Subscriber access provided by READING UNIV
Article
Ultraviolet rays induced sea cucumber (Stichopus japonicus) melting is mediated through the caspase-dependent mitochondrial apoptotic pathway Li Su, Jing-Feng Yang, Xi Fu, Liang Dong, Da-Yong Zhou, Li-Ming Sun, and Zhenwei Gong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03888 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 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 43
Journal of Agricultural and Food Chemistry
1
Ultraviolet rays induced sea cucumber (Stichopus japonicus) melting is mediated
2
through the caspase-dependent mitochondrial apoptotic pathway
3 4
Li Su†, Jing-Feng Yang†*, Xi Fu†, Liang Dong†, Da-Yong Zhou†, Li-Ming Sun†,
5
Zhenwei Gong‡*
6 †
7
School of Food Science and Technology, Dalian Polytechnic University, National
8
Engineering Research Center of Seafood, No.1 Qinggongyuan, Ganjingzi district,
9
Dalian 116034, P. R. China
10 11
‡
Division of Pediatric Endocrinology, Department of Pediatrics, Children's
12
Hospital of Pittsburgh of the University of Pittsburgh Medical Center, University of
13
Pittsburgh School of Medicine, 4401 Penn Ave, Pittsburgh, Pennsylvania 15224,
14
USA
15 16 17
*Corresponding authors: Dr. Jing-Feng Yang
18
E-mail address:
[email protected], Tel: +86-411-86318729
19
and
20
Dr. Zhenwei Gong
21
E-mail address:
[email protected], Tel: +1-412-692-9601
22 23
1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
24
Abstract
25
Sea cucumber body wall melting occurs under certain circumstances. We have
26
shown that apoptosis, but not autolysis, plays a critical role in the initial stage.
27
However, it is still unclear how apoptosis is triggered in this process. In this study, we
28
examined the reactive oxygen species (ROS), levels of B-cell lymphoma-2 (Bcl-2)
29
and Bcl-2 Associated X Protein (Bax) and the depolarization of mitochondria
30
transmembrane potential and Cytochrome c (Cyt c) release during the sea cucumber
31
melting induced by ultraviolet (UV) exposure. We also investigated the contribution
32
of caspase in this process by injecting pan-caspase inhibitor. Our data showed that UV
33
exposure stimulates the ROS production, dysfunction of mitochondria and the release
34
of Cyt c in sea cucumber coelomic fluid cells and body wall. We found a decrease of
35
Bcl-2 and increase of Bax in the mitochondria after UV exposure. We also
36
demonstrated that these changes are associated with the elevated activity of caspase-9
37
and -3. Finally, our data showed that inhibition of caspase -9 and -3 using inhibitor
38
suppresses the UV induced sea cucumber melting. These results suggest that the
39
apoptosis during sea cucumber melting is mediated through the mitochondria
40
dysfunction and following activation of caspase signaling pathway. This study
41
presents a novel insight into the mechanism of sea cucumber melting.
42 43
Key words: Stichopus japonicus, melting, apoptosis, autolysis, mitochondria
44
2
ACS Paragon Plus Environment
Page 2 of 43
Page 3 of 43
Journal of Agricultural and Food Chemistry
45
Introduction
46
Sea cucumber (Stichopus japonicus) is a well-known aquatic product in China and
47
its production reached more than 200,000 tons in 2015 with an industrial output value
48
exceeding 6 billion US dollars 1. In severe cases, such as expose to high temperature,
49
salt concentration, nutrient deficiency and exposure to sunlight or UV irradiation, the
50
sea cucumber body melting may occur, leading to the death of the animals 2. This
51
phenomenon is a major concern for sea cucumber production, particularly during
52
cultivation, transportation, handling and processing 3. Thus, understanding the sea
53
cucumber melting mechanism could contribute to the sea cucumber cultivation and
54
processing industry.
55
The sea cucumber melting has long been recognized as a mechanism of autolysis4-6,
56
which is commonly known as self-digestion of cells through the actions of their own
57
enzymes. Our previous study has demonstrated that the initiation of the sea cucumber
58
melting triggered by UV exposure is attributing to apoptosis but not autolysis 7. But
59
the mechanism by which apoptosis is triggered remains unknown.
60
Apoptosis can be triggered through two main pathways: the extrinsic (death
61
receptor-dependent) and intrinsic (mitochondrial-dependent) pathways 8. The intrinsic
62
pathway is a response to endogenous and exogenous factors, which causes the
63
mitochondrial membrane becoming permeable and depolarized. Subsequently, the Cyt
64
c is released from mitochondria to the cytosol leading to the elevation of cytosolic Cyt
65
c level 9. The cytosolic Cyt c then binds to apoptotic protease activating factor 1
66
followed by the activation of caspase
10
. Cytosolic Cyt c is a key component and 3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
11
67
indicator of an apoptotic event
. Mitochondrial membrane permeability, i.e.
68
depolarization of mitochondrial membrane potential, has also been proposed to play a
69
role in the mitochondrial apoptotic cascade 12.
70
In the current study, we sought to further investigate the molecular mechanism of
71
apoptosis in sea cucumber melting phenomenon and reveal its signaling pathway. We
72
examined the Cyt c release from mitochondria and the change of mitochondrial
73
transmembrane potential in sea cucumber coelomic fluid cells after UV exposure. In
74
addition, we investigated the changes in levels of ROS, Bcl-2 and Bax and activity of
75
caspase-9 and -3 during sea cucumber melting after UV exposure.
