Food Chemistry 141 (2013) 847–852

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Food Chemistry

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Protective effect of whey protein hydrolysates on H2O2-induced PC12cells oxidative stress via a mitochondria-mediated pathway

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.03.076

⇑ Corresponding author. Tel.: +86 0510 85329061; fax: +86 0510 85912155.E-mail address: lurr@jiangnan.edu.cn (R.-R. Lu).

Man-Man Jin a, Li Zhang b, Hui-Xin Yu b, Jun Meng a, Zhen Sun a, Rong-Rong Lu a,⇑a State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, Chinab Jiangsu Institute of Nuclear Medicine, Key Laboratory of Nuclear Medicine, Ministry of Health, 20 Qian Rong Road, Wuxi, Jiangsu 214063, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 December 2012Received in revised form 22 March 2013Accepted 24 March 2013Available online 4 April 2013

Keywords:Antioxidant peptidesH2O2

PC12ApoptosisMitochondria-mediated pathway

Whey protein hydrolysates (WPHs) were prepared with pepsin and trypsin. A PC12 cell model was builtto observe the protective effect of WPHs against H2O2-induced oxidative stress. The results indicated thatWPHs reduced apoptosis by 14% and increased antioxidant enzyme activities. Flow cytometry was usedto assess the accumulation of reactive oxygen species (ROS), Ca2+ levels and the mitochondrial membranepotential (MMP). The results showed that WPHs suppressed ROS elevation and Ca2+ levels and stabilisedMMP by 16%. The anti-apoptosis/pro-apoptosis proteins Bcl-2/Bax and poly (ADP-ribose) polymerase(PARP) were investigated by Western-blot analysis, which indicated that WPHs increased the expressionof Bcl-2 while inhibiting the expression of Bax and the degradation of PARP. WPHs also blocked Caspase-3activation by 62%. The results demonstrate that WPHs can significantly protect PC12 cells against oxida-tive stress via a mitochondria-mediated pathway. These findings indicate the potential benefits of WPHsas valuable food antioxidative additives.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Apoptosis, or programmed cell death, is a highly regulated pro-cess that involves the activation of a series of molecular events.Reactive oxygen species (ROS) destroy neurons by inducing apop-tosis, which may perturb the cell’s natural antioxidant defence sys-tem, resulting in damage that is implicated in several biologicaland pathological processes, such as ageing. Antioxidant peptides,which may inhibit oxidative stress, have generated considerableresearch interest. In particular, attention has been paid to highantioxidant activity, absorptivity and safety. Antioxidant peptidesfrom certain foods such as milk protein have been found to havestrong antioxidant activities. Peptides could inhibit lipoxygenase-catalysed lipid autoxidation (Laakso, 1984) and may also havestrong radical-scavenging activities (Qian, Jung, & Kim, 2008;Suetsuna, Ukeda, & Ochi, 2000). Daniel (2004) found that a smallnumber of peptides were absorbed into the intestinal epithelialcells and transported to the inner system without being hydrolysedinto amino acids through transporters in the apical membrane ofenterocytes. Furthermore, because of their remarkable safety,antioxidant peptides are much more favourable than syntheticantioxidants such as butyl hydroxyanisole (BHA) and butylatedhydroxytoluene (BHT). Due to their excellent characteristics,

antioxidant peptides have great potential for future developmentsin the food and healthcare fields.

Antioxidant peptides can be obtained by hydrolysing whey pro-tein. It is well known that whey protein hydrolysates (WPHs) pre-pared by various enzymes, such as pepsin, papain, trypsin,chymotrypsin or Protease N ‘Amano’ G, have antioxidant activities(Cheison, Wang, & Xu, 2007; Peña-Ramos, Xiong, & Arteaga, 2004).Although there are various methods for researching antioxidantactivities based on chemical experiments in vitro, the results ofthese chemical assays should only be extrapolated to real food orbiological systems with caution (Samaranayaka & Li-Chan, 2011).To the best of our knowledge, there have been no intensive studieson how the antioxidant peptides of WPHs affect the intrinsic anti-oxidant system in vivo.

