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Article
Anthocyanins from Chinese bayberry extract activate transcription factorNrf2 in beta cells and negatively regulate oxidative stress-induced autophagy
Bo Zhang, Miranbieke Buya, Wenjie Qin, Chongde Sun, Haolei Cai, Qiuping Xie, Bing Xu, and Yulian WuJ. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf4012399 • Publication Date (Web): 11 Aug 2013
Downloaded from http://pubs.acs.org on September 4, 2013
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Title page
Anthocyanins from Chinese bayberry extract activate transcription factor
Nrf2 in beta cells and negatively regulate oxidative stress-induced
autophagy
Bo Zhang††††﹟, Miranbieke Buya
†﹟, Wenjie Qin
†﹟, Chongde Sun
‡, Haolei Cai
†,
Qiuping Xie†, Bing Xu
†, Yulian Wu
*,†
†Department of Surgery (Key Laboratory of Cancer Prevention and Intervention,
China National Ministry of Education), Second Affiliated Hospital, School of
Medicine, Zhejiang University. No.88 Jiefang Road, Hangzhou, Zhejiang Province,
310009, P.R.China
‡Laboratory of Fruit Quality Biology/The State Agriculture Ministry Laboratory of
Horticultural Plant Growth, Development and Quality improvement, Zhejiang
University, Huajiachi Campus, Hangzhou, Zhejiang Province, 310029, P.R. China
﹟ These authors contribute to this work equally.
* Address correspondence and reprint requests to
Yulian Wu, M.D., Ph.D. (Tel: 86-0571-87784604; Fax: 86-0571-87784604; E-mail:
wuyulian@medmail.com.cn)
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Abstract 1
Islet replacement is the promising cure for insulin-dependent diabetes, but is 2
limited by a massive early cell death following transplantation. Overburden oxidative 3
stress is one of the major factors causing cell damage. We have shown previously that 4
anthocyanins in Chinese bayberry extract protected beta cells (INS-1) from hydrogen 5
peroxide (H2O2)-induced apoptosis and decreased grafts’ apoptosis after 6
transplantation partially through heme oxygenase-1 (HO-1) upregulation. In the 7
present study, we observed that H2O2 stimulation induced autophagy in beta cells. 8
Inhibition of autophagy increased cell viability and decreased cell death. 9
Anthocyanins pretreatment attenuated oxidative stress-mediated autophagic cell death. 10
Anthocyanins activated antioxidant transcription factor Nrf2 in INS-1 cells, and 11
Nrf2/HO-1 negatively regulated autophagy process. Furthermore, we here 12
demonstrated that autophagy also took place in beta-cell grafts during the early 13
post-transplantation phase. Beta cells pretreated with anthocyanins displayed 14
decreased extent of autophagy after transplantation. Taken together, these findings 15
further supported the conclusion that anthocyanins could serve as a protective agent of 16
beta cells and suggested that autophagy might play a role in beta cells during 17
transplantation. 18
19
Keywords: Anthocyanins; Nrf2; Heme oxygenase-1; Islet transplantation; 20
Autophagy; Oxidative stress. 21
22
23
24
25
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Introduction 26
Islet transplantation is currently the effective treatment of type 1 diabetes that 27
achieves insulin-independence. However, the grafts will experience a progressive 28
deterioration of cellular viability and function overtime, leading to a large proportion 29
of subjects returning to insulin dependence several years later (1). This outcome is 30
mainly attributed to (1) early graft damage during islet isolation, preservation and 31
early post-transplantation phase. During this stage, hypoxia, interrupted blood supply, 32
ischemia/reperfusion injury and non-specific inflammation are the main causes of 33
beta-cell death, with the highest percentage of islet graft loss and dysfunction 34
occurring just days after transplantation; (2) late phase loss. Immunoreactions play 35
major roles in this stage, which are capable of inflicting beta-cell damage and 36
impairing beta-cell function. Because of reduced antioxidant defenses, beta cells are 37
increasingly vulnerable to oxidative/nitrosative damage arising during these stages (2). 38
Increasing antioxidant capacity to render islets more resistance to free radical damage 39
is theoretically a promising strategy to delay cell failure. Antioxidant treatment is 40
reported to possess functions of decreasing free radical-induced beta cell damage in 41
