Zeaxanthin was purchased from Zelong Biotechnology (Shanxi China) and stored at −20 °C. It was dissolved in DMSO for in vitro and in vivo experiments. High-fat diet (HFD: protein 18.1%, fat 61.6%, carbohydrate 20.3%) and normal diet (ND: protein 18.3%, fat 10.2%, carbohydrate 71.5%) were purchased from Trofe Feed Technology (Jiangsu, China).
All animal experiments were performed according to the principles and guidance of the Harbin Medical University Committee on the Use and Care of Animals. 15 Eight-week-old C57BL/6 male mice (20-25 g body weight) were used in this study and housed in SPF conditions (22 ± 2.0◦C, 50 ± 5% humidity, 12-h light/12-h dark cycle, free access to water and food). To establish the myocardial injury model using HFD, mice were divided into three groups: ND, HFD, and HFD + Zeaxanthin. Zeaxanthin was given by oral gavage at a dose of 100 mg/kg once a day. ND and HFD groups were gavage with vehicle only. Mice were sacrificed after 8 weeks of feeding. All animals were euthanized with CO2 and ventricular tissues were harvested and stored at -80 °C for further experiments.
H9C2 cardiomyocytes were purchased from the Cell Bank of Chinese Academy of Sciences. H9C2 cardiomyocytes were maintained in DMEM containing 10% FBS and 1% penicillin-streptomycin and cultured in a standard humidified incubator at 37 °C with 5% CO2. After starvation in serum-free medium for 12 h, cardiomyocytes were treated with zeaxanthin (60 µM) for 24 h and stimulated with H2O2 (200 µM) for 12 h. And in the H9C2 cardiomyocyte experiments, the ctrl groups were untreated as the blank control. At the end of the experimental period, all used materials were treated innocuously.
Primary CFs were isolated from the heart tissue of Kunming mice. CFs were cultured in DMEM with 10% FBS and 1% penicillin-streptomycin and then placed in an incubator maintained at 37 °C with 5% CO2 and 95% air. Before TGF-β1 treatment, CFs were cultured in a serum-free medium for 6 h. The concentration of TGF-β1 used was 10 ng/mL. CFs were stimulated for 24 h with TGF-β1 and treated with zeaxanthin for 24 h, followed by extraction of RNA or protein for further analysis. In the CF experiments, the ctrl groups were untreated as the blank control. At the end of the experimental period, all used materials were treated innocuously.
An Annexin V-FITC/PI apoptosis detection kit (Solarbio, Beijing, China) was used to check for apoptosis in H9C2 cells. After the cells were grown and subjected to the appropriate treatment, an appropriate amount of cells were collected in the logarithmic growth phase and washed twice with PBS. The harvested cells were then incubated with a buffer solution containing annexin V-FITC for 10 min, followed by treatment with PI for another 10 min at room temperature. Flow cytometry was used to measure cellular apoptosis levels.
A DCFH-DA (10 µM) probe was used to detect ROS formation in cardiomyocytes. Cells were incubated with a probe for 30 min at 37 ℃. The culture medium was discarded, and cells were fixed with 4% paraformaldehyde, followed by three steps of washing with PBS. Images were acquired using a fluorescence microscope and analyzed using ImageJ.
Blood lipid measurement
Serum from animals in each group was collected and centrifuged at 3000 rpm for 10 min. The resultant supernatant was directly monitored using an automatic biochemical instrument (Rayto, Shenzhen, China). Triglyceride (TG) (C061-a), total cholesterol (TC) (C063-a), and low-density lipoprotein (LDL) (C062-b) standards were obtained from Huili Biotechnology (Changchun, China).
H&E and Masson staining
The ventricular tissues of 3 mice in each group were taken out for experiment. Myocardial tissues were fixed with 4% paraformaldehyde at 4 °C for 24 h. Samples were dehydrated to transparency, and paraffin embedding was performed. The embedded tissue was cut into 5-μm thick slices using a paraffin slicer and fixed on an adhesive slide. Sections were then stained using a Hematoxylin-Eosin (H&E) staining kit (Solarbio, Shanghai, China). Masson’s trichrome staining kit (Solarbio, Shanghai, China) was used to examine collagen deposition in myocardial tissues.
The ventricular tissues of 3 mice in each group were taken out for experiment. The paraffin-embedded myocardial tissue was sectioned into slices of 5-μm thickness. Sections were dewaxed and rehydrated, and permeabilized by treatment with 0.1% Triton X-100 (4 μL Triton X-100 and 0.1 g BSA in 1 mL PBS) for 1 h. Subsequently, the permeabilized sections were blocked with 50 % normal goat serum at 37 ℃ for 1 h. Then, 50 μL of rabbit anti-Bax primary antibody (1:100, Abcam) was added to each tissue section and incubated overnight at 4 ℃. The primary antibody was recovered the next day. Tissue sections were washed with PBS, followed by incubation with a goat anti-rabbit secondary antibody labeled with FITC (1:500, Alexa Fluor 594, Life Technology) at room temperature for 1 h. Nuclei were stained with DAPI for 5 min. Immunofluorescence images were acquired in a fluorescence microscope.
