Food and Chemical Toxicology 59 (2013) 643–649

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Food and Chemical Toxicology

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Octaphlorethol A, a novel phenolic compound isolated from Ishigefoliacea, protects against streptozotocin-induced pancreatic b celldamage by reducing oxidative stress and apoptosis

0278-6915/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.fct.2013.07.011

⇑ Corresponding author at: Department of Marine Life Science, Jeju NationalUniversity, Jeju 690-756, Republic of Korea. Tel.: +82 64 754 3475; fax: +82 64 7563493.

E-mail address: youjinj@jejunu.ac.kr (Y.-J. Jeon).

Seung-Hong Lee a, Sung-Myung Kang b, Seok-Chun Ko b, Min-Cheol Kang b, You-Jin Jeon b,c,⇑a Division of Food Bioscience, Konkuk University, Chungju Chungbuk 380-701, Republic of Koreab Department of Marine Life Science, Jeju National University, Jeju 690-756, Republic of Koreac Marine and Environmental Research Institute, Jeju National University, Jeju 695-814, Republic of Korea

a r t i c l e i n f o

Article history:Received 17 April 2013Accepted 7 July 2013Available online 16 July 2013

Keywords:Octaphlorethol AProtective effectPancreatic b cellsOxidative stressApoptosis

a b s t r a c t

Pancreatic b cells are extremely sensitive to oxidative stress, which probably has an important role in bcell damage in diabetes. The protective effect of octaphlorethol A (OPA), a novel phenolic compound iso-lated from Ishige foliacea, against streptozotocin (STZ)-induced pancreatic b cell damage was investigatedusing a rat insulinoma cell line (RINm5F pancreatic b cells). Pretreatment with OPA decreased the deathof STZ-treated pancreatic b cells at concentrations of 12.5 lg/ml or 50 lg/ml, and reduced the generationof thiobarbituric acid reactive substances and intracellular reactive oxygen species in a dose-dependentmanner in STZ-treated pancreatic b cells. In addition, the OPA pretreatment increased the activities ofantioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase in STZ-treatedpancreatic b cells. Moreover, OPA treatment elevated the level of insulin, which was reduced by STZ treat-ment, and protected pancreatic b cells against damage under STZ-treated conditions. These effects weremediated by suppressing apoptosis and were associated with increased anti-apoptotic Bcl-xL expressionand reduced pro-apoptotic Bax and cleaved caspase-3 expression. These findings indicate that OPA maybe useful as a potential pharmaceutical agent to protect against pancreatic b cell damage caused by oxi-dative stress associated with diabetes.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Pancreatic b cell dysfunction has a crucial role in the pathogen-esis of type 2 diabetes. Although the exact mechanism underlying bcell destruction remains unknown, oxidative stress has been sug-gested as one of the major factors contributing to the destructionof pancreatic b cells (Rashid et al., 2010). Pancreatic b cells are par-ticularly sensitive to damage by reactive oxygen species (ROS), be-cause they contain extremely low levels of antioxidant enzymes(Lenzen et al., 1996). Streptozotocin (STZ) is a potent DNA-methy-lating agent, which generates ROS that induce oxidative stress inpancreatic b cells (Szkudelski, 2001). Several studies have demon-strated that STZ increases ROS production, lipid peroxidation, andpancreatic b cell damage, and that radical scavengers reduce theseverity of STZ-induced oxidative damage (Szkudelski, 2001; Chenet al., 2001; Eum et al., 2004; Coskun et al., 2005). The results ofseveral studies suggest that supplementation with non-toxic ROS

scavengers and antioxidants can protect against the pathologicaldamage induced by the oxidative stress associated with diabetes(Kang et al., 2008; Lee et al., 2012a). Thus, it is important to searchfor methods to attenuate the oxidative stress in order to reduce therisk of pathological damage related to diabetes.