several animal and human studies (3). 42
Autophagy is the major cellular pathway for the degradation of long-lived 43
proteins and cytoplasmic organelles in animal cells(4). During autophagy, cytoplasmic 44
materials are sequestered into double-membrane vesicles, ‘autophagosome’, which 45
then fuse with lysosomes to form autolysosomes, where degradation of cellular 46
structures occurs (5, 6). Autophagy could be stimulated by a change of environmental 47
conditions such as nutrient deprivation. Hypoxia, ischemia/reperfusion and excessive 48
free radicals, which are also the stress stimulations during islet transplantation as 49
mentioned above, are considered as the inducers of autophagy in certain 50
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circumstances. However, up to now, limited information has been reported about 51
whether autophagy appears in beta cells during transplantation procedure. 52
Chinese bayberry is one of six Myrica species native to China and possesses 53
various biological activities including notable radical-scavenging ability(7-9). The 54
fruit extract is rich in anthocyanins, and cyanidin-3-O-glucoside (C3G) is identified as 55
a major anthocyanin component (9, 10). We previously showed that anthocyanins 56
pretreatment protected pancreatic beta cells (rat insulinoma cell line INS-1) against 57
hydrogen peroxide (H2O2)-induced necrosis and apoptosis via HO-1 induction, and 58
decreased graft apoptosis after transplantation(11) . In the present study, we explore 59
the occurrence of autophagy in beta cells during oxidative stress stimulation in vitro 60
and at early post-transplantation phase, and further investigate the protective effect of 61
anthocyanin with the emphasis on its impact on autophagy. 62
63
Materials and methods 64
Chinese bayberry fruit extract preparation 65
Chinese bayberry fruits were obtained from Yuyao County, Zhejiang Province, 66
China. The fruit was then ground and extracted with extract solvent (methanol 67
acidified with 0.05% HCl) as described previously(11). After being extracted for 3 68
times and dried by evaporation at low temperature, the extract was dissolved in 80% 69
ethanol solvent (4.5% formic acid) to remove impurities such as sugar. After being 70
dried by evaporation again, anthocyanins were dissolved in double-distilled water, 71
and underwent further cryo-concentration. HPLC analysis of the extract was carried 72
out (10), and the content of C3G was calculated. HPLC analysis demonstrated that 73
C3G accounted for 95% of total anthocyanins with a dominant peak at 520nm. The 74
concentration of C3G was 2.77 mmol/L (1.245 mg/ml), determined by peak area 75
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measurement with pure C3G as standard sample (FigureS1). Since C3G is the major 76
component, the usage dose of anthocyanins was standardized by the concentration of 77
C3G.. 78
Cell culture conditions, reagents and plasmid 79
Rat pancreatic beta-cell line INS-1 were grown in RPMI-1640 supplemented 80
with 10% fetal bovine serum at 37 ℃ in a humidified atmosphere (5% CO2 and 95% 81
air). MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide], Trypan 82
blue dye, 3-Methylamphetamine (3-MA), monodansylcadaverine (MDC, a specific 83
autophagic vacuole marker) and Hoechst 33258 (H33258) were the products of Sigma 84
Chemical Co. (St. Louis, MO, USA). The commercially available in situ cell 85
apoptosis detection kit (used for TUNEL test) was purchased from Promega 86
(Madison,WI,USA). Annexin V-FITC/PI apoptosis detection kit was obtained from 87
Abcam Public Limited Company (Abcam, Cambridge, MA, USA). LipofectamineTM
88
2000 transfection reagent was obtained from Invitrogen (Carlsbad,CA, USA). 89
GFP-mRFP-LC3 plasmid was kindly provided by Prof. T. Yoshimori (Osaka 90
University, Osaka, Japan). The pLNCX2-HO-1 vector carrying the coding sequence 91
of the rat HO-1 gene and INS-1 cells which were transfected with pLNCX2-HO-1 92
stably overexpressing HO-1 (INS-1/HO1) were established in our previous study(11) . 93
Cell viability assay 94
Cell viability was determined using MTT dyes as previously described(12). 5 95
×103 INS-1 cells per well were seeded in 96-well plates, after 1mM H2O2 stimulation 96
for 2 hours, 20 µL of MTT solution was added to each well. After 4 hours’ incubation, 97