76 77
Materials and Methods Materials and Chemicals
78
Sexually mature adult sea cucumbers (S. japonicus, 100-120 g; 15-20 cm total body
79
length) were obtained from Dalian Liujiaqiao Seafood Market. Antibodies used for
80
western blotting experiments are listed as following. Bax rabbit monoclonal antibody
81
(Rabbit Antibody, react with Mouse, Rat, Human, Chinese Hamster and predicting
82
react with Cow), anti-COX IV rabbit polyclonal, β-actin rabbit polyclonal antibody
83
and a secondary anti-rabbit IgG (HRP-linked antibody) were purchased from Cell
84
Signaling Technology (Beverly, MA, USA). The Bax antibody was generated using
85
synthetic Human Bax peptide aa 1-100 (N terminal) as antigens. Anti-Bcl-2 rabbit
86
polyclonal antibody (Rabbit Antibody, react with Human and predicting react with
87
Mouse, Rat, Chicken, Cow, Cat and Dog) was bought from Abcam (Abcam,
88
Cambridge, Mass, USA). The Bcl-2 antibody was generated using synthetic peptide 4
ACS Paragon Plus Environment
Page 4 of 43
Page 5 of 43
Journal of Agricultural and Food Chemistry
89
within Human Bcl-2 aa 1-239 as antigen. The Enhanced BCA Protein Assay Kit was
90
obtained from Beyotime Institute of Biotechnology (Shanghai, China). Sodium
91
dodecyl sulphate (SDS), N,N,N,N-tetramethylethylenediamine (TEMED), Coomassie
92
Brilliant Blue R-250, Bovine serum albumin (BSA) and the Tissue or Cell Total
93
Protein Extraction Kit were from Sangon Biotech Co., Ltd. (Shanghai, China). The
94
PVDF membrane was obtained from Immobilon (Millipore, Billerica, USA). All other
95
chemicals used in this study were of analytical grade.
96
Inducing sea cucumber melting by UV radiation
97
Sea cucumber melting induced by UV was performed as previously described 7.
98
Briefly, six groups of sea cucumber (seven samples per group) were exposed to UV
99
light (wavelength 253.7nm, Cnlight, ZW20S19W, Jiangyin, Jiangsu, China) for half
100
hour at intensity of 0.056 mw/cm2. After UV exposure, the sea cucumbers were
101
transported to incubator with surroundings at 20 °C in dark (covered with Aluminum
102
foil) and held for 0, 60, 120, 240 and 360 min respectively followed by apoptotic
103
analysis. Untreated fresh sea cucumbers were used as control.
104
Tissue preparation
105
The UV-treated sea cucumbers were cut at the end of cloacal aperture. Midsection
106
of sea cucumber body wall was sampled to tissue blocks (1 cm×1 cm×0.5 cm) and
107
fixed in 10% neutral formalin in phosphate buffered saline (PBS, pH 7.4), embedded
108
in paraffin and processed as previously described 7. Briefly, after fixation for 24 h,
109
tissues were dehydrated, paraffin embedded, sectioned for histological experiments.
110
Sections of 5 µm thick were heated in a distilled water bath (45 °C), collected on a 5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
111
glass slides, deparaffinised and rehydrated stepwise through an ethanol series and
112
processed for routine histological analysis.
113
Cyt c immunohistochemistry
114
Sections from above were subjected to an antigen retrieval procedure by heating the
115
samples at 65 °C for 30 min followed by cooling down for 10 min and then rinsing
116
with dimethylbenzene, ethanol and distilled water each for 5 min. The sections were
117
incubated in 3% hydrogen peroxide for 8 min to inactivate endogenous peroxidase in
118
samples. Antigen retrieval was done by boiling sections in 0.01 M citrate (Sigma
119
Aldrich, St. Louis, MO, USA, pH 6.0) and then rinsing with PBS for 5 min at room
120
temperature followed by immersion in 5% bovine serum albumin (BSA, Sangon
121
Biotech, Shanghai, China) at room temperature for 10 min. The processed sections
122
were incubated with Cyt c antibody (BioLegend, San Diego, CA, USA) at 4°C
123
overnight and were held in room temperature for 30 min followed by washing with
124
PBS for 3 times. Sections were then incubated with goat anti-rabbit IgG-HRP (1/500
125
in PBS, SE13, solarbio, Beijing, China) for 60 min at 37°C followed by PBS washing
126
for 3 times. Sections were subsequently treated with 0.05% diaminobenzidine (DAB,
127
Beyotime, Shanghai, China) for 10 min at room temperature, rinsed in PBS for 5 min
128
and allowed to dry in the air.
129
Mitochondrial membrane potential analysis
130
The Jiamay BiotechTM JC-1 Assay Kit (Beijing, China) was used to quantify
131
mitochondrial transmembrane potential. The cationic lipid fluorescent dye JC-1 was
132
used to study depolarization of mitochondria transmembrane potential (decreasing red 6
ACS Paragon Plus Environment
Page 6 of 43
Page 7 of 43
Journal of Agricultural and Food Chemistry
133
fluorescence density) and release of the JC-1 monomer into the cytoplasm (green
134
fluorescence). Coelomic fluid cells (500 µL) extracted from treated samples were
135
incubated with 500 µL of JC-1 (diluted 200 times according to the instructions) at 4°C
136
for 10 minutes. The cells were washed with PBS and then analyzed using a flow
137
cytometer (FACSVerse, Becton, Dickinson and Company, USA) at excitation 585 nm
138
and emission 590 nm for the JC-1 aggregate (red) and at excitation 515 nm and
139
emission 529 nm for the JC-1 monomer (green).
140
ROS generating analysis
141
DCFDA Cellular ROS Detection Assay Kit (Abcam, Sydney, Australia) was used
142
for quantitative measurement of cellular ROS in sea cucumber coelomic fluid cells.
143
After incubating with 5 µM DCFDA at room temperature for 30 min, coelomic fluid
144
cells obtained from control and treated sea cucumbers were subjected to laser
145
scanning confocal (SP8, Leica, Heidelberg, Germany) for ROS detection.
146
Western Blotting analysis
147
Western blotting analysis was done to determine the protein levels of Bax and Bcl-2
148
in sea cucumber coelomic fluid cells after UV treatment. Cytoplasmic and
149
mitochondrial proteins were extracted using cytoplasmic and mitochondrial protein
150
extraction kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s
151
instructions. Briefly, samples from different groups were homogenized in ice-cold
152
cytoplasmic extraction buffer using glass pestle. After incubating for 15 min on ice,
153
the pellets were collected and centrifuged at 900 g for 10 min at 4 °C. The
154
supernatants were transferred to new tubes and then centrifuged at 13,800 g at 4 °C 7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
155
for 30 min to obtain the cytoplasmic protein in the supernatants. The pellets were
156
re-suspended in cytoplasmic extraction buffer and centrifuged at 16,200 g at 4 °C for
157
10 min. The pellets containing mitochondrial fraction were re-suspended in
158
mitochondrial lysis buffer for 30 min on ice and then centrifuged at 16,200 g at 4 °C
159
for 10 min to obtain the mitochondrial protein in the supernatants. Protein
160
concentration was determined by the modified Bradford protein assay kit (Sangon
161
Biotech, Shanghai, China).
