Phycocyanin-Functionalized Selenium Nanoparticles Reverse

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Phycocyanin-functionalized selenium nanoparticles reverse palmitic acid-induced pancreatic beta cells apoptosis by enhancing cellular uptake and blocking ROS-mediated mitochondria dysfunction Chang Liu, Yuanting Fu, Chang-e Li, Tianfeng Chen, and Xiaoling Li J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36

Journal of Agricultural and Food Chemistry

1

Phycocyanin-functionalized selenium nanoparticles reverse palmitic

2

acid-induced pancreatic beta cells apoptosis by enhancing cellular uptake and

3

blocking ROS-mediated mitochondria dysfunction

4 5

Chang Liua#, Yuanting Fua#, Chang-e Lia, Tianfeng Chen a*, Xiaoling Li b*

6 7

a

Department of Chemistry, Jinan University, Guangzhou, China

8

b

Institute of Food Safety and Nutrition, Jinan University, Guangzhou, China

9 10

* Corresponding author. Jinan University, Guangzhou 510632, China. Tel.: +86 20

11

85227082. E-mail addresses: [email protected] (X LI). [email protected] (T Chen)

12 13 14 15 16 17 18 19 20 21 22

ACS Paragon Plus Environment


23

ABSTRACT: Accumulation of palmitic acid (PA) in human bodies could cause

24

damage to pancreatic beta cells and lead to chronic diseases by generation of reactive

25

oxygen species (ROS). Therefore, it is of great significance to search for nutrition

26

available agents with antioxidant activity to protect pancreatic islet cells against

27

PA-induced damage. Phycocyanin (PC) and selenium (Se) have been reported to have

28

excellent antioxidant activity. In this study, PC-functionalized selenium nanoparticles

29

(PC-SeNPs) were synthesized to investigate the in vitro protective effects on INS-1E

30

rat insulinoma beta cells against PA-induced cell death. Potent protective effect was

31

achieved by regulation of particle size and PC content. Among three PC-SeNPs (165

32

nm, 235 nm and 371 nm), PC-SeNPs-235 nm showed the highest cellular uptake and

33

the best protective activities. For cell cycle analysis, PC-SeNPs showed better

34

protective effect on PA-induced INS-1E cells apoptosis than PC or SeNPs, and

35

PC-SeNPs-235 nm exhibited the best effect. Further mechanistic studies demonstrated

36

that PA induced overproduction of intracellular reactive oxygen species (ROS),

37

mitochondria fragmentation, activation of caspase-3/-8/-9 and cleavage of PARP.

38

However, pre-treatment of the cells with of PC-SeNPs effectively blocked these

39

intracellular events, which suggest that PC-SeNPs could protect INS-1E cells against

40

PA-induced cell apoptosis via attenuating oxidative stress and downstream signalling

41

pathways. This finding provides a great promising nutritional approach to protect

42

diseases related with islet damage.

43 44

KEYWORDS: PC-functionalized selenium nanoparticles (PC-SeNPs), Oxidative


Page 2 of 36

Page 3 of 36


45

stress, Plamitic acid (PA), Antioxidant activities

46 47

INTRODUCTION

48

Obesity and hyperglycemia have been major problems exercising the minds of

49

modern people around the world and they were reported have relate to the damage of

50

islet beta cells (β cells)1,2. Many researchers have reported that the damage of β cells

51

is usually accompanied by the increasing production of reactive oxygen species (ROS)

52

and impaired antioxidant defenses3,4. Moreover, overproduction of ROS and reactive

53

nitrogen species (RNS) result in oxidative stress, which is a deleterious process that

54

can be important mediator of damage to cell structures5,6. The levels of antioxidant

55

enzymes in islet β cell was low, thus oxidative stress is the core sensitivity of

56

diabetes7,8. Therefore the β cells have a long-term chance of survival with the reduced

57

ROS level. Palmitic acid (PA), a saturated fatty acid, can cause the dysfunction of

58

pancreatic or isolated islets by generation of ROS9.

59

Phycocyanin (PC), a natural blue photosynthetic pigment purified from Spirulina, is

60

a highly active natural antioxidant, which could significantly activate SOD, GSH-PX

61

activity, increase the GSH content and enhance the intracellular antioxidant

62

capacity10-12. PC also shows good therapeutic values, such as antioxidant,

63

immunomodulation, anti-cancer, anti-inflammatory, blood vessel-relaxing and blood

64

lipid-lowering activities and so on11,13-16. Despite the widespread use of PC, there are

65

also some limitations in the application of PC for its instability, poor solubility and

66

poor penetrability into cells. Beside, PC is sensitive to moisture, light, temperature



67

and pH due to the degradation of the protein fraction15,17. Many studies have reported

68

that the modification of the protein conformation itself can improve the stability of

69

proteins18,19. Our previous studies have indicated that selenium-containing

70

allophycocyanin had hepatoprotective effect against the apoptosis induced by

71

t-BOOH20.

72

Selenium (Se) is one of the essential trace minerals in human and animals, which

73

attracts increasing interest both in pharmaceutical and food industry in recent

74

years21-23. Studies have identified that sodium selenite could improve glucose

75

homeostasis in type 1 and type 2 diabetic animals24,25. Previous studies also proved

76

that Se-PC could inhibit human islet amyloid polypeptide (hIAPP) fibrillation,

77

suppress the generation of ROS, and thus show protective effect on hIAPP-mediated

78

cell apoptosis and this effect was achieved by attenuating oxidative stress and

79

mitochondrial dysfunction26-28. Human erythrocytes could be protected by

80

selenium-containing allophycocyania from AAPH-induced oxidative damage through

81

inhibition of ROS generation29.

82

In the past few decades, emerging studies have indicated the potential application

83

of SeNPs and PC in food industry and pharmaceutical industry. However, little

84

information about the combination usage of PC and SeNPs was available in protecting

85

islet beta cells against diabetes. Therefore, it is of great interested to investigate

86

whether there was synergistic action between PC and SeNPs in protecting diseases

87

related with islet damage. In this present study, PC-SeNPs with different sizes were

88

constructed by selenite/GSH chemical reduction method to improve the antioxidant of


Page 4 of 36

Page 5 of 36


89

SeNPs. INS-1E rat insulinoma cell line was selected as the cell model to evaluate the

90

in vitro protective effects of PC- SeNPs against PA-induced cell damage (Scheme 1).

