García-Ramírez M, Revuelta-Cervantes J, Hernández C, Simó R, Valverde ÁM (2012) Bene fi cial effects of feno fi brate in retinal pigment epithelium by the modulation of stress and survival signaling under diabetic conditions. J Cell Physiol 227:2352-2362.

Mizisin AP, Powell HC (1997) Schwann cell changes induced as early as one week a■er galactose intoxication. Acta Neuropathol 93:611-618.

Mizukami H, Takahashi K, Inaba W, Tsuboi K, Osonoi S, Yoshida T, Yagihashi S (2014) Involvement of oxidative stress-induced DNA damage, endoplasmic reticulum stress, and autophagy de fi cits in the decline of β-cell mass in japanese type 2 diabetic patients. Diabetes Care [Epub ahead of print]

Murakawa Y, Zhang W, Pierson CR, Brismar T, Ostenson CG, Efendic S, Sima AA (2002) Impaired glucose tolerance and insulinopenia in the GK-rat causes peripheral neuropathy. Diabetes Metab Res Rev 18:473-483.

Olaleye M, Crown O, Akinmoladun A, Akindahunsi A (2013) Rutin and quercetin show greater efficacy than nifedipin in ameliorating hemodynamic, redox, and metabolite imbalances in sodium chloride-induced hypertensive rats. Hum Exp Toxicol 33:602-608.

Ost A, Svensson K, Ruishalme I, Brännmark C, Franck N, Krook H, Sandström P, Kjolhede P, Strålfors P (2010) Attenuated mTOR signaling and enhanced autophagy in adipocytes from obese patients with type 2 diabetes. Mol Med 16:235-246.

Pan Liangchun, Yin Zongsheng, Wang Wei (2005) Experimental study of methods of culturing and purifying newborn and adult rat Schwann cells. Zhongguo Kangfu Yixue Zazhi 20:817-819.

Peng KY, Horng LY, Sung HC, Huang HC, Wu RT (2011) Hepatocyte growth factor has a role in the amelioration of diabetic vascular complications via autophagic clearance of advanced glycation end products: Dispo85E, an HGF inducer, as a potential botanical drug. Metabolism 60:888-892.

Prasath GS1, Subramanian SP (2013) Fisetin, a tetra hydroxy fl avone recuperates antioxidant status and protects hepatocellular ultrastructure from hyperglycemia mediated oxidative stress in streptozotocin induced experimental diabetes in rats. Food Chem Toxicol 59:249-255.

Qu L, Liang XC, Zhang H (2008) Recent advance in the studies of the e ff ect of high glucose on Schwann cells in vitro. Jichu Yixue yu Linchuang 28:1324-1328.

Quan W, Hur KY, Lim Y, Oh SH, Lee JC, Kim KH, Kim GH, Kim SW, Kim HL, Lee MK, Kim KW, Kim J, Komatsu M, Lee MS (2012) Autophagy de fi ciency in beta cells leads to compromised unfolded protein response and progression from obesity to diabetes in mice. Diabetologia 55:392-403.

Shi Y, Liang XC, Wu QL, Sun LQ, Qu L, Zhao L, Wang PY (2013) Effects of Jinmaitong Capsule on ciliary neurotrophic factor in sciatic nerves of diabetes mellitus rats. Chin J Integr Med 19:104-111.

Shi Y, Liang XC, Zhang H, Wu QL, Qu L, Sun Q (2013) Quercetin protects rat dorsal root ganglion neurons against high glucose-induced injury in vitro through Nrf-2/HO-1 activation and NF-κB inhibition. Acta Pharmacol Sin 34:1140-1148.

Tsapas A, Liakos A, Paschos P, Karagiannis T, Bekiari E, Tentolouris N, Boura P (2014) A simple plaster for screening for diabetic neuropathy: A diagnostic test accuracy systematic review and meta-analysis. Metabolism 63:584-592.

Uchiyama Y, Shibata M, Koike M, Yoshimura K, Sasaki M (2008) Autophagy-physiology and pathophysiology. Histochem Cell Biol 129:407-420.

Wang K, Liu R, Li J, Mao J, Lei Y, Wu J, Zeng J, Zhang T, Wu H, Chen L, Huang C, Wei Y (2011) Quercetin induces protective autophagy in gastric cancer cells: involvement of Akt-mTOR-and hypoxia-induced factor 1α-mediated signaling. Autophagy 7:966-978.

Wang Q, Liang B, Shirwany NA, Zou MH (2011) 2-Deoxy-D-glucose treatment of endothelial cells induces autophagy by reactive oxygen species-mediated activation of the AMP-activated protein kinase. PLoS One 6:e17234.

Wu CY, Tang ZH, Jiang L, Li XF, Jiang ZS, Liu LS (2012) CSK9 siRNA inhibits HUVEC apoptosis induced by ox-LDL via Bcl/Bax-caspase9-caspase3 pathway. Mol Cell Biochem 359:347-358.

Wu WH, Zhang MP, Zhang F, Liu F, Hu ZX, Hu QD, Yan XY, Huang SM (2011) ■e role of programmed cell death in streptozotocin- induced early diabetic nephropathy. J Endocrinol Invest 34:e296-301.

Wu X, Qu X, Zhang Q, Dong F, Yu H, Yan C, Qi D, Wang M, Liu X, Yao R (2014) Quercetin promotes proliferation and di ff erentiation of oligodendrocyte precursor cells a■er oxygen/glucose deprivation-induced injury. Cell Mol Neurobiol 34:463-471.

Xie Z, Lau K, Eby B, Lozano P, He C, Pennington B, Li H, Rathi S, Dong Y, Tian R, Kem D, Zou MH (2011). Improvement of cardiac functions by chronic metformin treatment is associated with enhanced cardiac autophagy in diabetic OVE26 mice. Diabetes 60:1770-1778.

Yamahara K, Yasuda M, Kume S, Koya D, Maegawa H, Uzu T (2013)■e role of autophagy in the pathogenesis of diabetic nephropathy. J Diabetes Res 2013:193757.

Yin Y, Lin Q, Sun H, Chen D, Wu Q, Chen X, Li S (2012) Cytotoxic effects of ZnO hierarchical architectures on RSC96 Schwann cells. Nanoscale Res Lett 7:439.

Youl E, Magous R, Cros G, Oiry C (2014) MAP Kinase cross-talks in oxidative stress-induced impairment of insulin secretion. Involvement in the protective activity of quercetin. Fundam Clin Pharmacol doi: 10.1111/fcp.12078.

Younce CW, Wang K, Kolattukudy PE (2010) Hyperglycaemia-induced cardiomyocyte death is mediated via MCP-1 production and induction of a novel zinc- fi nger protein MCPIP. Cardiovasc Res 87:665-674.

Zemp C, Bestetti G, Rossi GL (1981) Morphological and morphometric study of peripheral nerves from rats with streptozotocin-induced diabetes mellitus. Acta Neuropathol 53:99-106.

Zychowska M, Rojewska E, Przewlocka B, Mika J (2013) Mechanisms and pharmacology of diabetic neuropathy - experimental and clinical studies. Pharmacol Rep 65:1601-1610.

Copyedited by Slone-Murphy J, Li CH, Song LP, Zhao M

10.4103/1673-5374.135328

http://www.nrronline.org/

Accepted: 2014-05-16