162
Protein samples (20 µg) from each group were resolved on 12% sodium dodecyl
163
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto
164
polyvinyl difluoride (PVDF) membranes (Millipore, Billerica, America). The
165
membranes were blocked with 5% non-fat milk in Tris Buffered Saline with 0.05%
166
Tween 20 (TBS-T) and then incubated with primary antibodies (Bcl-2, 1:1,000, Bax,
167
1: 10,000, β-Actin, 1:1,000, COX IV, 1:1,000) overnight at 4 °C. Membranes were
168
washed three times with 1×TBS-T for 15 min, and exposed to secondary antibody
169
(1:5,000) for 1 h at room temperature. Proteins were detected by Enhanced
170
chemiluminescence (ECL) reagents (Nacalai Tesque, Kyoto, Japan).
171
The influence of inhibitor on sea cucumber melting and the activity of Caspase-9 and
172
Caspase-3
173
The pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone
174
(Z-VAD-FMK, KeyGEN Biotech, Nanjing, China) was dissolved in dimethyl
175
sulfoxide (DMSO) and diluted to 20 µM with PBS. The inhibitor (2 mL) was injected
176
to each sea cucumber from cloacal aperture and exposed to UV light and processed as 8
ACS Paragon Plus Environment
Page 8 of 43
Page 9 of 43
Journal of Agricultural and Food Chemistry
177
indicated above. Caspase-9 and caspase-3 activities were determined using the
178
Caspase-9 and Caspase-3 Colorimetric Assay Kit (KeyGEN Biotech, Nanjing, China)
179
followed the manufacturer’s instruction. Briefly, sea cucumber intestine or coelomic
180
fluid cells (0.3 g wet weight) were homogenized in ice-cold lysis buffer and
181
centrifuged at 9,600 g for 5 min at 4°C to collect the supernatants for the assay. Fifty
182
microliter of 2X Reaction Buffer (containing 10 mM DL-Dithiothreitol) was mixed
183
with 50 µL of sample in a 96-well plate followed by addition of 5 µL of caspase-9 or
184
caspase-3 substrate (50 µM final concentration) and 4 hours of incubation at 37 °C.
185
The absorbance was read at 405 nm in a Microplate reader (Tecan, Männedorf,
186
Switzerland) and the results were calculated as fold increases compared with
187
untreated samples.
188
Statistical Analysis
189
All experiments were done at least 3 times independently with three replicates (n=3)
190
for each time. Data are presented as mean ± standard deviation (SD). Mean values
191
were compared by One-factor Analysis of Variance (ANOVA) and the differences
192
between means were evaluated by using S-N-K test as well as T-test. The statistical
193
analysis was performed by using SPSS 16.0 software (SPSS Inc. Chicago, IL, USA).
194
Comparisons that yielded p values < 0.01 were considered significant.
195 196
Results UV exposure induces Cyt c release from the mitochondria
197
The distribution of Cyt c in the body wall during sea cucumber melting induced by
198
UV was analyzed by the immunohistochemistry. We showed that the Cyt c in samples 9
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 10 of 43
199
without UV exposure are uniformly dispersed in the body wall with clear edge (Figure
200
1a). Nevertheless, the Cyt c in UV-exposed tissue samples had no clear edge (Figure
201
1b-1f). These results demonstrated that the release of Cyt c from mitochondria to
202
cytoplasm occurs within 0-6 hours after the UV exposure. The immunohistochemistry
203
analysis was also performed using the inner body wall tissues and we found a similar
204
pattern as in epidermis of the body wall tissues (Figure 2).
205
UV irradiation causes depolarization of the mitochondrial transmembrane potential
206
The change of mitochondrial membrane potential (∆Ψm) is a marker of 13
207
mitochondrial function and is associated with apoptosis at early stage
. A cationic
208
dye JC-1 was used to monitor the change of ∆Ψm in the sea cucumber coelomic fluid
209
cells. Flow cytometry was used to measure the red/green fluorescence ratio (JC-1
210
aggregate/JC-1 monomer ratio) as an indication of ∆Ψm. We showed that the ratios of
211
red/green fluorescence intensity gradually decrease in the UV treated groups
212
compared to the control group (0.1% DMSO) (Figure 3A and 3B). The ratios
213
decreased from 5.05 to 3.97, 1.90, 1.65 and 1.69 in samples holding for 0h, 1h, 2h, 4h
214
and 6 h post UV exposure. These observations suggest that the depolarization of ∆Ψm
215
occurred in coelomic fluid cells of sea cucumber after UV treatment.
216
UV exposure increases ROS production in sea cucumber coelomic fluid cells
217
The ROS level in coelomic fluid cells after UV exposure were detected by DCFDA
218
Cellular ROS Detection Kit and the ROS was labeled in green as shown in Figure 4A.
219
The quantification data demonstrated that compared to the controls, UV exposure
220
causes significantly increase in intracellular ROS and it continues to increase when 10
ACS Paragon Plus Environment
Page 11 of 43
Journal of Agricultural and Food Chemistry
221
hold in room temperature. We observed that the ROS level peaks at 2 hours after UV
222
treatment and declines at 4 and 6 hours after UV treatment (Figure 4B). These results
223
indicated that UV exposure leads to the increase in ROS levels in coelomic fluid cells
224
at early time points.