91

Particle size, PC content, cellular uptake, antioxidant activities, caspase activities,

92

ROS generation, mitochondria fragmentation and western blot were evaluated in this

93

study. The results showed that PC functionalization could enhance the protective

94

effect of SeNPs against PA-induced apoptosis through decreasing oxidative stress.

95

This finding demonstrates an effective nutritional approach to protect diseases related

96

with islet damage.

97 98

MATERIALS AND METHODS

99

Materials. Palmitic acid (PA), Propidium iodide (PI), thiazolyl blue tetrazolium

100

bromide

(MTT),

glutathione

(GSH),

4’,6-diamidino-2-phenylindole

101

bicinchoninic acid (BCA), sodium selenite and all other chemicals were bought from

102

Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO). fetal bovine serum (FBS),

103

RPMI-1640 medium and the antibiotic mixture (penicillin-streptomycin) were

104

purchased from Invitrogen (Carlsbad, CA). Antibody cleaved caspase-3, Caspase-3,

105

Caspase-8, Caspase-9 and cleaved caspase-9 were purchased from Cell Signaling

106

Technology (Beverly, MA). Caspase-3, caspase-8 and caspase-9 substrate were

107

obtained from Biomol (Germany).

108

Synthesis of PC-SeNPs. PC-SeNPs were prepared by the selenite/GSH chemical

109

reduction approach as reported30. Briefly, 0.5 mL of PC with a serious concentration

110

(5, 25, 50, 100, 150 mg/L) was mixed with 0.5mL of Na2SeO3 (100 mM), then 2 mL


(DAPI),


111

of GSH (100 mM) was drop-wise added into the mixture under stirring. After that,

112

deionized water was added to the mixture until the volume was 10 mL. 24 h later, the

113

mixture was dialyzed for 72 h in water and lyophilized for using.

114

Characterization of PC-SeNPs. The particle size distribution and zeta potential of

115

PC-SeNPs in aqueous solution was measured by Malvern Zetasizer Nano ZS

116

(Malvern Instruments Limited, Columbia, USA). The sizes and morphologies of SeNPs

117

and PC-SeNPs in the dry state were observed by transmission electron (TEM). FT-IR

118

(Equinox 55 IR spectrometer, Thermo Fisher, Waltham, USA) and UV-vis (Carry 5000

119

spectrophotometer, Palo Alto, USA) were applied to confirm the chemical composition

120

of nanoparticles.

121

Stability Assay. PC-SeNPs with the PC added concentration of 25, 50 and 100 mg/L

122

were selected to evaluate the stability of PC-SeNPs. At 37 ℃, particle size of

123

PC-SeNPs in PBS or DMEM were measured by Malvern Zetasizer Nano at

124

determined time intervals, respectively.

125

ABTS·+ Free Radical Scavenging Activity. The antioxidant activities of PC and

126

PC-SeNPs were measured by ABTS+ free radical scavenging assay as previously

127

described10. Briefly, the tested samples (50 µL) were mixed with ABTS+ reagent (1

128

mL) with absorbance of 0.70 ± 0.02 at 734 nm. After mixing for 6 min, the

129

absorbance of sample was measured.

130

Cellular Uptake Assay. Quantitative analysis of cellular uptake of PC-SeNPs was

131

carried out by testing the absorption of Se at determined time points. Briefly, INS-1E

132

cells (1.0 × 105 cells /well) were plated in 6-well plates for 24 h, then the culture


Page 6 of 36

Page 7 of 36


133

medium was replaced by DMEM medium without phenol red. 2 h later, different

134

PC-SeNPs were added to incubate with cells. At 0, 0.5, 1, 2, 3, 4, 6, 8, 10 and 12 h,

135

cells were collected and the content of Se in cells was measured by ICP-MS,

136

respectively.

137

Cell Culture and MTT Assay. INS-1E rat insulinoma cell line was bought from

138

American Type Culture Collection (ATCC, Manassas, VA, USA). With fetal bovine

139

serum (FBS, 10 %), L-glutamine (2 mM), sodium pyruvate (1 mM), HEPES (10 mM),

140

mercaptoethanol (50 µM), penicillin (100 units/mL) and streptomycin (100 µg/ mL),

141

the cells were cultured in RPMI-1640 medium at 37 °C in humidified atmosphere (5%

142

CO2). Cell viabilities were measured by using MTT assay as described previously31.

143

Briefly, INS-1E cells (6 × 104 cells/well) were seeded in 96-well plate at 37 °C for 24

144

h. Firstly, the cell cytotoxicity of PC-SeNPs or PA alone was carried out. For

145

determine the protective effect of PC, SeNPs and PC-SeNPs on INS-1E cells, the cells

146

were pre-incubated with PC, SeNPs and PC-SeNPs for 12 h, and then cells were

147

treated with PA, respectively (referred to PC + PA, SeNPs + PA and PC-SeNPs + PA

148

in the following description, respectively). After 48 h, the medium was removed and

149

MTT reagent was added. At 570 nm, the absorbance was determined by a microplate

150

reader (Spectra Max M5, Bio-Tek, Winooski, USA).

151

Cell Cycle Analysis. The cell cycle analysis was performed as described previously32.

152

The INS-1E cells were treated with different concentration of PC-SeNPs-235 nm for

153

12 h and then treated with PA for 2h. At 4 °C, using PBS solution containing 70 %

154

cold ethanol, the cells were washed, suspended and fixed for 24 h. Protected from



155

light, the cells were then incubated with PI/RNase staining solution (Cell Signaling)

156

for 30 min afterwards removing the fixation solution. The staining solution was

157

removed by washing 3 times with PBS, and then the cell cycle was analyzed using

158

flow cytometry (Beckman Coulter, Miami, FL, 1×106 cells/mL) with cells untreated

159

drugs as negative control. Similarly, for determining the protective effect of

160

PC-SeNPs, INS-1E cells were pre-incubated with different PC-SeNPs, PC and SeNPs

161

for 12 h, and then cells were treated with PA for 2 h.