It is possible to screen antioxidative compounds rapidly andinexpensively in vitro using cultured cell model systems toinvestigate their bioavailability, metabolism and bioactivity. Elias,Kellerby, and Decker (2008) confirmed that H2O2, as a major sourceof ROS, could induce oxidative stress in cell lines, causing apoptosisand signalling pathway changes. The mitochondria-mediated path-way is one of the important signalling pathways in apoptosis, andis associated with the activation of aspartate-specific cysteine pro-teases, such as caspase-3, and the cleavage of poly(ADP-ribose)polymerase (PARP) and certain other proteins (Fernandes-Alnemri,Litwack, & Alnemri, 1994; Tewari et al., 1995). Examining theeffects of antioxidants on these cell lines should facilitate anunderstanding of the antioxidant mechanism.

848 M.-M. Jin et al. / Food Chemistry 141 (2013) 847–852

It has been reported that the potential mechanisms of curcu-min’s neuroprotective effect against H2O2-induced oxidative stressin mouse neuroblastoma Neuro-2A cells are exerted through theinhibition of apoptosis and inactivation of the NF-jB signallingpathway (Zhao et al., 2011). The antioxidant ability of persimmonpeel polyphenol was studied in an investigation into the mechnismof oxidative stress in the LLC-PK1 cell line (Yokozawa, Kim, Kim,Lee, & Nonaka, 2007).

In this study, a neuronal PC12 cell model was built to observethe interaction between oxidative stress and the antioxidant pep-tides of WPHs. We explored the possible molecular mechanismsand investigated whether these peptides exert their protective ef-fects against oxidative cytotoxicity by preventing the cleavage ofpoly (ADP-ribose) polymerase (PARP) and/or by inactivating cas-pase-3, due to their antioxidant properties or other signalling path-ways. In attempting to explain the mechanisms of antioxidantpeptides, we aim to provide information that will contribute tothe food and healthcare industries.

2. Materials and methods

2.1. Materials

Heat-stable ALACEN392 whey protein concentrate was pur-chased from Fonterra (Auckland, New Zealand). The rat pheochro-mocytoma line 12 (PC12) cells were purchased from the Instituteof Biochemistry and Cell Biology (Shanghai, China). Dulbecco’smodified Eagle’s medium (DMEM) and foetal bovine serum wereobtained from Gibco (Grand Island, NY). Monoclonal mouse anti-Bcl-2 antibody, mouse anti-Bax antibody and mouse anti-PARPantibody were purchased from the Beyotime Institute of Biotech-nology (Shanghai, China). Fluo-3/AM was purchased from DojinLaboratories (Kumamoto, Japan). Propidium iodide, Hoechst33342 and rhodamine 123 anti-b-actin antibody were purchasedfrom Sigma (St. Louis, MO). All other chemicals were of the highestanalytical grade and purchased from commercial suppliers.

2.2. Preparation of whey protein hydrolysates (WPHs)

Preparation of the whey protein hydrolysates (WPHs) followedthe method described by Zhang et al. (2012). Enzymatic hydrolysiswas carried out using pepsin (pH 1.5, 37 �C) and trypsin (pH 7.6,50 �C) with an enzyme-to-substrate ratio of 2% (w/w). The pH ofhydrolysis was kept constant by continuous addition of 0.1 MNaOH. The reaction was stopped after 90 min by placing in boilingwater and then cooling immediately.

2.3. Cell culture and drug treatment

The PC12 cell cultures were prepared according to the methoddescribed by Lu et al. (2010). Briefly, PC12 cells were cultured inDulbecco’s modified Eagle’s medium (DMEM) with 10% (v/v) foetalbovine serum, 100 U penicillin/mL and 100 mg streptomycin/mLunder 5% CO2 at 37 �C. To produce oxidative stress, H2O2 wasfreshly prepared from a 30% stock solution prior to each experi-ment. PC12 cells were plated in cell culture plates at a density of3 � 105 per well (Corning Incorporated Life Sciences, Lowell, MA).

In our earlier study (Zhang et al., 2012), 25 lmol/L to 200 lmol/L H2O2 samples were treated with PC12 cells, and 100 lmol/L wasfound to be the most suitable concentration for use in the injurymodule, achieving around 50% cell survival rate. In this study,the H2O2-group cells were treated with 100 lmol/L H2O2 for24 h, while the WPHs-group cells were pretreated with WPHs(100 lg/mL or 200 lg/mL) for 2 h. The cells were then incubatedwith WPHs (100 lg/mL or 200 lg/mL) and 100 lmol/L H2O2 for

an additional 24 h. The control-group cells were treated with thesame medium without H2O2 or WPHs.