There is much interest in the identification of antioxidant com-pounds that do not cause side effects or exhibit toxicity. Phlorotan-nins, which constitute one of the most diverse and widespreadgroups of natural compounds, are probably the most abundant nat-ural phenolics found in marine algae. These compounds exhibit abroad spectrum of chemical and biological activities, includingantioxidant properties (Ahn et al., 2007; Heo and Jeon, 2009). Ishigefoliacea is found throughout the temperate coastal zone of the Kor-ean peninsula, generally forming highly persistent populations inclear waters (Lee and Kang, 1986). This species is abundant alongthe coast of Korea’s Jeju Island and is considered to be an ediblebrown alga. In our previous study, we isolated a new active com-pound, octaphlorethol A (OPA) (a type of phlorotannin), from thebrown alga I. foliacea and demonstrated the effect of its glucose up-take in skeletal muscle cells (Lee et al., 2012b). However, only fewbiological studies have studied this compound. Therefore, the pres-ent study first examined whether OPA, a novel phenolic compound

644 S.-H. Lee et al. / Food and Chemical Toxicology 59 (2013) 643–649

isolated from I. foliacea, could protect against STZ-induced damagein a rat insulinoma cell line (RINm5F pancreatic b cells) and theninvestigated the mechanism underlying this protection.

2. Materials and methods

2.1. Materials

The brown alga I. foliacea (Phylum: Phaeophyta, Class: Phaeophyceae, Order:Ishigeales, Family: Ishigeaceae, Lee and Kang, 1986) was collected from the coastof Jeju Island, Korea. The sample was washed 3 times with tap water to removethe salt, sand, and epiphytes attached to its surface, followed by careful rinsing withfresh water and maintenance in a medical refrigerator at �20 �C. Next, the frozensample was lyophilized and homogenized with a grinder prior to extraction. Allchemicals and reagents used were of analytical grade and were obtained from com-mercial sources.

2.2. Extraction and isolation

Dried I. foliacea powder was extracted 3 times with 80% methanol and filtered.The filtrate was evaporated at 40 �C to obtain the methanol extract, which was sus-pended in distilled water and partitioned with ethyl acetate. The ethyl acetate frac-tion was subjected to silica gel and Sephadex-LH 20 column chromatography. Theactive compound was finally purified by high-performance liquid chromatography(HPLC), and the structure of the active compound (Fig. 1) was determined by thenuclear magnetic resonance (NMR) spectral data (Lee et al., 2012b).

2.3. Cell culture

RINm5F pancreatic b cells (insulin-secreting rat insulinoma cell line) were cul-tured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with10% fetal bovine serum (FBS), streptomycin (100 lg/ml), and penicillin (100 units/ml) at 37 �C in a 5% CO2 humidified atmosphere.

2.4. Assay of cell viability

Cell viability was assessed by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which is based on the conversion ofMTT to MTT-formazan by mitochondrial enzymes (Fautz et al., 1991). Cells(1 � 105 cells/well) were added to 96-well culture plates and incubated with differ-ent concentrations of OPA for 3 h, followed by incubation with 10 mM STZ for 24 h.Next, 100 ll of the MTT solution (1 mg/ml) was added to each of the wells and incu-bated for 4 h at 37 �C, after which the medium containing MTT was removed. Theincorporated formazan crystals in the viable cells were dissolved in 100 ll dimethylsulfoxide, and the absorbance of each well was measured at 540 nm using a micro-plate reader.

2.5. Assay of lipid peroxidation

Lipid peroxidation was measured by thiobarbituric acid reactive substances(TBARS) production (Fraga et al., 1988). Cells (1 � 105 cells/well) were added to24-well culture plates and incubated with different concentrations of OPA for 3 h,followed by incubation with 10 mM STZ for 24 h. The cells were then washed withcold phosphate-buffered saline (PBS) and homogenized. The TBARS concentrationswere determined using a TBARS assay kit (ZeptoMetrix, Buffalo, NY, USA). TBARSvalues were expressed in equivalent nanomoles of malondialdehyde (MDA).