the supernatant was removed and 150 µL of DMSO was added to each well. The 98
plates were shaken for 10 minutes, and Optical Density was measured by an ELISA 99
reader (BIO-Tek ELx800, Winooski, VT, USA) at a wavelength of 570 nm. Cell 100
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viability was also measured using the trypan blue dye exclusion test as previously 101
described(11). After trypan blue dye was added to treated INS-1 cells for 5 minutes, 102
the number of staining cells (unviable cells) were counted and calculated as a percent 103
of total number of cells. 104
Flow cytometric detection of apoptosis 105
Cellular apoptosis was detected quantitatively by flow cytometry (FCM) as 106
previously described(12). After treatment, 1×106 INS-1 cells were suspended in 1 mL 107
1×PBS and centrifuged (1200 rpm×5 minutes), then the supernatant was removed and 108
cells were resuspended in 500 µL 1×Binding Buffer. 10 µL Annexin V-FITC and 5 µL 109
PI were added and kept in darkness with incubation of 15 minutes. The cellular 110
apoptosis was assessed by flow cytometry using the Cell Quest program (Becton 111
Dickinson, San Jose, CA, USA). 112
Transmission electron microscope (TEM) 113
After experimental manipulations, cells or tissues were fixed in 2.5% glutaraldehyde 114
and 1% osmic acid for 1h respectively, followed by 4% uranyl acetate for 30 minutes 115
and gradient ethanol dehydration. At last, the fixed samples were infiltrated with 116
absolute acetone and the final resin mixture. Then the materials were embedded in 117
Epon 812, cut with Ultracut UCT ultramicrotome (Leica, Bensheim, Germany), and 118
photographed by electron microscopy (JEM-1230). 119
Protein extraction and western blot analysis 120
Whole cell lysates were obtained by resuspending cells in a cell lysis buffer 121
containing a protease inhibitor cocktail as previously described(12). Nuclear and 122
cytoplasmic extracts were prepared using commercial NE-PER Nuclear and 123
Cytoplasmic Extraction Reagents (Pierce Biotechnology, USA; Cat: 78833). Protein 124
concentration was determined using the BCA protein assay kit (Pierce Biotechnology, 125
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USA). Protein samples (equal amounts) were separated by SDS-PAGE (sodium 126
dodecylsulfate-polyacrylamide) and transferred to PVDF membranes, which were 127
then blocked with 5% non-fat milk, and probed overnight with primary antibody at 128
4℃. After undergoing washes for 3 times, the membranes were incubated with 129
secondary antibodies. The blots were visualized by enhanced chemiluminescence. 130
Primary antibodies against beclin-1(BECN1), Nrf2 and actin were purchased from 131
Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-LC3 antibody was obtained 132
from MBL International (MBL, Nagoya, Japan). Antibody against HO-1 was 133
purchased from Cell Signaling Technology, Inc. (Beverly, MA,USA). Caspase-3 and 134
Lamin A/C antibodies were obtained from Abcam Public Limited Company (Abcam, 135
Cambridge, MA,USA). 136
Electrophoretic Mobility Shift Assay (EMSA) 137
To determine whether anthocyanins increase Nrf2 DNA-binding activity, we 138
implemented electrophoretic mobility shift assay (EMSA) using the Viagene EMSA 139
kit (Viagene Biotech Inc., China) following the manufacturer’s protocol. Briefly, 140
equal amounts of nuclear protein (5 µg) were incubated with the biotin-labeled Nrf2 141
oligonucleotide and poly dI:dC for 20 min at room temperature in binding reaction 142
buffer. The DNA-protein complexes were resolved on a 6.5% polyacrylamide gel 143
pre-electrophoresed in 0.25×TBE solution at 120V for 1h. Then the gel was 144
transferred on to positively charged nylon membrane. The transferred DNA was 145
cross-linked to the membrane and detected with horseradish peroxidase-conjugated 146
streptavidin. 147
Monodansylcadaverine (MDC) staining and Nrf2 immunofluorescence 148
INS-1 cells were grown on glass coverslips. After being treated with the 149
indicated agents, cells were incubated with 0.05mM MDC for 20 minutes at 37°C. 150
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Cells were washed 2 times with PBS and observed under fluorescence microscope. 151
For immunofluorescence detection of Nrf2, treated cells were fixed with 4% 152
paraformaldehyde (PFA) for 30 minutes at room temperature. After treatment with 153