2.4. T-AOC, SOD, CAT, MDA and LDH assays

To analyse the total antioxidant capacity (T-AOC), superoxidedismutase activity (SOD), catalase activity (CAT) and malondialde-hyde (MDA), the cells were washed with PBS twice then collectedand lysed for 30 min at 4 �C. The cell lysates were kept in ice for30 min and then centrifuged at 12,000g for 5 min at 4 �C. The pro-tein content was measured using a BCA kit (Beyotime Institute ofBiotechnology, Nantong, Jiangsu, China). The medium was col-lected to analyse the level of lactate dehydrogenase (LDH). Theactivities of T-AOC, SOD, CAT, MDA and LHD were tested by assaykits based on colorimetric methods (Institute of Biological Engi-neering of Nanjing Jian-cheng, Nanjing, China).

2.5. Nuclear staining for assessment of apoptosis

PC12 cells were stained with a combination of two fluorescentdyes, propidium iodide and Hoechst 33342 (Lieberthal, Menza, &Levine, 1998). PC12 cells plated in 24-well plates with or withoutWPH pretreatment were incubated with 100 lmol/L H2O2 for24 h. The cells were then washed with phosphate-buffered saline(PBS, 0.01 M, pH 7.4) and stained with Hoechst 33342 (10 lg/ml)and propidium iodide (10 lg/ml) at 37 �C for 10 min.

Each group of cells was photographed twice (Olympus Optical,Japan; magnification 400�). The number of apoptotic cells wascounted in five independent microscopic fields for each group (Gio-vannini et al., 2000).

2.6. Measurement of intracellular Ca2+

A Ca2+-sensitive fluorescence probe, Fluo-3/AM, was used tomonitor changes in the intracellular Ca2+ level by flow cytometry.PC12 cells were cultured as previously described, collected by pip-ette and washed twice with PBS. The intracellular Ca2+ level wasdetermined according to the method described by Huang, Lin,and Chiang (2008).

2.7. Assessment of mitochondrial membrane potential (MMP)

After staining the cells with the cationic lipophilic fluorochrome,rhodamine 123, the level of MMP was determined by flow cytome-try. Rhodamine 123 is used to determine the level of MMP on the ba-sis of its preferential binding with active mitochondria (Zhang et al.,2007). The loss of rhodamine 123 from the mitochondria and a de-crease in intracellular fluorescence is observed when MMP depola-rises (Takumi, Yasushi, Hitoshi, Yasuo, & Hiroshi, 1997).

Rhodamine 123 (10 lmol/L) was added to the cells after treat-ment with WPHs and H2O2, as described above. After incubationat 37 �C for 30 min, the cells were collected, washed twice withPBS and then analysed by flow cytometry.

2.8. Assessment of caspase-3 activities

After the treatments described above, the cells were collectedand the protein concentrations in the supernatants were deter-mined using the BCA protein assay kit (Beyotime Institute of Bio-technology, Nantong, Jiangsu, China).

The activity of caspase-3 was evaluated by a 200 mmol/L Ac-DEVD-MCA fluorogenic substrate in the assay buffer, followingthe assay kit instructions (Beyotime Institute of Biotechnology,Nantong, Jiangsu, China). Fluorescence intensity was measured at460 nm using a fluorescence spectrofluorometer (Hitachi, Ltd. Ja-pan) with excitation at 360 nm.

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2.9. Western blot analysis

After the treatments described above (3 � 105 cells/ml in a 6-well tissue culture plate for 24 h), PC 12 cells were collected andcentrifuged (at 8000g for 5 min). A lysis buffer [150 mmol/L NaCl,1% NP-40, 0.02% sodium azide, 10 lg/mL PMSF, 50 mmol/L Tris–HCl (pH 8.0); 30 lL per tube] was added to the cells. The lysatewas incubated on ice for 30 min and centrifuged at 12,000g for4 min at 4 �C. The supernatant was collected and the protein con-centration was analysed by BCA protein assay kit (Beyotime Insti-tute of Biotechnology, Nantong, Jiangsu, China).