2.6. Assay of intracellular ROS levels

The 20 ,70-dichlorfluorescein (DCF)-diacetate (DA) method was used to detect theintracellular ROS levels (Rosenkranz et al., 1992). DCF-DA diffuses into cells, whereit is hydrolyzed by intracellular esterase to polar 20 ,70-dichloro-dihydrofluorescein.This non-fluorescent fluorescein analog is trapped inside the cells and oxidized byintracellular oxidants to yield the highly fluorescent 20 ,70-dichloro-fluorescein. Thecells were treated with OPA at 12.5, 25, and 50 lg/ml for 3 h, followed by the addi-tion of STZ at 10 mM for an additional 24 h at 37 �C. Next, 10 lM of the DCF-DA

Fig. 1. Chemical structure of octaphloretho

solution was added to each well, and the fluorescence of 20 ,70-dichlorofluoresceinwas estimated using the FACSCalibur flow cytometer (Becton Dickinson, San Jose,CA, USA).

2.7. Antioxidant enzyme assays

Cells (1 � 106) were added to 10-mm dishes and incubated with different con-centrations of OPA for 3 h, followed by incubation with 10 mM STZ for 24 h. Themedium was removed and the cells were washed twice with PBS. Next, 1 ml of50 mM potassium phosphate buffer with 1 mM EDTA (pH 7.0) was added, andthe cells were scraped. The scraped cells were then homogenized using the Nuc-Buster™ Protein Extraction Kit (Novagen, San Diego, CA, USA) for 10 min and cen-trifuged at 10,000g for 5 min at 4 �C. The cell supernatant was used to estimatethe antioxidant enzyme activities. The protein concentrations were determinedusing the BCA™ Protein Assay Kit. Superoxide dismutase (SOD), catalase (CAT),and glutathione peroxidase (GSH-px) activities were determined using chemicalkits. Briefly, the determination of SOD activity (SOD Assay Kit-WST, DojindoMolecular Technologies, Inc., Rockville, MD, USA) was based on the production ofO2� anions by the xanthine/xanthine oxidase system. The determination of CATactivity (Catalase Assay Kit, Cayman Chemical, Ann Arbor, MI, USA) was basedon the reaction of the enzyme with methanol in the presence of an optimalconcentration of hydrogen peroxide. GSH-px activity (Glutathione Peroxidase AssayKit, Sigma, St. Louis, MO, USA) was estimated by the analysis of GSH in theenzymatic reaction.

2.8. Assay of insulin levels

Cells (1 � 106) were added to 10-mm dishes and incubated with different con-centrations of OPA for 3 h, followed by incubation with 10 mM STZ for 24 h. Theamount of insulin was determined using the Rat/Mouse Insulin ELISA Kit (Millipore,Billerica, MA, USA).

2.9. Cell cycle analysis

Cell cycle analysis was performed to determine the proportion of apoptotic sub-G1 hypodiploid cells (Nicoletti et al., 1991). Cells (1 � 105 cells/ml) were placed in a6-well plate; 16 h after seeding, the cells were pretreated with different concentra-tions of OPA for 3 h and then incubated with STZ for 24 h. Next, the cells were har-vested at pre-defined times and fixed in 1 ml of 70% ethanol for 30 min at 4 �C. Thecells were washed twice with PBS and incubated in the dark in 1 ml of PBS contain-ing 100 lg propidium iodide (PI) and 100 lg RNase A for 30 min at 37 �C. Flow cyto-metric analysis was performed using the FACSCalibur flow cytometer. The effect oncell cycle was determined by changes in the percentage of cell distribution at eachphase of the cell cycle and assessed by histograms generated by the computer pro-gram Cell Quest and Mod-Fit (Wang et al., 1993).