Triton X-100 (0.1%) for 10 minutes and normal goat serum for 30min at room 154
temperature, anti-Nrf2 antibody (Santa Cruz, CA,USA; 1:200 in 2%BSA) was added 155
and incubated overnight at 4°C. After an additional incubation for 1 hour at room 156
temperature with Alexor 488-conjugated goat-anti-rabbit antibody (Molecular Probe, 157
Invitrogen, USA) (1:200 in PBS), the slides were mounted and observed under 158
fluorescence microscope. 159
Plasmid transfection and RNA interference 160
For detection of autophagosomes, INS-1 cells were transfected with 161
GFP-mRFP-LC3 plasmid using LipofectamineTM
2000 reagent, and the transfected 162
cells were treated with H2O2 for 2 hours and observed under fluorescence microscope. 163
For RNA interference studies, the target sequence for HO-1-specific small 164
interference RNA (siRNA) was 5’- CCGUGGCAGUGGGAAUUUAUGCCAU-3’, 165
BECN1 siRNA sequence was5’- GUCCCUGACAGACAAAUCU-3’, both of which 166
and the control siRNA (no silencing) were synthesized by GenePharma Co. (Shanghai, 167
China). Nrf2 siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, 168
USA; Cat: sc-156128). Transfection was performed using LipofectamineTM
2000 169
reagent according to the manual instructions. 170
Animal studies 171
ICR mice of 8 weeks in age were used as the recipients in beta-cell 172
transplantation experiment and the experimental procedures were described in our 173
previous study(11). Pretreated INS-1 cells (2×106 cells with 1µM anthocyanins for 24 174
hours) and untreated INS-1 cells (2×106 cells) were transplanted under renal capsules 175
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of ICR mice respectively. 72 hours after transplantation, beta cell grafts with a part of 176
kidneys were harvested and fixed with 4% PFA, embedded in paraffin for 177
immunohistological analysis. Immunohistological staining for LC3, BECN1, and 178
TUNEL staining were performed according to the manual instructions. Some samples 179
were fixed in glutaraldehyde for further transmission electronic microscopic analysis 180
as described above. All experiments were carried out in accordance with the ethical 181
guidelines of Animal Experimentation Committee in our institute. 182
Statistical analysis 183
When necessary, data were expressed as mean±SD. Statistical analysis was 184
performed using SPSS version 13.0. One-way ANOVA with Bonferroni’s post hoc 185
test and Student’s t test were used to evaluate statistical significance and p<0.05 was 186
considered significant. 187
188
Results 189
H2O2 stimulation induced autophagic cell death in beta cells 190
We previously demonstrated that H2O2 injured INS-1 cells in a dose- and time- 191
dependent manner, and we selected 1mM H2O2 stimulation for 2 hours as stress 192
condition, which was also chosen for the present study. Since oxidative stress was 193
also an autophagy inducer, we further explored whether autophagy was activated in 194
H2O2-treated beta cells. As shown in Figure1A, in comparison to control cells, 195
extensive swelling mitochondria and autophagic vacuolization could be observed in 196
H2O2-treated cells under TEM. Microtubule-associated protein 1 light chain 3 (MAP 197
LC3 or LC3) is a specific marker for autophagosome formation. The GFP-mRFP-LC3 198
vector, carrying the coding sequence of LC3 gene, was transfected into INS-1 cells, 199
and cells with GFP-mRFP-LC3 vacuoles were observed under fluorescent microscope. 200
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Figure1B clearly indicated that GFP-mRFP-LC3 is localized in vacuoles (dots) after 201
2 hours’ H2O2 treatment, whereas in control cells, LC3 fusion protein distributed in a 202
diffuse pattern. These results supported the occurrence of autophagy in beta cells 203
under oxidative stress condition. 204
Since autophagy has two different effects (pro-survival or pro-death), we next 205
investigate the role of autophagy in H2O2-induced cell death. The expression of 206
BECN1 was down-regulated through siRNA transfection (about 70% downregulation), 207
leading to decreased extent of autophagy after H2O2 stimulation (Figure1C, D). We 208
found that H2O2-induced cell death was mitigated after BECN1 downregulation 209
(Figure1E, F) and the mitigation was independent of apoptosis (FigureS2), 210
suggesting the pro-death role of H2O2-induced autophagy (in other words, 211