Western-blot analysis was carried out as described previously(Zhao et al., 2011). Briefly, protein samples were electrophoresedon a 15% SDS–PAGE and subsequently transferred onto a nitrocellu-lose membrane (Millipore, Billerica, MA). The membrane was incu-bated in fresh blocking buffer at room temperature for 1 h and thenprobed with the following antibodies [all diluted at 1:1000, v/v(Beyotime Institute of Biotechnology, China)]: anti-b-actin,anti-poly (ADP-ribose) polymerase (PARP), anti-Bcl-2 and anti-Bax in blocking buffer [0.1% (v/v) Tween 20 in Tris-buffered saline,pH 7.4, containing 5% (w/v) skimmed milk] at 4 �C overnight. Afterwashing three times with TBST buffer for 5 min each time [Tris-buffered saline with 0.1% (v/v) Tween 20], the membrane was incu-bated with the appropriate HRP-conjugated secondary antibody(goat anti-mouse IgG, 1:2000, Kangchen Biotechnology, Shanghai,China; goat anti-rabbit IgG, 1:2000, Santa Cruz Biotechnology, San-ta Cruz, CA) at room temperature for 1 h and then washed againthree times in TBST buffer.

The membrane was incubated with enhanced chemilumines-cence substrate solution (Santa Cruz Biotechnology) for 5 minaccording to the manufacturer’s instructions and visualised withautoradiography film.

2.10. Statistical analysis

Results are expressed as the mean ± SD of at least three inde-pendent experiments. Statistical analyses were performed usingSPSS (SPSS 13.0, SPSS Inc., Chicago, IL). Differences between themeans were determined by Duncan’s multiple range test and wereconsidered statistically significant when p < 0.05.

3. Results

3.1. WPHs increased intracellular antioxidase system load

The effect of WPHs on antioxidant peptides was evaluated bydetecting different parts of the antioxidase system in cells. Antiox-idant ability was clearly evident: the WPHs altered the levels of T-AOC, SOD, CAT and LDH on the H2O2-induced PC12 cells, as shownin Table 1.

As illustrated in Table 1, the CAT and SOD levels of the H2O2 groupdeclined significantly compared with those of the control group(p < 0.01). Compared with the H2O2 group, the T-AOC (p < 0.05)

Table 1Effect of WPHs on T-AOC, MDA, LDH, CAT and SOD on H2O2-induced PC12 cells(Mean ± SD).

Control H2O2 WPHs 200 lg/mL + H2O2

T-AOC (U/mg) 2.25 ± 0.17 1.97 ± 0.15⁄ 2.37 ± 0.18#

SOD (U/mg) 37.2 ± 2.66 25.1 ± 2.45⁄⁄ 30.3 ± 2.17##

CAT (U/mg) 2.58 ± 0.20 1.17 ± 0.09⁄⁄ 2.44 ± 0.19##

MDA (nmol/mg) 2.49 ± 0.15 5.16 ± 0.43⁄⁄ 3.39 ± 0.29##

LDH (U/L) 565 ± 11.66 1073 ± 68.0⁄⁄ 977 ± 29.8

⁄p < 0.05.⁄⁄p < 0.01 vs. control.#p < 0.05.##p < 0.01 vs. H2O2-group.

and (p < 0.01) levels of the WPHs group (200 lg/mL) showed a sig-nificant increase. The MDA and LDH levels of the H2O2 group weremuch higher than those of the control group (p < 0.01). The MDAlevel was lower in the WPHs group than in the H2O2 group(p < 0.01), but there was no overt difference between the LDH levelsof the WPHs group and the H2O2 group (p > 0.05).

The T-AOC reflects the enzyme and nonenzyme antioxidase sys-tem as a whole. Catalase helps to scavenge intracellular H2O2. TheCAT level reflects catalase activity, which indicates oxidative dam-age to the cells. The SOD level reflects superoxide dismutase activ-ity, which indirectly indicates the level of intracellular radicals.These results suggest that WPHs maintained the antioxidant sys-tem, increased the clearance rate of H2O2 and protected the cellsagainst oxidative damage.