162

Caspase Activity Assay and Western blot analysis. Caspase activity was evaluated

163

by meaning of fluorescence intensity by using specific caspase-3, -8 and -9 substrates

164

as previous reported33. Briefly, after treatment with PA alone or PC + PA, SeNPs +

165

SeNPs, and different size of PC-SeNPs + PA, collecting the cells by centrifugation

166

and suspending them with cell lysis buffer. The cell proteins were collecting by

167

centrifugation (12000 g, 30 min). Finally, to determine the fluorescence intensity of

168

cell lysates, Total cell lysates (100 µg/well) were placed in 96-well plates and then

169

specific caspase-3, -8 and -9 substrates were added and incubated at 37℃ for 2 h in

170

darkness. Finally, the fluorescence intensity was measured by using a microplate

171

reader (Spectra Max M5, Bio-Tek, Winooski, USA) with ex/em wavelengths of

172

380/460 nm. The effects of PA, PC, SeNPs and PC-SeNPs on expression levels of the

173

protein related to the apoptosis effects were determined by western blot analysis34.

174

Measurement of ROS Generation. In order to evaluate ROS accumulation in

175

INS-1E cells, the effects of PA and PC-SeNPs + PA on intracellular ROS generation in

176

INS-1E cells were detected by DCFH-DA assay and DHE assay34,35. Briefly, INS-1E


Page 8 of 36

Page 9 of 36


177

cells were seeded in 96-well plates (1×106 cells/mL). After pre-incubated with 0.8 µM

178

PC-SeNPs for 12 h, PA was added into plates and incubated for 2 h. Then, incubating

179

the cells with H2DCF-DA (10 µM) or DHE (100 µM) for 30 min at 37 °C. By using a

180

microplate reader (Spectra Max M5), the ROS level was determined at 488/525 nm

181

for DCF and 300/600 nm for DHE, respectively.

182

Fragmentation Analysis. Mitochondrial fragmentation analysis was carried out as

183

reported. INS-1E cells were pre-incubated with different PC-SeNPs (0.8 µM) for 12 h,

184

then 0.4 mM of PA was added. 2 h later, mitochondria and nucleuses of the cells were

185

stained with Mito Tracker Red CMXRos (50 nM, 2 h) and DAPI (1 µg/mL, 20 min),

186

respectively. After that, the cells were washed three times with PBS and re-cultured in

187

fresh medium. Then the cells were photographed under a monochromatic Cool

188

SNAPFX camera (Roper Scientific, New Jersey, USA).

189

Western blot analysis. Statistical Analysis. All samples were carried out at least

190

three times and data were presented as mean ± SD. Two-tailed Student’s t-test was

191

performed for the comparison among the different groups. Statistical significance was

192

defined as * p < 0.05.

193 194 195

RESULTS AND DISCUSSION

196

Preparation, Characterization and Stability of PC-SeNPs. Different concentration

197

of PC modified SeNPs (PC-SeNPs) were prepared under a simple redox system of

198

sodium selenite and glutathione (GSH) in this study. The sizes and morphologies of



199

PC-SeNPs were characterized by transmission electron microscope (TEM) and

200

Zetasizer Nano ZS particle analyzer. As shown in Figure 1A, SeNPs without PC were

201

unstable and accumulated into clumps in aqueous solution. At the concentration of 5

202

mg/mL, there also were large gathering of PC-SeNPs. With the increase of

203

concentration of PC (25, 50, 100 and 150 mg/L), the nanoparticles had uniform

204

spherical shapes and homogeneous particle sizes, which were 165, 235, 371 and 815

205

nm, respectively. With the increasing concentration of PC, the size of PC-SeNPs

206

became larger. PC-SeNPs with the PC concentration of 25, 50 and 100 mg/mL

207

showed better disperse and morphology, so the three PC-SeNPs were chosen to

208

evaluate the antioxidant activity. The particle size measured by Malvern Zetasizer

209

Nano ZS showed similar tendency with the results of TEM (Figure 1B). The zeta

210

potential was increased with the increasing PC content on the surface of SeNPs in a

211

certain range (Figure 1C).

212

Considering the dimensions of PC

36

, ≈10.2 nm × ≈10.2 nm × ≈10.9 nm, the

213

thickness of PC layer was used to estimate the number of molecules present in the

214

shell of the SeNPs by using the equation as below37:

215 216

Where N is the number of PC molecules per SeNPs; VPC is the PC’s volume

217

(3207.2 nm3)38; rSeNPs + PC shell and rSeNPs are the radius of the SeNPs and PC-SeNPs

218

respectively.

219

As the values obtained from TEM, rSeNPs, rpc-SeNPs-a, rpc-SeNPs-b and rpc-SeNPs-c

220

were about 63, 82.5, 117.5 and 185.5 nm, respectively. Thus, the molecules of PC


Page 10 of 36

Page 11 of 36


221

layer were 421.9, 1806.5 and 8021.3 for PC-SeNPs with particle size of 165, 235 and

222

371 nm, respectively. Furthermore, the content of PC on the surface of PC-SeNPs

223

were evaluated by BSA method and the results were shown in Figure 1D. With the

224

increasing PC concentration, the PC content on the surface of SeNPs were 3.54, 9.82,

225

19.78, 33.45, 38.82 µg/L, respectively.

226

FT-IR and UV-vis were employed to characterize the structure and formation of

227

PC-SeNPs. As shown in Figure 2A, the present of two special peaks around 1656 and

228

1204 cm-1 in the spectrum of PC and PC-SeNPs were assigned to the characteristic

229

absorption of amide group. The peaks around 1412 cm-1 in the spectrum were

230

confirmed the stretching vibration of carboxyl group. These indicated the success bind

231

of PC on the surface of SeNPs. Meanwhile, the UV absorption of PC-SeNPs at

232

wavelength of 268 and 623 nm in the spectrum of PC and PC-SeNPs also suggested

233

the success bind of PC on the surface of SeNPs (Figure 2B).