“autophagic cell death”). 212
Anthocyanins treatment attenuated H2O2-induced autophagic cell death and 213
HO-1 upregulation participated in this process 214
Our previous study demonstrated that anthocyanins could time- and 215
dose-dependently upregulate HO-1 expression, which was involved in the protective 216
mechanism of anthocyanins against H2O2-induced necrotic and apoptotic cell death. 217
We here further investigate its impact on oxidative stress-induced autophagic cell 218
death. As MDC accumulates in mature autophagic vacuoles, such as 219
autophagolysosomes, MDC staining can be used to detect autophagic vacuoles. As 220
shown in Figure 2A, compared with H2O2-treated group, cells pre-incubated with 221
anthocyanins displayed decreased accumulation of MDC. Immunoblotting analysis 222
demonstrated decreased LC3II generation in the presence of 0.5 and 1µM 223
anthocyanins, compared with cells treated with H2O2 alone (Figure 2B). 224
To explore whether HO-1 was involved in H2O2-induced autophagic cell death, 225
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we first transfected beta cells with HO-1 siRNA to knock down HO-1 expression 226
(Figure 2C). Figure 2D showed that INS-1 cells, with reduced HO-1 expression, 227
displayed much notable autophagic cell death, as reflected by increased LC3II 228
production compared with control siRNA transfection group. Whereas in INS-1 cells 229
constitutively overexpressing HO-1 protein (INS-1/HO1), H2O2-induced autophagy 230
decreased (Figure 2E). In addition, HO-1 knockdown also attenuated anthocyanins’ 231
autophagy reduction effects, suggesting that anthocyanins-induced HO-1 expression 232
participated in autophagy process. 233
Involvement of Nrf2 activation in anthocyanins-induced HO-1 upregulation and 234
autophagy process 235
Nrf2 is known as an important transcription factor involves in antioxidant 236
response, binding to antioxidant response elements (ARE) encoding detoxification 237
enzymes including HO-1. We therefore examined whether the Nrf2 is involved in 238
anthocyanins-induced HO-1 expression. Immunofluorescence staining of Nrf2 239
showed that Nrf2 translocated from cytoplasm to nucleus in response to anthocyanins’ 240
treatment (Figure 3A). Such translocation was further supported by immunoblotting 241
analysis (Figure 3B). To evaluate anthocyanins-mediated Nrf2 transcription 242
factor-binding activity in INS-1 cells, EMSA was performed. As shown in Figure 3C, 243
treatment of cells with 1 µM or 2 µM anthocyanins for 12 h and 24 h increased the 244
DNA binding activity of Nrf2 in nuclear extracts. There was no detectable DNA 245
binding complex without loading protein. To investigate whether 246
anthocyanins-induced HO-1 expression is mediated through Nrf2 activation, cells 247
were transfected with Nrf2 siRNA for 24 h followed by anthocyanins stimulation. 248
Transfection of Nrf2 siRNA reduced Nrf2 expression (Figure 3D), as well as 249
anthocyanins-induced HO-1 expression (Figure 3E). Furthermore, Nrf2 siRNA 250
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exacerbated H2O2-induced autophagy (Figure 3F), indicating that Nrf2/HO-1 plays 251
protective role under such circumstance. 252
The occurrence of autophagy in beta-cell graft at the early post-transplantation 253
phase 254
To observe whether autophagy took place in beta-cell grafts, INS-1 cells were 255
transplanted under left renal subcapsule. 72 hours later, the recipient was sacrificed 256
and the graft was resected. As shown in Figure 4A,B, TEM displayed autophagic 257
vacuolization in beta-cell grafts. Immunofluorescence staining for MAP LC3 also 258
showed detectable punctate dots of LC3 in the cytoplasma (Figure 4C). These 259
observations supported the occurrence of autophagy at the early phase of 260
transplantation (72 hours). In addition, TUNEL-positive staining verified that some 261
cells underwent apoptosis (Figure 4D). 262
Anthocyanins decreased the extent of autophagy in grafts 263
We previously found that grafts pretreated with anthocyanins displayed less 264
apoptosis, we here focused on its impact on autophagy process. Figure 5A,B showed 265
that there were extensive LC3 immunopositive dot-like structures in cytoplasma of 266
transplanted INS-1 cells (Figure 5B), whereas the extent of punctuate dots of LC3 in 267