2.10. Western blot analysis

Cells (1 � 106 cells/ml) were added to 10-mm dishes and pretreated with dif-ferent concentrations of OPA for 3 h, followed by incubation with 10 mM STZ for24 h. The cells were lysed in the NucBuster™ Protein Extraction Kit (Novagen, SanDiego, CA, USA) for 10 min and then centrifuged at 10,000g for 5 min at 4 �C. Theprotein concentrations were determined using the BCA™ Protein Assay Kit. Thelysates containing 30 lg of protein were subjected to electrophoresis on 10% so-dium dodecyl sulfate–polyacrylamide gels, and the gels were transferred ontonitrocellulose membrane. The membranes were incubated with antibody againstBax, Bcl-xL, cleaved caspase-3, and b-actin in TTBS (25 mM Tris–HCl, 137 mMNaCl, 0.1% Tween 20, pH 7.4) containing 5% skimmed milk for 2 h. The mem-branes were then washed with TTBS and incubated with secondary antibodies.Signals were developed using the ECL Western Blotting Detection Kit and exposedto X-ray films.

2.11. Data and statistical analysis

The data were represented as mean ± standard error (SE). The statistical analy-sis was performed using SAS software. The values were evaluated by one-way anal-ysis of variance (ANOVA), followed by post hoc Duncan’s multiple range tests.

l A (OPA) isolated from Ishige foliacea.

S.-H. Lee et al. / Food and Chemical Toxicology 59 (2013) 643–649 645

3. Results

3.1. Cell viability

The effects of OPA on the viability of RINm5F pancreatic b cellstreated with STZ, as determined by the MTT assay, are depicted inFig. 2. The cell viability was significantly reduced to 50.09% bytreating the RINm5F pancreatic b cells with STZ for 24 h only. How-ever, OPA protected against the cellular damage induced by STZ ina dose-dependent manner. In particular, treatment with 50 lg/mlof OPA together with the STZ treatment resulted in a significant in-crease in cell viability to 82.65%.

3.2. Lipid peroxidation

The effects of OPA on lipid peroxidation in the STZ-treatedRINm5F pancreatic b cells, determined by measuring TBARS, a lipidperoxidation product, are depicted in Fig. 3. Analysis of RINm5Fpancreatic b cells incubated for 24 h with STZ revealed that TBARSwas significantly higher relative to the control cells. Treatmentwith 50 lg/ml of OPA together with STZ significantly inhibitedTBARS formation, indicating protection against lipid peroxidation.Analysis of the cells treated with 50 lg/ml of OPA revealed thatTBARS was significantly reduced, by 1.24 nmol MDA.

Cel

l via

bilit

y (%

)

Control 0 12.5 25 50

OPA (μμg/ml) + STZ

ab

cd

e

Fig. 2. Effect of OPA on viability of streptozotocin (STZ)-treated RINm5F pancreaticb cells. Cells were pre-incubated with different concentrations of OPA for 3 h,followed by incubation with 10 mM STZ for 24 h. Each value is expressed asmean ± standard error (S.E.) from triplicate experiments. a–eValues not sharing acommon letter are significantly different among the groups (P < 0.05).

Control 0 12.5

OPA ( μμg/ml) + STZ

TB

AR

S (n

mol

MD

A)

d

a

c

bab

25 50

Fig. 3. Effect of OPA on the generation of thiobarbituric acid reactive substances(TBARS) in STZ-treated RINm5F pancreatic b cells. Cells were pre-incubated withdifferent concentrations of OPA for 3 h and then incubated with 10 mM STZ for24 h. Each value is expressed as mean ± S.E. from triplicate experiments. a–dValuesnot sharing a common letter are significantly different among the groups (P < 0.05).

3.3. Intracellular ROS generation

As demonstrated in Fig. 4, the effects of OPA on intracellularROS generation in RINm5F pancreatic b cells treated with STZ weredetermined using flow cytometry. The fluorescence intensity val-ues represent the level of ROS detected by the DCF-DA fluorescencedye. The fluorescence intensity value in RINm5F pancreatic b cellsincreased significantly after treatment with STZ compared to thatin the control cells, which did not contain either the sample orSTZ. When RINm5F pancreatic b cells were exposed to STZ, thefluorescence intensity values significantly increased to 216.95.However, OPA pretreatment reduced the fluorescence intensityvalues in a dose-dependent manner in the cells induced by STZtreatment. In particular, pretreatment with 50 lg/ml of OPA re-sulted in a significant reduction in fluorescence intensity values,to 166.43. These results indicate that OPA significantly reducedthe elevated ROS levels induced by STZ, and suggest that OPA pos-sesses intracellular ROS scavenging activity.