234

Stability studies of PC-SeNPs were also carried out under different conditions.

235

As shown in Figure 2C, there was little change in the size of PC-SeNPs (235 nm) in

236

the first 15 days and it became slightly larger on the 30th day in PBS. However, the

237

sizes of PC-SeNPs (165 nm) and PC-SeNPs (371 nm) became large on the 3 th or 5 th

238

day, respectively. These demonstrated that SeNPs modified with 50 mg/L of PC were

239

much stable than other PC-SeNPs. The possible reason was that the zeta potential of

240

PC-SeNPs (165 nm, -17.5 mV) was lower than PC-SeNPs (235 nm, -33.3 mV), so the

241

repulsion force was smaller and thus they were much easier to in aqueous solution.

242

Meanwhile, the massive of proteins adsorbed onto the surface of SeNPs were easy to



243

connect with each other, which caused the unstability of PC-SeNPs (371 nm). Besides,

244

under the physiological condition, with the present of 10 % fetal bovine serum (FBS)

245

in DMEM medium, the sizes of these three PC-SeNPs maintained constant during 48

246

h incubation (Figure 2D). The possible reason was that the negative-charged proteins

247

in FBS could be adsorbed to the surface of nanoparticles, which increased the stability

248

of the nanoparticles. In this study, GSH instead of ascorbic acid (Vc) was selected as

249

the reducing agent for preparing SeNPs, because GSH is weak alkaline in water

250

solution which is beneficial for the stability of PC 17.

251 252

ABTS+ Free Radical Scavenging Activities. The antioxidant activity of SeNPs and

253

PC-SeNPs with different sizes were evaluated by ABTS·+ assay. The antioxidant

254

activities of tested specimens were corresponding to inhibition percentage. As Figure

255

3A shown, the three different PC-SeNPs showed higher inhibition ability of ABTS

256

oxidation than SeNPs, which suggesting that PC-SeNPs had higher antioxidant

257

activity. With the highest PC content, PC-SeNPs with the size of 371 nm exhibited the

258

highest antioxidant among the three nanoparticles and the possible reason was that PC

259

also had strong antioxidant activity. To validate influence of PC content on the

260

antioxidant, different concentration PC-SeNPs (235 nm) were given to the cells and

261

the results showed that the higher concentration of PC-SeNPs, the stronger

262

antioxidant activity (Figure 3B).

263 264

Cellular Uptake of PC-SeNPs. Cellular uptake experiments were carried out on


Page 12 of 36

Page 13 of 36


265

INS-1E cells. In the first two hours, as shown in Figure 4, the cellular uptake of

266

PC-SeNPs-165 nm showed a rapid rate and then slowed down in the following time,

267

while the PC-SeNPs-235 nm and PC-SeNPs-371 nm showed similar cellular uptake

268

rate. The reason of this phenomenon might be particle size was the limiting factor for

269

cellular uptake in the first. As time went on, PC-SeNPs-235 nm showed the highest

270

cellular among the three nanoparticles. Nevertheless, the cellular uptake of

271

PC-SeNPs-371 nm had been persistently slowing. The possible reasons were as

272

follows: Firstly, the particle size is one of the important factors which influence the

273

cellular. Particles with small size have relatively large surface area and curvature,

274

which benefit to the cellular uptake39. Moreover, the zeta potential of particle is

275

another important influence factor40,41. Negative charged particles could enter the cell

276

easily and the stronger the negative, the more particles enter into cells which the

277

reason was that negative charged were hard to combined with BSA in circulation. In

278

summary, PC-SeNPs with 235 nm and charged -33.3 mV showed the highest cellular

279

uptake by INS-1E cells.

280 281

PC-SeNPs Inhibits PA-induced Cytotoxicity. As reported, islet β cells could be

282

damage by exposure to high concentration of free fatty acids (FFA) and palmitic acid

283

(PA) is one of the most representative one in FFA42. Consequently, the effect of

284

nanoparticles on PA-induced INS-1E cells damage was examined. As illustrated in

285

Figure 5 A, PC-SeNPs-235 nm slightly inhibited INS-1E cells growth even when the

286

concentration raised up to 4 µM, as indicated by the the cell viability of INS-1E cells



287

was 74.5% under this concentration treatments. PA significantly inhibited INS-1E

288

cells proliferation in a concentration dependent manner with its IC50 value less than

289

1µM. And 0.4 mM of PA induced INS-1E cells death was about 20%, which was

290

suitable for the next experiments (Figure 5B). PC and SeNPs could not reversed PA

291

induced INS-1E cells death when the concentration varied from 0.25 µM to 1µM

292

(Figure 5C). Interestingly, we found that, pre-incubating the cells with PC-SeNPs for

293

12 h dramatically restrained PA inhibition in the cells proliferation as can be seen in

294

the cell viability of PC-SeNPs-165 nm, PC-SeNPs-235 nm, PC-SeNPs-371 nm were

295

about 91.2%, 98.4% and 82.4%, respectively, which demonstrated that PC modified

296

SeNPs could decrease cell damage caused by PA. Moreover, among the three

297

PC-SeNPs, PC-SeNPs-235 nm displayed the best protective effect and the possible

298

reason might be PC-SeNPs-235 nm show the highest cellular uptake, the relatively

299

small particle size and the relatively higher PC content (Figure 5 D). In all, these

300

results indicated that PC-SeNPs could protect INS-1E cells from PA-induced

301

cytotoxicity in vitro.