grafts significantly decreased after anthocyanins’ pre-incubation (Figure 5A). The 268
intensity of immunohistochemical staining for BECN1 in anthocyanins-pretreated 269
group was also less positive than that in control cells (Figure 5C,D). Together with 270
our previous observations, these results suggest that treating grafts with anthocyanins 271
prior to transplantation could decrease cellular death at early phase of 272
post-transplantation through downregulating both apoptotic and autophagic processes. 273
274
Discussion 275
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This work is the further step of our previous study, we here show that (1) 276
anthocyanins protect INS-1 cells from autophagic cell death induced by exogenous 277
H2O2 stimulation; and (2) autophagy happens in beta-cell grafts at early 278
post-transplantation phase, and anthocyanins pretreatment decrease the extent of 279
autophagy; (3) antioxidant enzyme expression may be associated with autophagy 280
process. These findings, combined with our previous results, further support that 281
anthocyanins in Chinese bayberry extract may be served as an islet-protective agent. 282
Autophagy plays an important role in cellular metabolism and is usually 283
activated under stress conditions such as starvation, ischemia/reperfusion, pathogen 284
infection and certain chemical stimulus. Recent studies showed that oxidative stress 285
could elicit increased autophagy(13-15). Exogenous H2O2 treatment leads to oxidative 286
stress and mitochondrial damage, which induce autophagy. It is also reported that 287
H2O2 treatment can mimic tumor necrosis factor alpha-induced autophagy and trigger 288
cell death(16). In the present study, exogenous H2O2 stimulation was applied to 289
increase intracellular ROS production in beta cells, and we also observed the 290
activation of autophagy in pancreatic beta cells after H2O2 treatment. H2O2 caused 291
excessive intracellular ROS production(11), mitochondrial damage (indicated by 292
TEM), and activated autophagy and apoptosis simultaneously. Further research 293
showed that downregulating BECN1 expression by siRNA attenuated H2O2-induced 294
cell injury (which was independent of apoptotic process), suggesting its pro-death role 295
under this circumstance. Such conclusion was contrary to the previous report 296
displaying autophagy acted as a survival pathway in a similar situation(17). We 297
suspected that the different effects of autophagy might be attributed to different cell 298
type, considering the susceptibility of beta cells to ROS. 299
In recent years, there are increasing studies performed to investigate the role of 300
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autophagy in beta cells and diabetes(18). Altered autophagy was observed in beta 301
cells of Zucker diabetic fatty rats, diabetic db/db mice and high-fat-fed C57BL/6 mice, 302
also in beta cell lines following high glucose or palmitate treatment(19-22). 303
Unbanlanced autophagy process was considered to be associated with beta-cell 304
dysfunction. Masini et al(23) observed that beta cells in patients with type 2 diabetes 305
have signs of altered autophagy, which might contribute to the loss of beta-cell mass. 306
In the field of islet transplantation, the early occurrence of apoptosis and necrosis in 307
islet grafts was confirmed in previous studies, but the questions of whether autophagy 308
participates in this process and its role in islet dysfunction have not yet been 309
investigated. We here, for the first time, observed that autophagy also occurred at 310
early post-transplantation phase (72 hours) in subrenal capsular transplantation model, 311
supported by the TEM and MAP LC3 immunostaining. Anthocyanins in CBE 312
protected beta cells against H2O2-induced autophagic cell death in vitro, and 313
pre-treating with anthocyanins also reduced autophagosome accumulation in beta-cell 314
transplantation model. In this context, despite the role of autophagy in the fate of 315
beta-cell grafts (pro-survival or pro-death) was not determined here, combined with 316
our previous and present results, the decreased extent of both apoptosis and autophagy 317
reflected the improved endurance of INS-1 cells to the hypoxia and ischemic stress in 318
graft’s microenvironment resulting in the survival of more viable and functional cells. 319