Malondialdehyde (MDA) is one of the products of lipid peroxi-dation. MDA causes phospholipids and protein to denaturise andcrosslink, which consequently causes cells to contract. The degreeof lipid peroxidation in cells was confirmed by the MDA level. Theleakage rate of lactate dehydrogenase (LDH) was assayed to evalu-ate the level of cell injury. The LDH level of the H2O2 group (Table1) increased significantly compared with that of the control-group(p < 0.01), suggesting that the lipid system had been damaged. TheMDA level of the WPHs group was higher than that of the H2O2

group (p < 0.01). However, the LDH level of the WPHs group de-creased insignificantly compared with the H2O2 group (p > 0.05).The results indicated that the WPHs protected the cell membraneto a certain extent, by reducing the invasion of reactive oxygenand thus inhibiting lipid peroxidation.

3.2. Nuclear staining for assessment of apoptosis

Propidium iodide and Hoechst 33342 double-labelling wereused to measure apoptosis. A fluorescence microscope was usedto observe the karyomorphism of PC12 cells. As illustrated inFig. 1, apoptosis cells were a brilliant condensed blue1, normal cellswere homodisperse light blue and dead cells were red.

In Fig. 1A, there was no obvious apoptosis in the normal cellsafter 48-h cultivation in the control group (Fig. 1Aa). Cells turnedto a brilliant condensed blue after 24 h of exposure to 100 lmol/L H2O2, indicating multiple chromatin and fragmented apoptoticnuclei (Fig. 1Ab). When cells were treated with 100 and 200 lg/mL WPHs in the presence of H2O2 for another 24 h, the numberof apoptotic nuclei significantly reduced (Fig. 1Ac and Ad). WPHsalone did not induce apoptosis in PC12 cells (Fig. 1Ae and Af).

A quantitative evaluation of apoptosis was obtained by countingthe apoptotic cells following Hoechst 33342 and PI staining (Fig. 1B).Compared with the control group, the apoptotic rate of PC12 cells inthe H2O2 group significantly increased to 40.7% (p < 0.01). WhenPC12 cells were incubated with WPHs (100, 200 lg/mL), the per-centage of apoptotic cells decreased to 32.3% and 26.7%, respectively(p < 0.05). In addition, the apoptotic rate of the WPHs-alone groupwas similar to that of the control group (p > 0.05).

WPHs quenched excess free radicals and prevented DNA frombeing attacked by ROS. This antioxidant activity was enhanced asthe WPHs concentration increased. It has been shown that the cellapoptosis rate in stroke models detected by Hoechst staining de-creased from 35% to 20% after treatment by berberine (Zhou, Zeng,Kong, & Sun, 2008). Another study showed that H2O2 activatesnucleus condensation and DNA fragmentation in Neuro-2A cells,but pre-treatment with curcumin mitigated the induction of thesemorphological changes (Zhao et al., 2011). Our results confirm that

1 For interpretation of color in Fig. 1, the reader is referred to the web version ofthis article.

Fig. 1. WPHs protected PC12 cells against H2O2-induced apoptosis. Cells werepretreated with 100 and 200 lg/mL WPHs, respectively for 2 h, then exposed toboth WPHs (the same concentrations as before) and H2O2 (100 lmol/L) for 24 h. (A)Morphological apoptosis was determined by staining with Hoechst 33342 and PI.Arrowheads indicate apoptosis cells. (a) PC12 cells were treated with normalculture medium. (b) PC12 cells were treated with 100 lmol/L H2O2. (c) PC12 cellswere pre-treated with 100 lg/mL WPHs before 100 lmol/L H2O2 treatment. (d)PC12 cells were pre-treated with 200 lg/mL WPHs before 100 lmol/L H2O2

treatment. (e) PC12 cells were only treated with 100 lg/mL WPHs. (f) PC12 cellswere only treated with 200 lg/mL WPHs. (B) Apoptosis ratio was determined bycounting under fluorescence microscope after PC12 cells were stained with Hoechst33342 and PI. ⁄p < 0.05 vs. H2O2 alone, ##p < 0.01 vs. control; all data wererepresentative of three independent experiments.

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WPHs inhibited H2O2-activated apoptosis by reducing nuclearchromatin condensation.

3.3. WPHs inhibited H2O2-induced Ca2+ influx increase in PC12 cells

The intracellular Ca2+ level is a marked feature of cell apoptosisinduced by oxidative stress. It is widely accepted that the intracel-lular Ca2+ level is elevated by H2O2. A Ca2+-sensitive fluorescence

probe, Fluo-3/AM, was used to monitor changes in the intracellularCa2+ level by flow cytometry.