225
UV exposure induces Bax translocation
226
The changes of Bcl-2 and Bax in both cytoplasmic and mitochondrial fractions of
227
sea cucumber coelomic fluid cells were examined using western blotting. We
228
demonstrated that the Bcl-2 is undetectable and Bax decreases in cytoplasmic fraction
229
after the UV exposure (Figure 5A). We also found that the levels of Bax in
230
mitochondrial fraction can hardly be detected at the earlier time points, but increased
231
2 h after the UV exposure in a time dependent manner (Figure 5B). However, the
232
Bcl-2 in mitochondrial fraction dropped (Figure 5C). We quantified the ratio of Bax to
233
Bcl-2 in mitochondrial fraction and showed that the ratio of Bax/Bcl-2 in
234
mitochondrial fraction increased significantly from 2 to 6 hour (Figure 5D).
235
Pan-caspase inhibitor suppresses Caspase-3 and Caspase-9 activities induced by UV
236
exposure
237
After the UV treatment, the Caspase-3 activity increased significantly from 1 to
238
2.94-fold in the intestinal cells and from 1 to 2.83-fold in coelomic fluid cells within 6
239
hours (Figure 6A and 6B). Notably, the increase of caspase-3 activity induced by UV
240
treatment was greatly inhibited by injection of Z-VAD-FMK, a pan-caspase inhibitor.
241
A similar pattern was also observed in Caspase-9 activity both in coelomic fluid cells
242
and intestinal cells (Figure 7). 11
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
243
Page 12 of 43
Caspase inhibitor suppresses sea cucumber body wall melting
244
Z-VAD-FMK was injected into the fresh sea cucumbers from the cloacal aperture
245
prior to the UV treatment. After 30 min UV exposure and 47 h left at 20 °C, the
246
inhibitor treated sea cucumbers appeared identical to the fresh group sea cucumbers.
247
However, the UV treated sea cucumbers without inhibitor injection showed melting
248
phenomenon 20 h after UV treatment (Figure 8). This demonstrated that the
249
pan-caspase inhibitor blocks the sea cucumber melting and the UV induced sea
250
cucumber melting is mediated through the caspase-dependent pathway.
251
Discussion
252
Sea cucumbers initiate self melting upon environmental change, including changes
253
in temperature, and salt concentration, and exposure to sunshine and UV. It was
254
believed that autolysis plays key role in this process 4. However, our previous study
255
showed that apoptosis, but not autolysis is the major cause contributing to the sea
256
cucumber melting induced by UV exposure 7. We demonstrated that apoptosis appears
257
in more than 50% of total cells in the body wall of sea cucumber within 6 h after UV
258
exposure 7. The sea cucumber body wall is mainly composed of collagen with small
259
amounts of cells. In our study, the apoptosis was analyzed in multiple tissues and cells
260
such as the body wall, intestinal cells and body coelomic fluid cells. The evidence
261
supports that the apoptosis appears not only in cell level but also in tissue level.
262
Two major signaling pathways have been discovered to mediate the cellular
263
apoptosis, the intrinsic and extrinsic pathways 14. Mitochondria are well known to be
264
involved in the intrinsic pathway and play a critical role in apoptosis 12
ACS Paragon Plus Environment
15
. The
Page 13 of 43
Journal of Agricultural and Food Chemistry
265
mitochondrial pathway of cell apoptosis is regulated through caspase-9 16. It has been
266
reported that the release of Cyt c causes the activation of caspase-9 through the
267
apoptosome and then activates caspase-3 to induce cell death 17. The decrease in ∆Ψm
268
and the release of Cyt c are two key steps for activating mitochondria mediated
269
apoptosis 18. In the current study, UV exposure was used to trigger the sea cucumber
270
melting and the results demonstrated that UV exposure induces the release of Cyt c
271
and mitochondrial dysfunction in sea cucumbers (Figure 1, 2 and 3).
272
The loss of ∆Ψm is a hallmark and early event of apoptosis coinciding with caspase 19
273
activation
. Two hours after exposing to UV, sea cucumber coelomic fluid cells
274
exhibited a collapse of ∆Ψm, which may further lead to the release of Cyt c from
275
mitochondria into the cytosol. Indeed, we observed the increase of Cyt c release from
276
mitochondria and the collapse of ∆Ψm in sea cucumber cells after exposed to UV,
277
suggesting that both the Cyt c release and collapse of ∆Ψm play critical roles in UV
278
induced apoptosis in sea cucumber.
279
Cyt c works on the electron transport chain during respiration in the mitochondria.
280
It transfers an electron from respiratory Complex III to Complex IV in the inner
281
mitochondrial membrane
282
release of Cyt c. The release of Cyt c from mitochondria into the cytoplasm triggers a
283
cascade of reactions leading to apoptosis 18 ,21. The release of Cyt c from mitochondria
284
is generally preceded by mitochondrial stress that starts with receiving an apoptotic
285
signal from outside or internal factors
286
cucumber mitochondria likely through the oxidative stress and the collapse of ∆Ψm. It
20
. The apoptotic signals affect the ∆Ψm and lead to the
22
. The UV exposure may cause stress to sea
13
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
287
has been reported that sea cucumber tissue sample generates reactive oxygen species
288
(ROS) and oxidative stress after UV exposure, which might be involved in sea
289
cucumber melting 23. Our study also showed an increased ROS in coelomic fluid cells
290
after the sea cucumber exposed to UV and held in room temperature (Figure 4). ROS
291
induces cardiolipin peroxidation in the mitochondrial inner membrane, thus causing
292
Cyt c release due to the breach of electrostatic/hydrophobic interactions 24.
293
The Bcl-2 family members serve as either activators (e.g., Bax or Bad) or inhibitors 12
294
(e.g., Bcl-2, Bcl-xL) for cellular apoptosis
295
mitochondrial membrane, Cyt c release and activation of caspase-9 and caspase-3
296
cascade 25. It is demonstrated that the overexpression of Bcl-2 prevents Cyt c release
297
but Bax enhances it
298
level of Bcl-2 in mitochondria (Figure 5C), and the level of Bax decreased in
299
cytoplasm but increased in mitochondria after the UV exposure. These results
300
demonstrated that the Bax spontaneously translocates to the mitochondria from
301
cytoplasm 2 h after the UV exposure. Upon delivery of an apoptotic stimulus,
302
cytoplasmic Bax translocates to the outer mitochondrial membrane, where it forms
303
homodimers, creating pores that causes loss of ∆Ψm, Cyt c release from the
304
intermembrane space of the mitochondrion into the cytosol 28. The reduction of Bcl-2
305
in mitochondria and the translocation to the mitochondria of Bax in sea cucumber
306
cells lead to the Cyt c release from the mitochondria into the cytoplasm and activation
307
of caspase-9 and caspase-3.