For excluding the possibility of direct scavenging H2O2 by anthocyanins, the 320
culture medium containing anthocyanins which was used to pre-treat INS-1 cells was 321
removed before H2O2 stimulation in this study. We found that anthocyanins increased 322
transcriptive activity of Nrf2, which was associated with the upregulation of HO-1. It 323
is well established that the Nrf2-Keap1 pathway functions as a critical regulator of the 324
cell's defense mechanism against oxidative stress by inducing the expression of many 325
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cellular protective proteins(24). We here observed that HO-1 overexpression (via gene 326
transfection or anthocyanins inducing) rescued cells from oxidative stress-induced 327
autophagy. Knockdown of HO-1 or Nrf2 exacerbated H2O2-induced autophagy, 328
suggesting that Nrf2/HO-1 pathways involved in regulating (oxidative stress-mediated) 329
autophagy in beta cells. These results were, to some extent, consistent with previous 330
study, in which Rao et al(25) observed Nrf2-regulated enzyme NQO1 played an 331
essential role in controlling the level of autophagy, and knockdown of Nrf2 caused 332
increase in autophagy. Similarly, HO-1 was reported to protect against cigarette 333
smoke extract-induced cell death by concurrently downregulating apoptosis and 334
autophagy-related signaling(26). Bolisetty et al(27) observed that HO-1 deficient cells 335
displayed higher levels of basal autophagy, whereas overexpression of HO-1 336
decreased extent of cisplatin-induced cell autophagy. 337
Overexpression of antioxidant enzymes, in most situations, could reduce the 338
occurrence of autophagy as mentioned above. However, the detailed regulation 339
process has not been fully determined yet. It is reported that HO-1 expression could 340
change the autophagic level via directly modulating the association of Beclin-1 with 341
Bcl-xL and Rubicon, a novel negative regulator of autophagy(28). Some other 342
research displayed that direct interaction existed between Nrf2-Keap1 pathway and 343
p62(29), a crucial adaptor between LC3-decorated autophagosomes and 344
ubiquitin-conjugated protein aggregates. Keap1, a negative controller of Nrf2 345
activation, could directly regulate autophagy through its interaction with LC3 and 346
p62(30-32). For the present study, whether upregulated Nrf2 itself or its induced 347
HO-1 overexpression controlled autophagic process is largely unknown. Furthermore, 348
significance of interactions among oxidative stress, Nrf2-Keap1 pathway, autophagy 349
(p62) in beta cells (including transplantation) and diabetes remains to be further 350
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elucidated. 351
352
Abbreviations 353
C3G: cyanidin-3-O-glucoside; CBE: Chinese bayberry extracts; MAP LC3: 354
microtubule-associated protein 1 light chain 3; MDC: monodansylcadaverine; Nrf-2: 355
nuclear factor erythroid 2-related factor 2; HO-1: heme oxygenase-1; ROS: reactive 356
oxygen species; siRNA: small interference RNA; TEM: transmission electron 357
microscopy. 358
359
Conflict of interest statement 360
The authors declare that there are no conflicts of interest. 361
362
Acknowledgement 363
This work was supported by grants from the National Natural Science Foundation of 364
China (nos. 81100549, 81172158, 81001094, and 81272332) and the Ministry of 365
Science and Technology of People’s Republic of China (no. 2007AA02Z476).). 366
367
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2. Kajimoto, Y.; Kaneto, H., Role of oxidative stress in pancreatic beta-cell dysfunction. Ann N Y 371
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456
Figure captions: 457
Figure1. H2O2 stimulation induced autophagic cell death in beta cells. A. INS-1 cells were treated 458
with 1mM H2O2 for 2h and underwent TEM examination. B. Representative micrographs of 459
INS-1 cells showing the punctate dots of LC3 in the cytoplasm. Cells were transiently transfected 460
with GFP-mRFP-LC3 plasmid for 24h and treated with/without H2O2 for 2h. C. INS-1 cells were 461
transfected with BECN1 siRNA or control siRNA (non-silencing). BECN1 expression was 462
analyzed by immunoblotting with anti-BECN1 antibody 48h after transfection (nearly 70% 463
downregulation). D. Cells transfected with BECN1 siRNA displayed decreased expression of 464