3.4. Antioxidant enzyme activities

Cells are protected from activated oxygen species by endoge-nous antioxidant enzymes including SOD, CAT, and GSH-px. The ef-fects of OPA on antioxidant enzyme activities in STZ-treatedRINm5F pancreatic b cells are shown in Table 1. Only treatmentwith STZ for 24 h significantly attenuated the SOD activity of theRINm5F pancreatic b cells. Treating the cells with OPA increasedthe SOD activity in the STZ-treated cells; the SOD activity was sig-nificantly increased to 86.09% after the cells were treated with50 lg/ml of OPA. The STZ treatment of RINm5F pancreatic b cellsreduced CAT activity compared to the control cells; however,OPA treatment increased the CAT activity in a dose-dependentmanner in the STZ-treated cells. The CAT activity was increasedsignificantly to 1.06 lmol/mg protein after the cells were treatedwith 50 lg/ml of OPA. Similarly, the GSH-px activity in RINm5Fpancreatic b cells treated with STZ was significantly reduced com-pared to the control cells. Treating the STZ-treated cells with OPAincreased the GSH-px activity, as shown by a GSH-px activity of3.41 lmol/mg protein at a dosage of 50 lg/ml.

3.5. Insulin level

The effects of OPA on insulin level in STZ-treated RINm5F pan-creatic b cells are shown in Table 1. Only treatment with STZ for24 h significantly decreased the insulin level of the cells. However,OPA treatment increased insulin levels in a dose-dependent man-ner in the STZ-treated cells. Analysis of the cells treated with50 lg/ml of OPA revealed that the insulin level was significantlyincreased, by 3.85 ng/ml.

3.6. Protective effects against apoptosis

The results of flow cytometry analysis with PI staining of STZ-treated RINm5F pancreatic b cells are depicted in Fig. 5; the apop-totic sub-G1 DNA content was higher than that in the control cells.However, OPA pretreatment reduced the apoptotic sub-G1 DNAcontent in the STZ-treated cells in a dose-dependent manner. Inparticular, pretreatment with 50 lg/ml of OPA resulted in a signif-icant reduction in apoptotic sub-G1 DNA content. These data indi-cate that OPA may have notable apoptosis inhibition activityagainst STZ-treated RINm5F pancreatic b cells.

3.7. Expression of Bax, Bcl-xL, and cleaved caspase-3

To determine whether OPA could induce the expression of pro-teins related to STZ-induced apoptosis, RINm5F pancreatic b cells

Control

FI : 157.09

STZ

FI : 216.95

OPA12.5µg/ml + STZ

FI : 186.44 FI : 173.89

FI : 166.43

OPA25µg/ml + STZ

OPA50 µg/ml + STZ

0

50

100

150

200

250

Flu

ores

cenc

e in

tens

ity

a

d

OPA ( μg/ml) + STZCon

0 12.5 25 50

cb a

Fig. 4. Effect of OPA on intracellular reactive oxygen species (ROS) generation in STZ-treated RINm5F pancreatic b cells. Cells were pre-incubated with differentconcentrations of OPA for 3 h and then incubated with 10 mM STZ for 24 h. The intracellular ROS generated was detected by flow cytometry after 20 ,70-dichlorfluorescein(DCF)-diacetate (DA) treatment. Each value is expressed as mean ± S.E. from triplicate experiments. a–dValues not sharing a common letter are significantly different amongthe groups (P < 0.05).

Table 1The effects of OPA on antioxidant enzyme activities and insulin level in STZ-treated RINm5F pancreatic b cells.