308
. They regulate the permeability of the
26,27
. We showed in this study that the UV exposure reduces the
The activation of the initiators of apoptosis, caspase-3 and -9, were observed in the 14
ACS Paragon Plus Environment
Page 14 of 43
Page 15 of 43
Journal of Agricultural and Food Chemistry
309
UV treated sea cucumber cells (Figure 6 and 7). The caspase family proteins exist as
310
inactive precursor form in the cytosol. Upon activation by apoptotic signals, the
311
precursors of caspase are cleaved and processed to generate active enzymes through
312
proteolytic pathways
313
irreversibly binds to the catalytic site of caspase proteases and blocks up the apoptosis
314
19
. Z-VAD-FMK is a cell-permeable pan-caspase inhibitor that
29
. Our data demonstrated that the injection of inhibitor results in the suppression of
315
caspase-3 and caspase-9 activation in UV induced sea cucumber, indicating the
316
feasibility of applying Z-VAD-FMK to inhibit the activity of caspase-3 and -9 in vivo
317
in sea cucumber. In addition, we showed that Z-VAD-FMK injection mostly blocks
318
the UV induced sea cucumbers melting (Figure 8). Since activation of caspase
319
cascade is a pro-apoptotic signal 21, and Z-VAD-FMK is an effective inhibitor to the
320
caspase proteases, we conclude that both caspase-3 and -9 contribute to the apoptosis
321
in sea cucumber cells induced by UV exposure and the sea cucumber melting is
322
mediated through the caspase-dependent mitochondrial apoptotic pathway.
323
Taking together, our study demonstrated that the UV exposure increases the ROS
324
production, collapse of ∆Ψm and Cyt c release from the mitochondria into the cytosol,
325
therefore activating caspase-9 and caspase-3 dependent apoptosis in the sea cucumber
326
cells. Our study demonstrated that the apoptosis in sea cucumbers is mediated through
327
the reduction in Bcl-2 and induction Bax in the mitochondria after UV treatment. The
328
current studies further revealed that apoptosis plays critical roles in the sea cucumber
329
melting, and that UV induced apoptosis in sea cucumber is mediated through the
330
caspase-dependent mitochondrial apoptotic pathway. 15
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 16 of 43
331
Funding Sources
332
This work is supported by “The National Natural Science Foundation” (No.
333
31771998,No. 31301430), “Natural Science Foundation of Liaoning Province” (No.
334
20170540062), Support of National Engineering Research Center of Seafood from
335
National
336
2012FU125X03, Third batch of Liaoning Collaborative Innovation Center from
337
provincial department of public education LJF[2015]NO.99.
Ministry
of
Science
and
Technology
338
The authors declare no competing financial interest.
339
Reference
340 341
GKFJ[2013]NO.405
and
1. Bureau of Fisheries in Ministry of Agriculture, China Fishery Statistical Yearbook, China Agriculture Press, Beijing. 2016, 29.
342
2. Zhu, B.; Zheng, J.; Zhang, Z.; Dong, X.; Zhao, L.; Tada, M. Autophagy Plays a
343
Potential Role in the Process of Sea Cucumber Body Wall “Melting” Induced by
344
UV Irradiation. Wuhan Univ. J. Nat. Sci. 2008, 13, 232-238.
345
3. Wu, H.; Li, D.; Zhu, B.; Sun, J.; Zheng, J.; Wang, F.; Konno, K.; Jiang, X.
346
Proteolysis of noncollagenous proteins in sea cucumber, Stichopus japonicus,
347
body wall:
348
Food Chem. 2013, 141, 1287-1294.
Characterisation and the effects of cysteine protease inhibitors.
349
4. Yan, L.; Zhan, C.; Cai, Q.; Weng, L.; Du, C.; Liu, G.; Su, W.; Cao, M. Purification,
350
Characterization, cDNA Cloning and In Vitro Expression of a Serine Proteinase
351
from the Intestinal Tract of Sea Cucumber (Stichopus japonicus) with Collagen
352
Degradation Activity. J. Agric. Food Chem. 2014, 62, 4769-4777. 16
ACS Paragon Plus Environment
Page 17 of 43
Journal of Agricultural and Food Chemistry
353
5. Zhou, D.; Chang, X.; Bao, S.; Song, L.; Zhu, B.; Dong, X.; Zong, Y.; Li, D.; Zhang,
354
M.; Liu, Y.; Murata, Y. Purification and partial characterisation of a cathepsin
355
L-like proteinase from sea cucumber (Stichopus japonicus) and its tissue
356
distribution in body wall. Food Chem. 2014, 158, 192-199.
357 358
6. Wilkie, I. Is muscle involved in the mechanical adaptability of echinoderm mutable collagenous tissue? J. Exp. Biol. 2002, 205, 159-165.
359
7. Yang, J.; Gao, R.; Wu, H.; Li, P.; Hu, X.; Zhou, D.; Zhu, B.; Su, Y. Analysis of
360
Apoptosis in Ultraviolet-Induced Sea Cucumber (Stichopus japonicus) Melting
361
Using
362
End-Labeling Assay and Cleaved Caspase-3 Immunohistochemistry. J. Agric.
363
Food Chem. 2015, 63, 9601-9608.
364 365
Terminal
Deoxynucleotidyl-Transferase-Mediated
dUTP
Nick
8. Martin, D.; Baehrecke, E. Caspases function in autophagic programmed cell death in Drosophila. Development. 2004, 131, 275-284.
366
9. Sanz-Blasco, S.; Valero, RA.; Rodríguez-Crespo, I.; Villalobos, C.; Núñez, L.
367
Mitochondrial Ca2+ overload underlies Abeta oligomers neurotoxicity providing
368
an unexpected mechanism of neuroprotection by NSAIDs. PLoS One. 2008 7,
369
1-16.