As illustrated in Fig. 2, after incubating cells with 100 lmol/LH2O2 for 24 h, the peak of the H2O2 group (Peak 4) drifted higherthan that of the control group (Peak 1). However, WPHs (200 lg/mL) reduced the H2O2-induced elevation of the intracellular Ca2+

level. The peak of the WPHs group (Peak 3) drifted lower than thatof the H2O2 group (Peak 4).

Many studies have confirmed that apoptosis is generally accom-panied by a rising intracellular Ca2+ level. At the beginning of apop-tosis, the intracellular Ca2+ level rises rapidly with the release ofcytochrome c to cytosol, which leads to apoptosis (Ansari et al.,2006). Our result was consistent with this theory (Fig. 2, Peak 4).WPHs decreased the intracellular Ca2+ level (Fig. 2, Peak 2), illus-trating that WPHs could inhibit oxidative damage in the early stageof apoptosis. Zhao et al. (2011) found that curcumin (20 and 25 lg/ml) dose-dependently inhibited the H2O2-induced elevation ofintracellular Ca2+ levels in Neuro-2A cells.

3.4. WPHs inhibited H2O2-induced MMP change in PC12 cells

The cationic lipophilic fluorochrome, rhodamine 123, was usedto determine the level of MMP, based on its preferential bindingwith active mitochondria. After staining the cells with rhodamine123, various degrees of fluorescence were exhibited according tothe degree of oxidative damage.

Depolarisation of MMP resulted in the loss of rhodamine 123from the mitochondria, presenting a decrease in intracellular fluo-rescence (Takumi et al., 1997). As shown in Fig. 3B, after incubatingthe cells with 100 lmol/L H2O2 for 24 h, MMP levels rapidly de-creased. Rhodamine 123-negative cells in the H2O2 group(Fig. 3B) increased from 4.05 ± 1.21% to 21.56 ± 6.29%, comparedwith the control group (Fig. 3A) (p < 0.05). However, pretreatmentwith 200 lg/mL of WPHs (Fig. 3C) reduced rhodamine-negativecells from 21.56 ± 6.29% to 8.07 ± 2.77%, compared with theH2O2-group (p < 0.05). At the same time, WPHs alone (Fig. 3D)had no significant effect on MMP, compared with the control-group (p > 0.05).

Apoptosis can be suppressed by inhibiting the disruption ofMMP (Chipuk et al., 2004). Koya et al. (2000) found that gelsolininhibited apoptosis by blocking the potential loss of the mitochon-drial membrane and cytochrome c release. Our results illustratedthat WPHs (200 lg/mL) reduced H2O2-induced PC12 cell damageby 16%. This suggests that WPHs stabilised the intracellular Ca2+ le-vel (Fig. 2), which consequently reduced the disruption of MMP(Fig. 3) by controlling the opening of the mitochondrial permeabil-ity transition pores. Eventually, WPHs blocked the release of cyto-chrome c and apoptin, and maintained the cells’ normalphysiological functions.

The results imply that one of the mechanisms by which WPHsexerts its protective effects against oxidative stress is throughmaintaining the MMP balance, which inhibits the disruption ofmitochondrial functioning.

3.5. WPHs inhibited H2O2-induced activation of caspase-3

Caspase-3 is the key executor during apoptosis (Wang, Chi, Lin,Chen, & Shiao, 2001). The activity of caspase-3 was demonstratedby specific fluorogenic peptide substrates for caspases.

Fig 4 shows that after incubating cells with 100 lmol/L H2O2 for24 h, the proteolytic activity of caspase-3 increased by 187%(p < 0.05). However, when PC12 cells were incubated with H2O2

in the presence of WPHs at 100 and 200 lg/mL concentrations, cas-pase-3 activity decreased by 55.8 and 62.4%, respectively (p < 0.05).WPHs alone had little effect on caspase-3 activity (p > 0.05).

Fig. 2. Effects of WPHs on H2O2-induced intracellular Ca2+ increase in PC12 cells. Intracellular Ca2+ levels were determined using flow cytometry with Fluo-3/AM fluorescentdye. Cells were pretreated with 200 lg/mL WPHs for 2 h, then exposed to both WPHs and H2O2 (100 lmol/L) for 24 h. Data was representative of three independentexperiments.