Con OPA (lg/ml) + STZ

0 12.5 25 50

SOD activity (%) 90.72 ± 7.79d 55.36 ± 3.32a 74.93 ± 8.30b 80.51 ± 7.36c 86.09 ± 7.50d

CAT (lmole/mg of protein) 1.08 ± 0.05d 0.39 ± 0.02a 0.79 ± 0.03b 0.90 ± 0.03c 1.06 ± 0.04d

GSH-px (lmole/mg of protein) 3.81 ± 0.08c 1.33 ± 0.03a 2.57 ± 0.06b 2.73 ± 0.07bc 3.41 ± 0.09c

Insulin level (ng/ml) 4.11 ± 0.23e 1.36 ± 0.17a 2.23 ± 0.32b 3.18 ± 0.16c 3.85 ± 0.13d

Cells (1 � 106 cells) were added to 10-mm dishes and incubated with different concentrations of OPA for 3 h, followed by incubated with 10 mM STZ for 24 h.SOD: superoxide dismutase; CAT: catalase; GSH-px: glutathione peroxidase. Each value is expressed as the mean ± S.E. from triplicate experiments. a–eValues not sharing acommon letter are significantly different between the groups (P < 0.05).

646 S.-H. Lee et al. / Food and Chemical Toxicology 59 (2013) 643–649

were pretreated with 25 lg/ml and 50 lg/ml doses of OPA, and thelevels of Bax, Bcl-xL, and cleaved caspase-3 were determined bywestern blotting. The level of Bax pro-apoptotic protein expressionwas clearly higher in the STZ-treated cells than in the control cells(Fig. 6). However, the expression level after treatment with OPA

was markedly reduced. In addition, the expression of the anti-apoptotic protein Bcl-xL tended to be decreased in the STZ-treatedcells, whereas the cells pretreated with OPA showed higher Bcl-xLexpression than the cells treated with STZ only. The expression ofcleaved caspase-3 in RINm5F pancreatic b cells treated with STZ

Control STZ

OPA12.5µg/ml + STZ OPA25 µg/ml + STZ

OPA50 µg/ml + STZ

0

5

10

15

20

25

30

35

40

Sub-

G1

(%)

a

e

d

c

b

OPA (µg/ml) + STZCon

0 12.5 25 50

Fig. 5. Effect of OPA on STZ-induced apoptosis in RINm5F pancreatic b cells. The cells were pretreated with different concentrations of OPA for 3 h and then incubated with10 mM STZ for 24 h. The cells were stained with propidium iodide (PI) and analyzed by flow cytometry. Each value is expressed as mean ± S.E. from triplicate experiments. a–

eValues not sharing a common letter are significantly different among the groups (P < 0.05).

S.-H. Lee et al. / Food and Chemical Toxicology 59 (2013) 643–649 647

was much higher than that in the control cells. However, treatingthe RINm5F pancreatic b cells with OPA reduced the expressionof cleaved caspase-3 in the STZ-treated cells.

4. Discussion

Oxidative stress has been demonstrated to contribute to diabe-tes-associated pathological damages (Baynes and Thorpe, 1999).Acute STZ injection has been used to study cellular or tissue oxida-tive damage, because it produces ROS and reduces antioxidant en-zyme activity, especially in pancreatic tissues (Coskun et al., 2005).Several studies have demonstrated that exposure of b cells to STZresults in b cell dysfunction and apoptosis (Coskun et al., 2005;Kang et al., 2008). Pancreatic b cell dysfunction plays a key rolein the pathogenesis of type 2 diabetes. Thus, to reduce the risk ofpathological damage in diseases such as diabetes, it is importantto search for ways to protect b cells against damage induced byoxidative stress. There is much interest in the identification of anti-oxidative compounds that do not cause side effects or exhibit tox-icity. The phlorotannins, which constitute one of the most diverse

and widespread groups of natural compounds, are probably themost abundant natural phenolics found in marine algae. Thesecompounds exhibit a broad spectrum of chemical and biologicalactivities, including antioxidant properties (Kang et al., 2005;Ahn et al., 2007; Heo and Jeon, 2009). The present study demon-strates the protective effect of OPA, a type of phlorotannin, whichis a marine algal polyphenolic compound isolated from I. foliaceaagainst STZ-induced pancreatic b cell damage.