370
10. Lee, J.; Park, Y.; Pun, S.; Lee, S.; Lo, J.; Lee, L. Real-time investigation of
371
cytochrome c release profiles in living neuronal cells undergoing amyloid beta
372
oligomer-induced apoptosis. Nanoscale. 2015, 7, 10340-10343.
373
11. Hengartner , MO. The biochemistry of apoptosis. Nature. 2000 , 407, 770-776.
17
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
374
12. Wu, Z.; Sun, H.; Li, J.; Ma, C.; Zhao, S.; Guo, Z.; Lin, Y.; Lin, Y.; Liu, L. A
375
polysaccharide from Sanguisorbae radix induces caspase-dependent apoptosis in
376
human leukemia HL-60 cells. Int. J. Biol. Macromol. 2014, 70, 615-620.
377 378
13. Roy, S.; Hajnoczky, G. Calcium, mitochondria and apoptosis studied by fluorescence measurements. Methods. 2008, 46, 213-223.
379
14. Green, D.; Evan, G. A matter of life and death. Cancer Cell. 2002, 1, 19-30.
380
15. Adrain, C.; Martin, S. The mitochondrial apoptosome: a killer unleashed by the
381
cytochrome seas. Trends Biochem. Sci. 2001, 26, 390-397.
382
16. Norberg, E.; Orrenius, S.; Zhivotovsky, B. Mitochondrial regulation of cell death:
383
Processing of apoptosis-inducing factor (AIF). Biochem. Biophys. Res. Commun.
384
2010, 396, 95-100.
385 386
17. Nalepa, G.; Zukowska, E. Caspases and apoptosis: Die and let live. Wiad. Lek. 2002, 55, 100-106.
387
18. Garrido, C.; Galluzzi, L.; Brunet, M.; Puig, P.; Didelot, C.; Kroemer, G.
388
Mechanisms of cytochrome c release from mitochondria. Cell Death Differ. 2006,
389
13, 1423-1433.
390
19. Zhang, X.; Mei, W.; Tan, G.; Wang, C.; Zhou, S.; Huang, F.; Chen, B.; Dai, H.;
391
Huang, F. Strophalloside Induces Apoptosis of SGC-7901 Cells through the
392
Mitochondrion-Dependent Caspase-3 Pathway. Molecules. 2015, 20, 5714-5728.
393
20. Ardail, D.; Privat, J.; Egret-Charlier, M.; Levrat, C.; Lerme, F.; Louisot, P.
394
Mitochondrial contact sites. Lipid composition and dynamics. J. Biol. Chem. 1990,
395
265, 18797-18802. 18
ACS Paragon Plus Environment
Page 18 of 43
Page 19 of 43
Journal of Agricultural and Food Chemistry
396
21. Zhang, B. Xu, Z.; Zhang, Y.; Shao, X.; Xu, X.; Cheng, J.; Li, Z. Fipronil induces
397
apoptosis through caspase-dependent mitochondrial pathways in Drosophila S2
398
cells. Pestic. Biochem. Physiol. 2015, 119, 81-89.
399
22. Vladimirov, Y. A.; Proskurnina, E. V.; Alekseev, A.V. Molecular Mechanisms of
400
Apoptosis. Structure of Cytochrome c-cardiolipin complex. Biochemistry
401
(Moscow). 2013, 78, 1086-1097.
402
23. Qi, H.; Fu, H.; Dong, X.; Feng, D.; Li, N.; Wen, C.; Nakamura, Y.; Zhu, B.
403
Apoptosis induction is involved in UVA-induced autolysis in sea cucumber
404
Stichopus japonicus. J. Photochem. Photobiol., B. 2016, 158, 130-135 .
405 406 407 408
24. Orrenius, S.; Zhivotovsky, B. Cardiolipin oxidation sets cytochrome c free. Nat.Chem. Biol. 2005, 1, 188-189. 25. Shi, Y. A structural view of mitochondria-mediated apoptosis. Nat. Struct. Biol. 2001, 8, 394-401.
409
26. Zhang, H.; Huang, Q.; Ke, N.; Matsuyama, S.; Hammock, B.; Godzik, A.; Reed,
410
J.C. Drosophila pro-apoptotic Bcl-2/Bax homologue reveals evolutionary
411
conservation of cell death mechanisms. J. Biol. Chem. 2000, 274, 27303-27306.
412
27. Scorrano, L.; Korsmeyer, S.J. Mechanisms of cytochrome c release by
413
proapoptotic BCL-2 family members. Biochem. Biophys. Res. Commun. 2003,
414
304, 437-444.
415
28. Park, M.T.; Kim, M.J.; Kang, Y.H.; Choi, S.Y.; Lee, J.H.; Choi, J.A.; Kang, C.M.;
416
Cho, C.K.; Kang, S.; Bae, S.; Lee, Y.S.; Chung, H.Y.; Lee, S.J. Phytosphingosine
417
in combination with ionizing radiation enhances apoptotic cell death in 19
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
418
radiation-resistant cancer cells through ROS-dependent and -independent AIF
419
release. Blood. 2005.105.1724-1733.
420
29. Rajah, T.; Chow, S.C. The inhibition of human T cell proliferation by the caspase
421
inhibitor z-VAD-FMK is mediated through oxidative stress. Toxicol. Appl.
422
Pharmacol. 2014. 278. 100–106.
423 424 425
Figure 1. Cytochrome C immunostaining of sea cucumber at the epidermis edge of
426
body wall tissues before (a) and after UV exposure for half hour and held 20 C°
427
for 0 hour (b), 1 hour (c), 2 hour (d), 4 hour (e), 6 hour (f) with magnification of
428
200X.
429
Figure 2. Cytochrome C immunostaining of sea cucumber before (a) and after UV
430
exposure for half hour (b-f) at the inner of body wall tissues with magnification
431
of 400X. Cytochrome C immunostaining of sea cucumber body wall tissue at 0
432
hour (b), 1 hour (c), 2 hour (d), 4 hour (e), 6 hour (f) after UV exposure for half
433
hour.