Fig. 3. Effect of WPHs on H2O2-induced reduction of MMP. Cells in labelled zones represent rhodamine 123 negative, percentage represents apoptosis cells in total cells (A:Control, B: H2O2, C: 200 lg/mL WPHs + H2O2, D: 200 lg/mL WPHs).

M.-M. Jin et al. / Food Chemistry 141 (2013) 847–852 851

After caspase-3 activation, some specific substrates for caspase-3, such as PARP, which indicate the occurrence of apoptosis, werecleaved. Piceatannol has been found to reduce caspase-3 activationsignificantly (p < 0.01), thus inhibiting apoptosis and preventingoxidative stress damage in PC12 cells (Kim, Lee, Kim, & Lee,2008). Therefore, the H2O2-induced activation of caspase-3 wassignificantly inhibited by WPHs through antioxidant mechanisms.

3.6. Regulation of H2O2-induced protein expression: Bcl-2, Bax andPARP

The effects of WPHs on the Bax, Bcl-2 and PARP protein levelswere further confirmed by Western-blot analysis (Fig. 5).

The treatment of PC12 cells with 100 lmol/L H2O2 for 24 hcaused overexpression of Bax levels, but low expression of Bcl-2levels. When the PC12 cells were treated with H2O2 in the presence

of 100 and 200 lg/mL WPHs for another 12 h, WPHs dose-depen-dently increased Bcl-2, but decreased Bax protein levels. The re-sults showed that H2O2 and WPHs affected the expression of Bcl-2 family proteins.

The mitochondrial pathway contains many genes involved inregulating apoptosis. It has been reported that the Bcl-2 proteinis a channel protein located in the mitochondrial membrane. TheBcl-2 protein decomposes pro-apoptotic proteins, such as Bax,when it is over-expressed. The integrity of the mitochondrial mem-brane can be maintained by increasing the Bcl-2 protein level(Gross et al., 1999). Our findings suggest that treatment withH2O2 could have a significant effect on the upregulation of Baxand downregulation of Bcl-2, whereas WPHs reverse this trend.

PARP is required for DNA repair. Pro-PARP is cleaved by cas-pase-3 activation in apoptosis cells (Daniel, 2004; Ishikawa,2008). As shown in Fig. 5, the pro-PARP protein was significantly

Fig. 4. The effect of WPHs on caspase-3 activity in H2O2-induced PC12 cells. WPHsinhibited H2O2-induced activation of caspase-3. Cells were pretreated with WPHsfor 2 h, then exposed to both WPHs (the same concentrations as before) and H2O2

(100 lmol/L) for 24 h. ⁄p < 0.05 vs. H2O2 alone, #p < 0.05 vs. Control. All data wasexpressed as mean ± SD of three experiments and each included triplicate sets.

Fig. 5. Effects of WPHs (100 lg/mL, 200 lg/mL) on the Bax, Bcl-2 and PARP proteinexpression in H2O2-treated PC12 cells measured by Western blot analysis.

852 M.-M. Jin et al. / Food Chemistry 141 (2013) 847–852

cleaved in the H2O2 group of PC12 cells, whereas the cleaved PARPprotein increased. In contrast, incubating the cells with H2O2 in thepresence of WPHs appeared to attenuate PARP cleavage (Fig. 5). Asillustrated in Fig. 4, caspase-3 was activated in the H2O2 group,resulting in the cleavage of pro-PARP. However, a protective effectwas observed when WPHs were added to the medium, indicatingthat cleavage of pro-PARP was reduced.

4. Conclusion

In conclusion, WPHs protected cells from oxidative stress dam-age. WPHs increased the intracellular antioxidase system load andinhibited H2O2-activated apoptosis by maintaining Ca2+ and mito-chondrial membrane potential levels. The results also showed thatWPHs were essential in regulating the expression of Bcl-2 and Baxproteins and in suppressing Caspase-3 activation and PARP cleav-age. We conclude that WPHs protected PC12 cells against oxidativestress by regulating the mitochondrial apoptotic pathway.

Acknowledgement

This study was supported by the grants from the National Nat-ural Science Foundation of China (No.31071491), Program for NewCentury Excellent Talents in University (NCET-09-0436), the 863project (2013AA102207) and ‘‘Twelfth 5-year’’ National Key Tech-nology R&D Program of China (2012BAD33B05).

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