J Cancer Prev 2020; 25(4): 244-251
Published online December 30, 2020
© Korean Society of Cancer Prevention
Department of Food and Nutrition, BK21 FOUR, College of Human Ecology, Yonsei University, Seoul, Korea
Astaxanthin is the ketocarotenoid responsible for the red-orange pigmentation observed in aquatic organisms such as salmons and lobsters . Astaxanthin has attracted substantial interest due to its anti-inflammatory and anti-cancer effects . Our previous study has revealed that astaxanthin protects gastric epithelial cells from the harmful effects of
Male C57BL/6 mice (6 weeks of age), purchased from Orient Bio Inc. (Seongnam, Korea), were maintained in a controlled room in the animal facility of Yonsei University College of Medicine under the following conditions: temperature, 23.0°C ± 3.0°C; humidity, 50% ± 10%; 12-hour light/dark cycle. Animals were housed in polypropylene cages furnished with hardwood chip bedding (5 animals per cage) and had free access to food and water. All experimental procedures were approved by the Institutional Review Board (IRB) at Animal Ethical and Experimental Committee of Yonsei University College of Medicine, Seoul, Korea (IRB No. IACUC No. 2018-0127).
The mice were randomly assigned to the following 3 groups (n = 15 per group): (1) non-infected control (None) group comprising mice administered sterile PBS and fed standard chow (AIN-76A; Research Diets, New Brunswick, NJ, USA); (2) infected control (
Mice were weighed weekly during the study period, while food intake was evaluated three times a week. Mice were killed by carbon dioxide inhalation at the end of the 7th week. Upon death, gastric mucosal tissues were collected. One half of the tissues were subjected to histological analysis. The remaining tissues were homogenized in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 25 mM Tris pH 7.4) and used for the determination of LPO abundance, MPO activity, levels of total protein (using the Bradford assay; Bio-Rad Laboratories, Hercules, CA, USA), and protein and mRNA levels of IFN-γ, c-myc, and cyclin D1.
For evaluating the oxidative damage, the level of LPO was assessed by measuring the gastric mucosal concentration of malondialdehyde according to the method of Ohkawa et al. . Results were expressed as nmol/mg protein. For the determination of the gastric mucosal accumulation of neutrophilic components, the activity of MPO, a peroxidase enzyme abundantly expressed in neutrophils, was quantified according to the modified method of Krawisz et al.  and expressed in U/mg protein. One unit of MPO indicates the enzymatic activity needed to reduce 1 μmol of hydrogen peroxide per minute at 25°C.
The mRNA levels of IFN-γ, c-myc, and cyclin D1 were assessed using real-time PCR. Complementary DNA (cDNA) was generated from total RNA by reverse transcription using random hexamers and MuLV reverse transcriptase (Promega; Madison, WI, USA) using the following protocol: 23°C for 10 minutes, 37°C for 60 minutes, and 95°C for 5 minutes. The cDNA was amplified with the specific primers and FAM-BHQ-labeled probes described in Table 1 under the following conditions: 45 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 30 seconds. In the first cycle, the 95°C step was extended to 3 minutes. GAPDH was used as the reference gene for data normalization.
Western blot analysis was conducted as previously described . After electro-blotting, nitrocellulose membranes were probed using antibodies for c-myc (sc-40), cyclin D1 (sc-8396), IFN-γ (sc-52673), and β-actin (sc-47778; all purchased from Santa Cruz Biotechnology, Dallas, TX, USA) diluted in TBS-Tween solution containing 2% dry milk, and incubated overnight at 4°C. After washing three times with TBS-Tween, the primary antibodies were detected using horseradish peroxidase-conjugated anti-mouse secondary antibody and visualized with the enhanced chemiluminescence detection system (Santa Cruz Biotechnology). β-Actin was used as a loading control. Results were expressed as the percentage density ratio of each protein to β-actin.
Half of the stomach tissues dissected from mice were fixed in freshly prepared 10% neutral-buffered formalin, embedded in paraffin, cut into 4 μm slices, stained with hematoxylin and eosin, and subjected to morphological observation under an optical microscope. To evaluate pathological changes, microscopic images (200 ×) were examined by a single investigator who was blind to the group assignments.
All values were expressed as means ± SEM. One-way ANOVA was carried out to assess statistical significance, followed by Newman-Keul’s post-hoc test. Differences with
Body weight gain in the infected control group (
The level of LPO reflects the oxidative stress to lipids, and, by extension, is indicative of the oxidative injury to cells. On the other hand, MPO activity is used as an index for
The gastric mucosa of the uninfected (None) group displayed normal morphology, i.e., lack of inflammation or hyperplasia (Fig. 5A). In contrast, the infected control (
Oxidative stress in the gastric mucosa resulting from
Increased expression of IFN-γ after
Several studies have shown that
We have previously demonstrated that activation of NF-κB and AP-1 mediates hyperproliferation by inducing β-catenin and c-myc in
Jin et al.  and Luo et al.  demonstrated that 6- to 7-week-treatment of
In conclusion, astaxanthin may suppress oxidative gastric tissue damage and the expression of the inflammatory cytokine, IFN-γ, and the oncogenes, c-myc and cyclin D1, in
This study was supported financially by a grant from the National Research Foundation (NRF) of Korea, which is funded by the Korean Government (NRF-2018R1A2B2005575). Authors are grateful to Professor Joo Young Kim (Dept. Of Pharmacology, Yonsei University College of Medicine, Seoul, Republic of Korea) for her kind advice and technical assistance in animal experimentation.
No potential conflicts of interest were disclosed.
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