302 303

Effects of PC-SeNPs on the cell cycle distribution induced by PA. Cell cycle arrest

304

and apoptosis are two major action modes which cause cell death43,44. In order to

305

investigate the mechanisms of PA induced cells death the protective effects of

306

PC-SeNPs, we carried out flow cytometry assay to examine the cell cycle distribution

307

and apoptosis of PA, PC, SeNPs and PC-SeNPs. The premier cell cycle pattern of

308

INS-1E cells without PA-induced treated with PI was set as control group. As shown


Page 14 of 36

Page 15 of 36


309

in Figure 6, PA treatment increased the population in sub-G1 from 3.7% to 30.6%,

310

which indicated that PA induced INS-1E cells death mainly through inducing

311

apoptosis. PC and SeNPs alone slightly decreased the cell apoptosis from 30.6% to

312

23.1% and

313

PC-SeNPs-235 nm and PC-SeNPs-371 nm) pretreatment significantly protect PA

314

induced INS-1E cells apoptosis without other phases obviously changes and

315

PC-SeNPs-235 nm appears to exhibited the most potent protective effects, as reflected

316

by the decreased population of sub-G1 from 30.6 % to 22.6 %, 19.4 % and 22.3 %

317

respectively. To further determine the effect of PC-SeNPs-235 nm, the dose-course

318

effects of PC-SeNPs-235 nm were evaluated. The results demonstrated that

319

PC-SeNPs-235 nm pretreatment notably protected INS-1E cells apoptosis induced by

320

PA. Taken together, these results demonstrated that PC-SeNPs effectively blocked the

321

apoptosis effects induced by PA.

24.3% respectively.

However,

PC-SeNPs

(PC-SeNPs-165

nm,

322 323

PC-SeNPs Inhibits PA-induced Caspase Activation. Evidences have implicated that

324

caspases play an essential role in apoptosis regulation46,47, therefore, we next

325

conducted fluorimetric assay to determine whether caspases activation were

326

involved in PA-induced apoptosis. As indicated in Figure 7A, PA significantly

327

induced caspase-8/9 and caspase-3 activation in INS-1E cells, which indicated the

328

activation of both extrinsic and intrinsic mediated apoptosis signaling pathway.

329

However, these effects were significantly inhibited by the pretreatment of PC and

330

SeNPs. Moreover, pre-incubation of PC-SeNPs dramatically reversed PA-induced



331

caspase-8/-9 and caspase-3 activation, as indicated by the decreased fluorescence

332

intensity in PC-SeNPs pre-treatments. Among them, PC-SeNPs-235 nm appeared to

333

display the most protective effects against PA induced caspases activation. These

334

results were further certified Western blotting results. As illustrated in Figure 7B,

335

exposure of INS-1E cells to PA resulted in significant cleavage of Caspase-3/-8/-9 and

336

PARP. PC and SeNPs displayed slightly protective effects against PA induced INS-1E

337

cells apoptosis as can be seen in the activation of caspase-3/8/9 and PARP. However,

338

PC-SeNPs pretreatment strongly restrained the caspases activation and PARP

339

cleavage induced by PA which indicated the protective effects of PC-SeNPs against

340

the apoptosis inducing capacity of PA towards INS-1E cells. Together, these results

341

clearly demonstrated that PC-SeNPs exhibited strongly protective effects against

342

PA-induced death receptor and mitochondria mediated apoptosis towards INS-1 cells.

343 344

Protective effect of PC-SeNPs Against ROS Overproduction. ROS and RNS

345

generation have been found to play important role in the oxidative damage of islet

346

cells. The excess amount of ROS could attack many components of DNA, thus causes

347

DNA damage48. To evaluate the effect of PC-SeNPs on ROS scavenging, INS-1E

348

cells were pre-incubated with 0.4 mM PA to induce the overproduction ROS. As

349

shown in Figure 8A, ROS in PA-induced INS-1E cells increased to 153% as the

350

control group was 100%. However, when INS-1E cells pre-incubated with PC-SeNPs,

351

the generation of ROS in PA-induced INS-1E cells significantly reduced. It was

352

103.2% when treated with PC-SeNPs-235 nm, which had the best protective effect.


Page 16 of 36

Page 17 of 36


353

Similarly, they were 110.3% and 142.3% for PC-SeNPs-165 nm and PC-SeNPs-371

354

nm, respectively. Furthermore, the representative DHE fluorescence images also

355

showed the similar results as shown in Figure 8B. In general, PC-SeNPs could

356

protect INS-1E from the damage by PA by eliminating ROS and PC-SeNPs-235 nm

357

showed the best protective effect.

358 359

PC-SeNPs Inhibits PA-induced Mitochondrial Fragmentation. Mitochondria are

360

remarkably dynamic organelles and the importance of energy generated by

361

mitochondria has long been appreciated. Mitochondrial dynamics are important

362

indications for the quality control of mitochondria49,50. Oxidative stress could cause

363

mitochondrial malfunction and enhance metabolism, which promoted the release of

364

apoptogenic factors from mitochondria inner membrane space and initiates apoptotic

365

cascades51. The cells treated with PA for 2 h and washed twice by PBS, then stained

366

by mito-Red for 2 h, mitochondrial fragmentation were observed under fluorescence

367

microscopy. As shown in Figure 9, the mitochondria were in good condition without

368

any for the control group, while 0.4 mM PA incubation induced obvious disruption of

369

mitochondria structure. Significantly reduction in these changes were observed when

370

pre-treated different PC-SeNPs and PC-SeNPs-161 nm showed the best effect. The

371

morphological improvements indicated that PC-SeNPs could restrain the PA-induced

372

damage to mitochondria in INS-1E cells.

373 374

Funding



375

This work was supported by National High-level personnel of special support

376

program, National High Technology Research and Development Program of China

377

(SS2014AA020538), Science Foundation for Distinguished Young Scholars of

378

Guangdong Province (S2013050014667), YangFan Innovative & Entepreneurial

379

Research Team Project (201312H05), Guangdong Special Support Program and

380

Guangdong Frontier Key Technological Innovation Special Funds (2014B050505012)

381

and Fundamental Research Funds for the Central Universities.

382 383

Notes

384

The authors declared no competing financial interest.

385

# Chang Liu and Yuanting Fu contributes equally to this manuscript.

386 387

References:

388

(1). Robertson RP, Harmon JS. Pancreatic islet β-cell and oxidative stress: The

389

importance of glutathione peroxidase. FEBS Lett. 2007;581:3743-3748.