MTT assays were conducted to measure cell viability. The expo-sure of RINm5F pancreatic b cells to STZ resulted in significantreductions in cell viability. However, OPA pretreatment was shownto inhibit cell death, thereby suggesting that OPA protects RINm5Fpancreatic b cells against STZ-induced cytotoxicity.

Lipid peroxidation may be a form of cell damage mediated byfree radicals (Sevanian and Hochstein, 1985). STZ treatment hasbeen recently shown to induce lipid peroxidation in RINm5F pan-creatic b cells, and OPA was shown to effectively inhibit TBARS for-mation. One of the more serious consequences of lipidperoxidation is damage to biomembranes such as mitochondrialand plasma membranes. During lipid peroxidation, low molecu-lar-weight end products, especially MDA, are formed via oxidation

Bax

Bcl-xL

Cleaved caspase-3

β-actin

A

ConOPA (μμg/ml) + STZ

OPA (μg/ml) + STZ

0 25 50

0

1

2

3

4

5

6

7

Arb

itra

ry d

ensi

tom

etri

cuni

ts

Bax Bcl-xL Cleaved caspase-3B

Con0 25 50

a a a

d

d

d

c

c

c

b

b

b

Fig. 6. Effects of OPA on the expression of apoptosis-related proteins in STZ-treatedRINm5F pancreatic b cells. The cells were pretreated with the indicated concen-trations of OPA for 3 h and then incubated with 10 mM STZ for 24 h. Equal amountsof cell lysates (30 lg) were subjected to electrophoresis and analyzed for Bax, Bcl-xL, and cleaved caspase-3 expression by western blotting. Actin was used as aninternal control. (A) Bax, Bcl-xL, and cleaved caspase-3 protein expression; and (B)Quantification of Bax, Bcl-xL, and cleaved caspase-3 expression. Values areexpressed as mean ± S.E. from triplicate experiments. a–dValues not sharing acommon letter are significantly different among the groups (P < 0.05).

648 S.-H. Lee et al. / Food and Chemical Toxicology 59 (2013) 643–649

of polyunsaturated fatty acids; these end products can react withtwo molecules of thiobarbituric acid to generate a pinkish-redchromogen. The protective action of OPA on TBARS formation inthis study can be attributed to its antiperoxidative effects.

High ROS levels induce oxidative stress, which can result in avariety of biochemical and physiological lesions. Such cellulardamage frequently impairs metabolic function and results in celldeath (Finkel and Holbrook, 2000). Our results demonstrated thatthe treatment of RINm5F pancreatic b cells with STZ significantlyincreased intracellular ROS levels. However, OPA inhibited STZ-in-duced ROS generation, indicating that OPA alleviates oxidativestress via the inhibition of ROS generation induced by STZ treat-ment. Polyphenolic compounds from marine algae have beenshown to have strong antioxidative activities against free radicals(Kang et al., 2003; Ahn et al., 2007; Heo and Jeon, 2009). In addi-tion, previous studies have shown that polyphenols are electron-rich compounds that are inclined to enter into efficient electron-donation reactions and generate phenoxyl radicals (PhO�) as inter-mediates in the presence of oxidizing agents. PhO�s are stabilizedby resonance delocalization of the unpaired electron to the orthoand para positions of the ring. In addition to resonance stability,PhO� can also be stabilized via hydrogen bonding with an adjacenthydroxyl group. PhO� also undergoes dimerization (‘‘phenol cou-pling’’) to produce new C–C or C–O linkages (Larson, 1997). Thisintrinsic stability of polyphenol structures and their potency aselectron donors may be associated with the antioxidative activityof OPA. ROS may also have a major role as endogenous initiatorsand promoters of DNA damage and mutations, contributing tothe development of cancer, diabetes, and other age-related dis-eases. Therefore, we expect that OPA can protect against oxidativeDNA damage and reduce the risk of diabetic complications.