LC3II after H2O2 treatment compared with control group. E,F. After treatment of BECN1/control 465
siRNA-transfected cells with 1mM H2O2 for 2h, cell viability was measured by MTT assay and 466
trypan blue exclusion assay. *p<0.05 vs control siRNA-transfected cells. 467
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468
Figure2. Anthocyanins attenuated H2O2-induced autophagic cell death and HO-1 was involved in 469
regulation. A. INS-1 cells pretreated with/without anthocyanins were incubated with 1mM H2O2 470
for 2h, stained with MDC (0.05mM), and then visualized under fluorescence microscope. B. 471
Immunoblotting analysis showed LC3-II expression in INS-1 cells following treatment with H2O2, 472
in the absence or presence of anthocyanins pre-incubation. C. INS-1 cells were transfected with 473
control siRNA or HO-1 siRNA. 48 hours later, the expression of HO-1 protein was determined. D. 474
INS-1 cells were transfected with HO-1/control siRNA. 24~48h later, transfected cells were then 475
incubated with/without anthocyanins for another 24h, followed by 1mM H2O2 stimulation for 2h. 476
The expression of LC3II and the ratio of LC3II to LC3I were determined. E. INS-1 cells stably 477
overexpression HO-1 (INS-1/HO1) and control cells were stimulated with 1mM H2O2 for 2h. 478
Immunoblotting for LC3I/II were carried out.3-MA-pretreated (5 mM for 12h) group was serving 479
as a reference. 480
481
Figure3. Involvement of Nrf2 activation in anthocyanins-induced HO-1 upregulation and 482
H2O2–induced autophagy. A. After incubation with or without 1µM anthocyanins for 24 h, cells 483
were stained with anti-Nrf2 antibody and nuclei with H33258 dye. B. INS-1 cells were treated 484
with 1µM anthocyanins for 12 or 24 h, then the protein expression of Nrf2 in cytoplasmic and 485
nuclear extracts was determined by immunoblotting. C. After stimulation with or without 486
anthocyanins (1 or 2 µM) for 12h and 24h, the nuclear extracts of cells were obtained and EMSA 487
was performed to monitor Nrf2-ARE binding activity in the nuclear fraction. D. INS-1 cells were 488
transfected with Nrf2 siRNA, 48 h later, the Nrf2 expression was determined. E. Cells were 489
transfected with Nrf2 siRNA for 24 h, followed by stimulation with 1 µM anthocyanins for 490
another 24 h. Then, the protein level of HO-1 was observed by immunoblotting. F. After 491
transfection with or without Nrf2 siRNA for 24 h, followed by 1µM anthocyanins for 24 h, INS-1 492
cells were then stimulated with 1mM H2O2 for 2h. The expression of LC3I/II protein was 493
determined. Noted that Nrf2 down-regulation exacerbated H2O2–induced autophagic cell death. 494
495
Figure4. Autophagy occurred in beta-cell graft at the early phase of post-transplantation. INS-1 496
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cells were transplanted under renal capsule of mice and 72h later, the grafts were removed. A, B. 497
TEM examination displayed the presence of autophagosomes and autolysosomes in graft cells. C. 498
Punctate LC3 dots were observed in grafts detected by immunofluorescent for MAP LC3. D. A 499
portion of cells undergoing apoptosis were TUNEL positive. 500
501
Figure5. Anthocyanins pretreatment reduced the autophagy occurred in beta cells at the early 502
phase of post-transplantation. A~D. Immunochemical detection of LC3 and BECN1. INS-1 cells 503
pretreated with/without anthocyanins for 24h were transplanted under renal capsule, and 72h later, 504
the grafts were removed. Representative sections demonstrated that graft cells pretreated with 505
anthocyanins displayed less LC3 dots in cytoplasma (A) and less immunopositive for BECN1 (C) 506
compared with control cells (B,D). 507
508
Supplements: 509
FigureS1. HPLC profiles of the prepared sample display one major peak at a detection wavelength 510
of 520nm, which is identified as C3G by cochromatography with the standard C3G. The content of 511
C3G was determined by calculation of peak area. 512
513
FigureS2. The mitigation of H2O2-induced cell death after BECN1 downregulation was 514
independent of apoptosis. A. Apoptosis ratio of INS-1 cells in different groups was evaluated by 515
FCM after 1mM H2O2 stimulation for 2 hours. B. The protein expression of caspase-3 and PARP 516
were analyzed by immunoblotting. 517
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