390

(2). Andrikopoulos S. Obesity and Type 2 diabetes: Slow down!—Can metabolic

391

deceleration protect the islet beta cell from excess nutrient-induced damage? Mol Cell

392

Endocrinol. 2010; 316:140-146.

393

(3). Yu T, Robotham JL, Yoon Y. Increased production of reactive oxygen species in

394

hyperglycemic conditions requires dynamic change of mitochondrial morphology.

395

Proc Natl Acad Sci U S A. 2006; 103:2653-2658.

396

(4). Ha H, Hwang I, Park JH, Lee HB. Role of reactive oxygen species in the


Page 18 of 36

Page 19 of 36


397

pathogenesis of diabetic nephropathy. Diabetes Res Clin Pr. 2008; 82:S42-S45.

398

(5). Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and

399

antioxidants in oxidative stress-induced cancer. Chem-Biol Interact. 2006;160:1-40.

400

(6). Lenzen

401

2008;36:343-347.

402

(7). Robertson RP, Harmon J, Tran POT, Poitout V. β -Cell Glucose Toxicity,

403

Lipotoxicity, and Chronic Oxidative Stress in Type 2 Diabetes. Diabetes. 2004.

404

(8). Robertson RP. Chronic Oxidative Stress as a Central Mechanism for Glucose

405

Toxicity

406

2004;279:42351-42354.

407

(9). Ji J, Zhang L, Wang P, et al. Saturated free fatty acid, palmitic acid, induces

408

apoptosis in fetal hepatocytes in culture. Exp Toxicol Pathol. 2005;56:369-376.

409

(10) . Chen T, Wong Y. In vitro antioxidant and antiproliferative activities of

410

selenium-containing phycocyanin from selenium-enriched Spirulina platensis. J Agr

411

Food Chem. 2008;56:4352-4358.

412

(11). Thangam R, Suresh V, Princy WA, et al. C-Phycocyanin from Oscillatoria tenuis

413

exhibited an antioxidant and in vitro antiproliferative activity through induction of

414

apoptosis and G(0)/G(1) cell cycle arrest. Food Chem. 2013;140:262-272.

415

(12). Yan M, Liu B, Jiao X, Qin S. Preparation of phycocyanin microcapsules and its

416

properties. Food Bioprod Process. 2014;92:89-97.

417

(13) . Madhyastha HK, Sivashankari S, Vatsala TM. C-phycocyanin from Spirulina

418

fussiformis exposed to blue light demonstrates higher efficacy of in vitro antioxidant

in

S.

Oxidative stress: the vulnerable β-cell.

Pancreatic

Islet

Beta

Cells

in

Diabetes.


Biochem Soc

J

Biol

T.

Chem.


419

activity. Biochem Eng J. 2009;43:221-224.

420

(14). Zhu C, Ling Q, Cai Z, et al. Selenium-Containing Phycocyanin from

421

Se-Enriched Spirulina platensis Reduces Inflammation in Dextran Sulfate

422

Sodium-Induced Colitis by Inhibiting NF- κ B Activation. J. Agr. Food Chem.

423

2016;64:5060-5070.

424

(15). RISS J, DÉCORDÉ K, SUTRA T, et al. Phycobiliprotein C-Phycocyanin from

425

Spirulina platensis Is Powerfully Responsible for Reducing Oxidative Stress and

426

NADPH Oxidase Expression Induced by an Atherogenic Diet in Hamsters. J Agr

427

Food Chem. 2007;55:7962-7967.

428

(16).

429

Allophycocyanin Purified from Selenium-Enriched Spirulina platensis Attenuates

430

AAPH-Induced Oxidative Stress in Human Erythrocytes through Inhibition of ROS

431

Generation. J Agr Food Chem. 2011;59:8683-8690.

432

(17).

433

from Spirulina sp.: Influence of temperature, pH and preservatives. Process Biochem.

434

2012;47:659-664.

435

(18).

436

and

437

2004;63:89-94.

438

(19).

439

C-phycocyanin in a silica matrix. Res Chem Intermediat. 2009;35:607-613.

440

(20).

Zhang H, Chen T, Jiang J, Wong Y, Yang F, Zheng W. Selenium-Containing

Chaiklahan R, Chirasuwan N, Bunnag B. Stability of phycocyanin extracted

Fukui K, Saito T, Noguchi Y, et al. Relationship between color development protein conformation

in the

phycocyanin molecule. Dyes Pigments.

Li Y, Yang H, Cao FM. Effect of ultraviolet irradiation on photostability of

Fan C, Jiang J, Yin X, Wong K, Zheng W, Chen T. Purification of


Page 20 of 36

Page 21 of 36


441

selenium-containing allophycocyanin from selenium-enriched Spirulina platensis and

442

its hepatoprotective effect against t-BOOH-induced apoptosis. Food Chem.

443

2012;134:253-261.

444

(21).

445

mammalian selenoproteomes. SCIENCE. 2003;300:1439-1443.

446

(22).

447

Selenium-Containing Phycocyanin from Selenium-Enriched Spirulina platensis. J Agr

448

Food Chem. 2008;56:4352-4358.

449

(23).

450

zinc, selenium, and iron fertilizers on nutrients concentration and yield of rice grain in

451

China. J Agr Food Chem. 2008;56:2079-2084.

452

(24).

453

supranutritional selenate doses. In vivo and in vitro investigations with type II

454

diabetic db/db mice. J Nutr Biochem. 2006;17:548-560.

455

(25).

456

2000;57:1874-1879.

457

(26).

458

formation by selenium-containing phycocyanin and prevention of beta cell apoptosis.

459

Biomaterials. 2014;35:8596-8604.

460

(27).

461

against human islet amyloid polypeptide-induced apoptosis through attenuating

462

oxidative stress and modulating JNK and p38 mitogen-activated protein kinase

Kryukov GV, Castellano S, Novoselov SV, et al. Characterization of

CHEN T, WONG Y. In Vitro Antioxidant and Antiproliferative Activities of

Fang Y, Wang L, Xin Z, Zhao L, An X, Hu Q. Effect of foliar application of

Mueller AS, Pallauf J. Compendium of the antidiabetic effects of

Stapleton

SR.