H9C2 cells were plated in 96-well plates, and 200 μL of culture medium was added to each well. After the appropriate treatment, 20 μL of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) solution (5 mg/ml, Beyotime Biotechnology, China) was added to each well and cells were incubated at 37 °C for 4 h. Then, 150 μL of dimethyl sulfoxide was added to each well and incubated for 15 min. Absorbance was measured at a wavelength of 490 nm using a fluorescence microplate reader.
CCK-8 viability assay
Cell Counting Kit-8 (CCK-8) was used to assess the optimal time point for zeaxanthin treatment in H9C2 cells. H9C2 cells were seeded into 96-well plates. After pretreatment with 60 µM zeaxanthin for 0, 12, 24, and 48 h, H2O2 (200 μM) was added for 4 h to induce cardiomyocyte hypoxia. Then, the culture medium was replaced with CCK-8 solution. Absorbance was measured at 450 nm using a fluorescence microplate reader.
TUNEL apoptosis assay
H9C2 cells were grown to confluence after treatment and washed thrice with PBS. Cells were then treated with TUNEL solution, according to the manufacturer’s instructions. TUNEL apoptosis images were obtained using an Olympus microscope. The ratio of apoptosis-positive cell nuclei/total cell nuclei represents the rate of cardiomyocyte apoptosis. Cellular apoptosis events were quantified using the Image J software.
Total proteins from cells or tissues were extracted and lyzed with 40-60 μL of RIPA buffer (Beyotime Biotechnology, Jiangsu, China) containing protease inhibitors. Protein samples were separated on an SDS-PAGE gel and transferred to a pure PVDF membrane (Merck, Germany). The membrane was probed with primary antibodies overnight and incubated at 4 ℃. The primary antibodies used were GADPH antibody (1:5000, No. 66535-1-Ig), Fibronectin-1 (FN1) antibody (1:500, No. 15613-1-AP), Collagen-I (Col-I) antibody (1:500, No. 14695-1-AP), Bcl2-associated X protein (Bax) antibody (1:500, No. 50599-2-Ig), B-cell lymphoma-2 (Bcl2) antibody (1:500, No. 12789-1-AP), cleaved-caspase-3 antibody (1:500, No. 19677-1-AP), p-NF-κB antibody (1:500, No. 66535-1-Ig), p-p38MAPK antibody (1:500, No. 66234-1-Ig)), TGF-β1 antibody (1:500, No. 21898-1-AP), and Smad2/3 antibody (1:500, No. 12570-1-AP) (all from Proteintech, Rosemont, IL, USA). After washing with PBS, the membranes were further incubated with HRP-conjugated secondary antibodies at room temperature for 50 min. The Image Lab software was used to detect protein expression levels.
RNA extraction and qRT-PCR assay
Total RNA was extracted from cells using the TRIzol reagent. After TRIzol was used to lyze the cells, chloroform was added and cells were thoroughly vortexed to ensure stratification of the aqueous and organic phases. The upper aqueous phase was separated after centrifugation for 10 min and mixed gently with the same amount of isopropanol for 10 min. Afterwards, the samples were again centrifuged for 10 min at 4 ℃ at 12000 rpm. The supernatant was discarded and the extracted RNA was washed with ethanol. Finally, 20 μL of DEPC-treated water was added to dissolve the RNA.
A High-Capacity cDNA Reverse Transcription Kit was used to reverse transcribe the isolated RNA to cDNA. cDNA was used to detect the relative expression of mRNA on a QuantStudio 6 real-time PCR system (Applied Biosystems, Foster City, CA, USA) in the presence of 50X ROX reference dye 2 (Vazyme Biotech, Jiangsu, China). The relative mRNA expression level was calculated based on the Ct values and normalized to GAPDH levels. The primer sequences used were as follows: GAPDH (Forward:5′-ATGGGTGTGAACCACGAGA-3′, Reverse:5′-CAGGGATGATGTTCTGGGCA-3′);
CAATGCTCCCAGA-3′); Col-I (Forward:5′AAGAAGACATCCCTGAAGTCA-3′, Reverse:5′-TTGTGGCAGATACAGATCAAG-3′); TGF-β1(Forward:5′-TT
Matrix-metalloproteinase-9 (MMP-9) (Forward:5′-AAAGGCCATTCGAACACCAC-3′, Reverse:5′-GGATGACAATGTCCGCTTCG-3′); Connective tissue growth factor (CTGF) (Forward:5′-GGAAGACACATTTGGCCCAG-3′, Reverse:5′-TAGGTGTCC
All data were analyzed using GraphPad Prism 8.0 and expressed as mean ± SEM. The significance level was calculated by Student's t-test between two groups, and one-way analysis of variance (ANOVA) was used for comparisons across multiple conditions. Bonferroni correction was used to evaluate whether there were differences in each group. A value of p<0.05 was considered as statistically significant.