434
Figure 3. Depolarization of mitochondrial transmembrane potential in coelomic fluid
435
cells. Sea cucumbers exposed to UV light for half hour and then left in dark at
436
20 °C for 0 to 6 hours. The coelomic fluid cells were isolated and stained with
437
JC-1, followed by analysis with a flow cytometer. The ratio of red to green
438
fluorescence intensity was presented as means ± SD values of three experiments
439
in triplicate (A). Values in the same dose of different groups with different letters
20
ACS Paragon Plus Environment
Page 20 of 43
Page 21 of 43
Journal of Agricultural and Food Chemistry
440
(a and b) are significantly different at p < 0.01; Intracellular JC-1 accumulation
441
was observed by fluorescent microscopy (10×) (B).
442
Figure 4. ROS production in the coelomic fluid cells of the sea cucumbers after UV
443
exposure. Sea cucumbers exposed to UV for half hour and then left in dark at
444
20 °C for 0 to 6 hours. The coelomic fluid cells were subsequently extracted and
445
stained by DCFH-DA. (A) Laser confocal microscopy image of coelomic fluid
446
cells stained with ROS detecting probes. (B) Quantification of fluorescence
447
intensity using the Image J software. Values in the same dose of different groups
448
with different letters (a and c) are significantly different at p < 0.01.
449
Figure 5. The sea cucumbers were exposed to UV for half hour and left in room
450
temperature for 0, 1, 2, 4 and 6 hours, then the coelomic fluid cells were isolated
451
immediately for western blotting analysis. (A), The expression of Bax and Bcl-2
452
after UV exposure. COX IV is used as internal control for mitochondrial fraction
453
and β-actin for cytosolic fraction. (B and C), The densitometric analysis of the
454
blots for Bax and Bcl-2 in mitochondria. The quantification result of Bax and
455
Bcl-2 is presented as Bax/Bcl2 ratio (D). Values in the same dose of different
456
groups with different letters (a and b) are significantly different at p < 0.01.
457
Figure 6. Caspase-3 activity changes in sea cucumber intestinal cells (A) and
458
coelomic fluid cells (B) after sea cucumbers exposed to UV radiation.
459
UV+Z-VAD-FMK group sea cucumbers were injected with pan-caspase inhibitor
460
Z-VAD-FMK from cloacal aperture followed by UV exposure as in UV group.
461
Normal group sea cucumbers were placed at same environment for 30 min and 21
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
462
then left in dark at 20 °C. Results are expressed as n-fold increase in caspase
463
activity compared to the controls. Values in the same dose of different groups
464
with different letters (a to f) are significantly different at p < 0.01.
465
Figure 7. Caspase-9 activity changes in sea cucumber intestinal cells (A) and
466
coelomic fluid cells (B) after sea cucumbers exposed to UV radiation.
467
UV+Z-VAD-FMK group sea cucumbers were injected with pan-caspase inhibitor
468
Z-VAD-FMK from cloacal aperture followed by UV exposure as in UV group.
469
Normal group sea cucumbers were placed at same environment for 30 min and
470
then left in dark at 20 °C. Results are expressed as n-fold increase in caspase
471
activity compared to the controls. Values in the same dose of different groups
472
with different letters (a to f) are significantly different at p < 0.01.
473
Figure 8. The morphological change of sea cucumber melting. UV group sea
474
cucumbers were induced by UV irradiated in 0.056mw/cm2 for 30 min, then left
475
in dark at 20 °C. UV+Z-VAD-FMK group sea cucumbers were injected with
476
pan-caspase inhibitor Z-VAD-FMK from cloacal aperture followed by UV
477
exposure as in UV group. Normal group sea cucumbers were placed at same
478
environment for 30 min and then left in dark at 20 °C.
22
ACS Paragon Plus Environment
Page 22 of 43
Page 23 of 43
Journal of Agricultural and Food Chemistry
479
Figure 1
480 a
b
c
d
e
f
481 482 Cyto c
483 484 485 486 487
Cyto c
488 489 490 491 492 493 494 495 496 497 498 499 500 501 23
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
502
Page 24 of 43
Figure 2
503
a
b
c
d
e
f
504 505 506
Cyto c
507 508 509 Cyto c
510 511
24
ACS Paragon Plus Environment
Page 25 of 43
512
Journal of Agricultural and Food Chemistry
Figure 3
A
B
Fresh
0h
1h
2h
4h
6h
Green
Red
Green
Red
513
25
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
514
Figure 4 A
B
Fresh
0h
2h
4h
1h
6h
515 516
26
ACS Paragon Plus Environment
Page 26 of 43
Page 27 of 43
Journal of Agricultural and Food Chemistry
517 518
Figure 5 A
519 520
fresh
0h
1h
2h
4h
6h
521 β-actin 522 523 524 Bax (Cyt.)525 526 Bax (Mit.)527 528 529 530 Bcl-2 (Mit.) 531 532 COX IV533 534 535
B
C
D
536 537 538 539 540
27
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
541 542
Figure 6
A
543 544
B
545
28
ACS Paragon Plus Environment
Page 28 of 43
Page 29 of 43
Journal of Agricultural and Food Chemistry
546
Figure 7
A
547 548
B
549
29
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
550
Page 30 of 43
Figure 8 Fresh
2h
6h
20h
47h
Normal
UV+Z-VAD-FMK
Body wall melting
UV
551
30
ACS Paragon Plus Environment
Page 31 of 43
552 553
Journal of Agricultural and Food Chemistry
TOC Graphic Inactivation Caspase 9↓
Caspase 3↓
UV
ROS ↑
Pan-caspase Inhibitor
Bcl-2/Bax↓
∆Ψm↓
Mitochondria
Melting Apoptosis UV
Cyto c↑
Activation Caspase 9↑
554 555
31
ACS Paragon Plus Environment
Caspase 3↑
20 h later
Journal of Agricultural and Food Chemistry
Graphics for Manuscript
1 2 3
Figure 1. Cytochrome C immunostaining of sea cucumber at the epidermis edge of
4
body wall tissues before (a) and after UV exposure for half hour and held 20 C°
5
for 0 hour (b), 1 hour (c), 2 hour (d), 4 hour (e), 6 hour (f) with magnification of
6
200X.