Cells are protected from activated oxygen species by endoge-nous antioxidant enzymes such as CAT, SOD, and GSH-px. We ob-served that the treatment of STZ-treated RINm5F pancreatic b cellswith OPA resulted in increases in CAT, SOD, and GSH-px activities.Thus, OPA may increase the expression level of antioxidant en-zymes in pancreatic b cells. SOD, the endogenous scavenger, cata-lyzes the dismutation of the highly reactive superoxide anion toH2O2 (Husain and Somani, 1998). GSH-px catalyzes the reductionof H2O2 at the expense of reduced GSH. H2O2 is also scavengedby CAT (Runnegar et al., 1987). The reduced activities of bothCAT and GSH-px in the RINm5F pancreatic b cells treated withSTZ has been demonstrated to result in a substantially reducedcapacity to scavenge H2O2 produced in the cells, leading to in-creases in ROS and oxidative stress in response to STZ treatment(Alptekin et al., 1996). High superoxide anion radical productioninhibits CAT activity (Kono and Fridovich, 1982). Thus, the excessof superoxide anion radical, as a consequence of a reduction inSOD activity, may be responsible for the reduction in the activitiesof CAT in STZ-treated RINm5F pancreatic b cells. These effects weremediated by reducing the oxidative stress, which is associated withreduced ROS generation and increased cell survival.

Pancreatic b cells perform an important function in maintainingglucose homeostasis via the secretion of insulin (Hou et al., 2008).Oxidative stress has been reported to exert a variety of harmful ef-fects, including the inhibition of insulin secretion, the impairmentof insulin gene expression, and the induction of cell death in b cells(Kaneto et al., 2005). STZ treatment has recently been shown to re-duce insulin levels in RINm5F pancreatic b cells, and OPA has beenshown to effectively elevate insulin levels. These results demon-strate that OPA increases insulin secretion in pancreatic b cells,and that this mechanism may be involved in the protection of insu-lin-secreting cells by reducing oxidative stress and increasing cellsurvival.

ROS are by-products of normal cellular oxidative stress pro-cesses and are generated in the mitochondria and other sources.ROS inflict serious damage on nucleic acids and on protein andmembrane lipids, and they have been suggested to regulate pro-cesses involved in the initiation of apoptotic signaling. Several re-cent studies have demonstrated that ROS generation performs acrucial function in pro-apoptotic activities. Members of the Bcl-2family (e.g., Bcl-xL) of proteins are critical regulators of the apopto-tic pathway (Zanke et al., 1996). Bcl-2 and Bcl-xL are upstreammolecules in the apoptotic pathway and have been identified aspotent suppressors of apoptosis (Szatrowski and Nathan, 1991).Previous reports have demonstrated that Bcl-2 family-mediatedcaspase-3 activation was responsible for ROS-induced apoptosis(Chen and Chang, 2009). Caspase-3 is one of the key executionersof apoptosis, because it is involved in the cleavage of key proteinssuch as the nuclear enzyme poly (ADP-ribose) polymerase (PARP)(Fernandes-Alnemri et al., 1995). In this study, we demonstratedthat OPA protects RINm5F pancreatic b cells from damage underSTZ treatment. This effect was mediated by suppressing apoptosisand was associated with increased anti-apoptotic Bcl-xL expres-sion and reduced pro-apoptotic Bax and cleaved caspase-3 expres-sion. In addition, cell cycle analysis was performed to determinethe proportion of apoptotic sub-G1 hypodiploid cells. The exposureof RINm5F pancreatic b cells to STZ resulted in an increased rate ofapoptosis. However, OPA pretreatment was shown to reduce thelevel of apoptosis in the cells. The results of these two assays pro-vide substantial evidence to support the protective role of OPA inSTZ-induced apoptosis.

Lee et al. (2012b) observed antidiabetic effects of OPA related toglucose transport in in vitro tests. Our present findings demon-strate that OPA can also be used as an effective natural antidiabe-togenic material, owing to its protective effect against pancreatic bcell damage. In conclusion, we demonstrated in this study that OPA

S.-H. Lee et al. / Food and Chemical Toxicology 59 (2013) 643–649 649

protects pancreatic b cells from cell damage by reducing oxidativestress and apoptosis.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgement

This work was supported by Konkuk University.

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