Selenium: an insulin-mimetic.

Cell Mol Life Sci.

Li X, Ma L, Zheng W, Chen T. Inhibition of islet amyloid polypeptide fibril

Li X, Xu G, Chen T, et al. Phycocyanin protects INS-1E pancreatic beta cells



Page 22 of 36

463

pathways. Int J Biochem Cell B. 2009;41:1526-1535.

464

(28).

465

human islet amyloid polypeptide-induced apoptosis in INS-1E pancreatic beta cells:

466

An effect attenuated by phycocyanin. Int J Biochem Cell B. 2011;43:525-534.

467

(29).

468

Allophycocyanin Purified from Selenium-Enriched Spirulina platensis Attenuates

469

AAPH-Induced Oxidative Stress in Human Erythrocytes through Inhibition of ROS

470

Generation. J Agr Food Chem. 2011;59:8683-8690.

471

(30).

472

size effect in the induction of seleno-enzymes in both cultured cells and mice. Life Sci.

473

2004;75:237-244.

474

(31).

475

apoptosis

476

2013;34:7106-7116.

477

(32).

478

Derivatives to Antagonize Hyperglycemia-Induced Drug Resistance in Cancer Cells.

479

Chem-Asian J. 2015;10:642-652.

480

(33).

481

cells growth by Selenium nanoparticles through Akt/Mdm2/AR controlled apoptosis.

482

Biomaterials. 2011;32:6515-6522.

483

(34).

484

MCF-7 human breast carcinoma cells with involvement of p53 phosphorylation and

Li X, Chen T, Wong Y, et al. Involvement of mitochondrial dysfunction in

Zhang H, Chen T, Jiang J, Wong Y, Yang F, Zheng W. Selenium-Containing

Zhang JS, Wang HL, Bao YP, Zhang L. Nano red elemental selenium has no

Huang Y, He L, Liu W, et al. Selective cellular uptake and induction of of

cancer-targeted

selenium

nanoparticles.

Biomaterials.

Liu Y, Luo Y, Li X, Zheng W, Chen T. Rational Design of Selenadiazole

Kong L, Yuan Q, Zhu H, et al. The suppression of prostate LNCaP cancer

Chen T, Wong Y. Selenocystine induces caspase-independent apoptosis in


Page 23 of 36


485

reactive oxygen species generation. Int J Biochem Cell B. 2009;41:666-676.

486

(35).

487

Prevents Glutamate-Induced Cell Death by Blocking Mitochondrial Fragmentation

488

and Permeability Transition Pore Opening. Int J Biol Sci. 2016;12:688-700.

489

(36).

490

rubidum sp. A09DM.

491

(37).

492

Albumin-coated SPIONs: an experimental and theoretical evaluation of protein

493

conformation, binding affinity and competition with serum proteins. Nanoscale.

494

2016:14393-14406.

495

(38).

496

fromSynechocystis PCC 6803 reveals the structural basis for the extreme redshift of the

497

terminal emitter in phycobilisomes. Acta Crystallographica Section D Biological

498

Crystallography. 2014;70:2558-2569.

499

(39).

500

targeting based on the effect of enhanced permeability and retention (EPR)

501

mechanism of receptor-mediated endocytosis (RME). Int J Pharm. 2004;277:39-61.

502

(40).

503

zeta-potential

504

2010;8:279-285.

505

(41).

506

Delivery Systems - A Review (Part 2). Trop J Pharm Res. 2013;12:265-273.

Kumari S, Mehta SL, Milledge GZ, Huang X, Li H, Li PA. Ubisol-Q10

Crystal structure of Phycocyanin from marine cyanobacterium Phormidium

Yu S, Perálvarez-Marín A, Minelli C, Faraudo J, Roig A, Laromaine A.

Peng P, Dong L, Sun Y, et al. The structure of allophycocyanin B

Tanaka T, Shiramoto S, Miyashita M, Fujishima Y, Kaneo Y. Tumor and the

Tantra R, Schulze P, Quincey P. Effect of nanoparticle concentration on measurement

results

and

reproducibility.

Particuology.

Honary S, Zahir F. Effect of Zeta Potential on the Properties of Nano-Drug



507

(42).

Haber EP, Procópio J, Carvalho CRO, Carpinelli AR, Newsholme P, Curi R.

508

New Insights into Fatty Acid Modulation of Pancreatic β‐Cell Function. Int. Rev.

509

Cytology. Vol. Volume 248: Academic Press; 2006:1-41.

510

(43).

511

chemoprevention and chemotherapy by selenium compounds. Curr Cancer Drug Tar.

512

2004;4:13-28.

513

(44).

514

against human islet amyloid polypeptide-induced apoptosis through attenuating

515

oxidative stress and modulating JNK and p38 mitogen-activated protein kinase

516

pathways. Int. J. Biochem. Cell Biol. 2009;41:1526-1535.

517

(45).

518

an in situ

519

cells in healing full-thickness cutaneous wounds. J Control Release. 2016.

520

(46).

521

critical regulators of signaling pathways and targets for anti-cancer therapy. Exp.

522

Oncology. 2012.

523

(47).

524

Disease. Csh Perspect Biol. 2013;5:a8656.

525

(48).

526

in chemical carcinogenesis. Toxicol Appl Pharm. 2011;254:86-99.

527

(49).

528

quality control. Redox Biology. 2015;4:6-13.

Sinha R, Ei-Bayoumy K. Apoptosis is a critical cellular event in cancer

Li X, Xu G, Chen T, et al. Phycocyanin protects INS-1E pancreatic beta cells

Choi SK, Park JK, Kim JH, et al. Integrin-binding elastin-like polypeptide as gelling delivery matrix enhances the therapeutic efficacy of adipose stem

de Almagro MC, Vucic D. The inhibitior of apoptosis (IAP) proteins are

McIlwain DR, Berger T, Mak TW. Caspase Functions in Cell Death and

Klaunig JE, Wang Z, Pu X, Zhou S. Oxidative stress and oxidative damage

Ni H, Williams JA, Ding W. Mitochondrial dynamics and mitochondrial


Page 24 of 36

Page 25 of 36


529

(50).