7
Figure 2. Cytochrome C immunostaining of sea cucumber before (a) and after UV
8
exposure for half hour (b-f) at the inner of body wall tissues with magnification
9
of 400X. Cytochrome C immunostaining of sea cucumber body wall tissue at 0
10
hour (b), 1 hour (c), 2 hour (d), 4 hour (e), 6 hour (f) after UV exposure for half
11
hour.
12
Figure 3. Depolarization of mitochondrial transmembrane potential in coelomic fluid
13
cells. Sea cucumbers exposed to UV light for half hour and then left in dark at
14
20 °C for 0 to 6 hours. The coelomic fluid cells were isolated and stained with
15
JC-1, followed by analysis with a flow cytometer. The ratio of red to green
16
fluorescence intensity was presented as means ± SD values of three experiments
17
in triplicate (A). Values in the same dose of different groups with different letters
18
(a and b) are significantly different at p < 0.01; Intracellular JC-1 accumulation
19
was observed by fluorescent microscopy (10×) (B).
20
Figure 4. ROS production in the coelomic fluid cells of the sea cucumbers after UV
21
exposure. Sea cucumbers exposed to UV for half hour and then left in dark at
22
20 °C for 0 to 6 hours. The coelomic fluid cells were subsequently extracted and
23
stained by DCFH-DA. (A) Laser confocal microscopy image of coelomic fluid 1
ACS Paragon Plus Environment
Page 32 of 43
Page 33 of 43
Journal of Agricultural and Food Chemistry
24
cells stained with ROS detecting probes. (B) Quantification of fluorescence
25
intensity using the Image J software. Values in the same dose of different groups
26
with different letters (a and c) are significantly different at p < 0.01.
27
Figure 5. The sea cucumbers were exposed to UV for half hour and left in room
28
temperature for 0, 1, 2, 4 and 6 hours, then the coelomic fluid cells were isolated
29
immediately for western blotting analysis. (A), The expression of Bax and Bcl-2
30
after UV exposure. COX IV is used as internal control for mitochondrial fraction
31
and β-actin for cytosolic fraction. (B and C), The densitometric analysis of the
32
blots for Bax and Bcl-2 in mitochondria. The quantification result of Bax and
33
Bcl-2 is presented as Bax/Bcl2 ratio (D). Values in the same dose of different
34
groups with different letters (a and b) are significantly different at p < 0.01.
35
Figure 6. Caspase-3 activity changes in sea cucumber intestinal cells (A) and
36
coelomic fluid cells (B) after sea cucumbers exposed to UV radiation.
37
UV+Z-VAD-FMK group sea cucumbers were injected with pan-caspase inhibitor
38
Z-VAD-FMK from cloacal aperture followed by UV exposure as in UV group.
39
Normal group sea cucumbers were placed at same environment for 30 min and
40
then left in dark at 20 °C. Results are expressed as n-fold increase in caspase
41
activity compared to the controls. Values in the same dose of different groups
42
with different letters (a to f) are significantly different at p < 0.01.
43
Figure 7. Caspase-9 activity changes in sea cucumber intestinal cells (A) and
44
coelomic fluid cells (B) after sea cucumbers exposed to UV radiation.
45
UV+Z-VAD-FMK group sea cucumbers were injected with pan-caspase inhibitor 2
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
46
Z-VAD-FMK from cloacal aperture followed by UV exposure as in UV group.
47
Normal group sea cucumbers were placed at same environment for 30 min and
48
then left in dark at 20 °C. Results are expressed as n-fold increase in caspase
49
activity compared to the controls. Values in the same dose of different groups
50
with different letters (a to f) are significantly different at p < 0.01.
51
Figure 8. The morphological change of sea cucumber melting. UV group sea
52
cucumbers were induced by UV irradiated in 0.056mw/cm2 for 30 min, then left
53
in dark at 20 °C. UV+Z-VAD-FMK group sea cucumbers were injected with
54
pan-caspase inhibitor Z-VAD-FMK from cloacal aperture followed by UV
55
exposure as in UV group. Normal group sea cucumbers were placed at same
56
environment for 30 min and then left in dark at 20 °C.
57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 3
ACS Paragon Plus Environment
Page 34 of 43
Page 35 of 43
79
Journal of Agricultural and Food Chemistry
Figure 1
80
a
b
c
d
e
f
81 82 Cyto c
83 84 85 86 87
Cyto c
88 89 90 91 92 93 94 95 96 97 98 99 100 101 4
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
102
Page 36 of 43
Figure 2
103
a
b
c
d
e
f
104 105 106
Cyto c
107 108 109 Cyto c
110 111
5
ACS Paragon Plus Environment
Page 37 of 43
112
Journal of Agricultural and Food Chemistry
Figure 3
A
B
Fresh
0h
1h
2h
4h
6h
Green
Red
Green
Red
113
6
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
114
Figure 4 A
B
Fresh
0h
2h
4h
1h
6h
115 116
7
ACS Paragon Plus Environment
Page 38 of 43
Page 39 of 43
117 118
Journal of Agricultural and Food Chemistry
Figure 5 A
119 120
fresh
0h
1h
2h
4h
6h
121 β-actin 122 123 124 Bax (Cyt.)125 126 Bax (Mit.)127 128 129 130 Bcl-2 (Mit.) 131 132 COX IV133 134 135
B
C
D
136 137 138 139 140
8
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
141 142
Figure 6
A
143 144
B
145
9
ACS Paragon Plus Environment
Page 40 of 43
Page 41 of 43
146
Journal of Agricultural and Food Chemistry
Figure 7
A
147 148
B
149
10
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
150
Page 42 of 43
Figure 8 Fresh
2h
6h
20h
47h
Normal
UV+Z-VAD-FMK
Body wall melting
UV
151
11
ACS Paragon Plus Environment
Page 43 of 43
Journal of Agricultural and Food Chemistry
TOC Graphic
Inactivation Caspase 9↓
Caspase 3↓
UV
ROS ↑
Bcl-2/Bax↓
ΔΨm↓
Pan-caspase Inhibitor Mitochondria
Melting Apoptosis UV
Cyto c↑
Activation Caspase 9↑
12
ACS Paragon Plus Environment
Caspase 3↑
20 h later