Knott AB, Perkins G, Schwarzenbacher R, Bossy-Wetzel E. Mitochondrial

530

fragmentation in neurodegeneration. Nat Rev Neurosci. 2008;9:505-518.

531

(51).

532

chemo-/radiotherapy through ROS-mediated signaling pathways. Biomaterials.

533

2015;51:30-42.

He L, Lai H, Chen T. Dual-function nanosystem for synergetic cancer

534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550



551

Scheme 1

552 553

Scheme 1. Rational design of PC-SeNPs to protect INS-1E cells from oxidative

554

damage and the underlining mechanisms. (A) The core-shell structure of

555

PC-SeNPs. (B) Proposed structure of PC-SeNPs. (C) and (D) The action mechanism

556

of PC-SeNPs-caused protective effect on PA-induced INS-1E cells.

557 558 559 560 561 562 563


Page 26 of 36

Page 27 of 36


564

Figure 1

565 566

Figure 1. The morphology and particle size of PC-SeNPs. (A) TEM images of

567

PC-SeNPs with different PC concentration, and the concentrations from a to f were 0,

568

5, 25, 50, 100, 150 mg/L, respectively. (B) The particle size distribution of PC-SeNPs

569

with different PC concentration. (C) Zeta potential of PC-SeNPs with different PC

570

concentration. (D) Effects of added PC concentration in the synthesis reaction on the

571

conjugated surface PC contents in PC-SeNPs.

572 573 574 575



576

Figure 2

577 578

Figure 2. The spectroscopy and stability of PC-SeNPs. (A) The FT-IR spectrum of

579

PC, SeNPs and PC-SeNPs. (B) The UV-vis spectrum of PC, SeNPs, and PC-SeNPs

580

with different size. (C) The stability of PC-SeNPs in PBS. (D) The stability of

581

PC-SeNPs in DMEM.

582 583 584 585 586 587 588


Page 28 of 36

Page 29 of 36


589

Figure 3

590 591

Figure 3. The antioxidant activities of PC-SeNPs determined by ABTS·+ assay. (A)

592

The comparison antioxidant activities of SeNPs and PC-SeNPs with different particle

593

size. (B) The antioxidant activities of different concentration PC-SeNPs (235 nm).

594 595 596



597

Figure 4

598 599

Figure 4. Cellular uptake of nanoparticles by INS-1E cells. INS-1E cells were

600

treated with 0.8 µM of PC-SeNPs, and cells were collected and the content of Se were

601

measured at determined time point with ICP-MS.

602 603 604 605 606 607 608 609 610


Page 30 of 36

Page 31 of 36


611

Figure 5

612 613

Figure 5. Cell viability assay of INS-1E cells under different condition by MTT

614

assay. (A) Cell viability of INS-1E cells treated with different concentration of

615

PC-SeNPs-235 nm. (B) Cell viability of INS-1E cells treated with different

616

concentration of PA. (C)

617

concentration of PC or SeNPs for 2 h before treated PA, respectively. (D) Cell

618

viability of cells pre-incubated with different PC-SeNPs for 12 h before treated with

619

PA for 2 h. n=3, p < 0.05.

Cell viability of the cells pre-incubated with different

620 621 622



623

Figure 6

624 625

Figure 6.Different effects of PC-SeNPs on the cell cycle induced by PA as

626

examined by propidium iodide (PI)-flow cytometric analysis. (A) Cells (2 × 104

627

cells/mL) were treated with PC, SeNPs, different form or concentreation of PC-SeNPs

628

and PA for indicated times. (B) and (C) Quantitative cell cycle distribution data for

629

INS-1E in every group. n=3.

630 631 632 633 634


Page 32 of 36

Page 33 of 36


635

Figure 7

636 637

Figure 7. PC-SeNPs Inhibits PA-induced Caspase Activation. (A) INS-1E cells

638

pre-incubated with PC, SeNPs and different PC-SeNPs (0.8 µM) for 12 h before

639

treated with 0.4 mM of PA for 2 h and the influence was determined by synthetic

640

fluorogenic substrate (n =3, P < 0.05). (B) Inhibition of PARP and caspase cleaved in

641

the apoptosis pathway and equal protein loading was confirmed by Western analysis

642

of β-actin in the protein extracts.

643 644 645 646 647 648 649 650 651 652



653

Figure 8

654 655

Figure 8. ROS generation of PA-induced INS-1E cells prevented by PC-SeNPs.

656

(A) Changes in intracellular ROS generation in INS-1E cells pre-incubated with 0.8

657

µM of PC-SeNPs for 12h and then exposed to 0.4 mM of PA for 2 h. Then cells were

658

stain with DHE-DA for 30min. (B) Representative DHE fluorescence images of

659

INS-1E cells exposed to PA after treated with different PC-SeNPs. n = 3.

660 661


Page 34 of 36

Page 35 of 36


662

Figure 9

663 664

Figure 9. PC-SeNPs Inhibits PA-induced Mitochondrial Fragmentation. The

665

representative images of mitochondrial fragmentation in INS-1E cells after treatment

666

with 0.8 µM of PC-SeNPs for 12 h and 0.4 mM PA for 2 h, after that cells were

667

stained by Red Mito-Tracker for 2 h and stained by DAPI for 20 min. Mitochondria

668

fragmentation was measured by fluorescence microscope.

669 670 671 672 673 674 675 676



677

TOC graphic

678 679

TOC: Rational design of PC-SeNPs to protect INS-1E cells from oxidative

680

damage and the underlining mechanisms. (A) The core-shell structure of

681

PC-SeNPs. (B) Proposed structure of PC-SeNPs. (C) and (D) The action mechanism

682

of PC-SeNPs-caused protective effect on PA-induced INS-1E cells.

683


Page 36 of 36

Phycocyanin-Functionalized Selenium Nanoparticles Reverse

Recommend Documents