Article pubs.acs.org/JAFC
Proteomic Analysis of the Effect of DHA-Phospholipids from Large Yellow Croaker Roe on Hyperlipidemic Mice Peng Liang,† Min Zhang,† Wenjian Cheng,† Wenxiong Lin,‡ and Lijiao Chen*,† †
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China Life Sciences College, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China
‡
S Supporting Information *
ABSTRACT: Previously, we found that phospholipids derived from large yellow croaker (Pseudosciaena crocea) roe had a higher level of docosahexaenoic acid (DHA−PL), which had beneficial effects on lipid metabolism. However, the mechanism by which DHA−PL from P. crocea roe exerts these effects has not yet been illuminated. Herein, we investigated the underlying molecular action of DHA−PL by examining changes in liver protein expression in control, hyperlipidemic, and DHA−PL-treated mice. A total of 16 proteins, 9 up-regulated and 7 down-regulated, were identified and classified into several metabolic pathways, such as fat digestion and absorption, peroxisome proliferator activated receptor (PPAR) signaling, and antigen processing and presentation; the largest functional class found was that of fat digestion and absorption. We revealed Apoa1 to be a biomarker of DHA−PL effects on hyperlipidemic mice by DHA−PL diet. These results not only improve our current understanding of hyperlipidemic regulation by DHA−PL, but also suggest that DHA−PL should be applied as a beneficial food additive. KEYWORDS: large yellow croaker, fish roe, phospholipid, lipid metabolism, proteomic
■
entire collection of proteins in a sample. Kalupahana et al.13 conducted proteomic analysis and identified several novel proteins in C57BL/6J male mice treated with EPA or arachidonic acid (ARA). The results showed that high saturated fat diets incorporated with EPA was associated with reduced lipogenesis and elevated the markers of fatty acid oxidation. Moreover, the mechanism by which fish oil regulates lipid metabolism has been shown to involve several unclear transcription factors [e.g., PPAR−α, −β/δ, and −γ] and metabolic pathways, such as fatty acid metabolism, lipid oxidation, and amino acid metabolism, based on two-dimensional (2-DE) proteomics techniques.14 These initial comprehensive and systematic studies of the fish oil on mitochondrial metabolism in mice have greatly promoted our understanding of the regulatory mechanisms of lipid metabolism when compared to the molecular biology discoveries. Previously, we investigated the effect of DHA−PL extracted from P. crocea roe on the reduction of blood lipid content in mice. The results showed that DHA−PL was capable of preventing body weight gain. Serum indices showed that middle- and high-dose DHA−PL groups had significantly lower cholesterol, triacylglycerol, low-density lipoprotein cholesterol (LDL), AI1, and AI2 levels compared to the model group, but increased high-density lipoprotein cholesterol (HDL) levels (P < 0.05). Based on the results of pathological liver tissue histology, low-dose DHA−PL had an insignificant effect on hepatocyte levels (P > 0.05).15 These results demonstrated that DHA−PL is able to prevent fatty degeneration of liver tissues
INTRODUCTION Large yellow croaker (Pseudosciaena crocea) is a common marine fish widely cultivated in China for its delicious taste and commercial value.1,2 However, utilization of P. crocea roe remains problematic due to its poor taste and sensory quality. Generally, fish roe has a lot of eicosapentaenoic acid (EPA)/ docosahexaenoic acid (DHA), which are primarily incorporated into phospholipids (PLs). Recently, these polyunsaturated fatty acids (PUFAs) have received much attention because of their health benefits. Some reports have shown that unsaturated PLs provide more advantages in the prevention and treatment of cardiovascular, inflammatory, autoimmune disorders and neurological functions.3−5 Previous reports have demonstrated that fish roe and krill contain high amounts of DHA−PL and EPA−PL.6−9 Hayashi et al.10 studied the ingestion of n−3 PUFA−enriched PC from salmon roe and found it could significantly accelerate lipid metabolism in humans with chronic liver disease. In addition, EPA−PL extracted from Cucumaria f rondosa was also discovered to have a positive effect on lipid metabolism through stimulating peroxisome proliferator−activated receptors (PPARα).11 The finding indicated that EPA−PL supplementation obviously accelerated the expression of genes related to β-oxidation in the liver and epididymal adipose tissue, such as UCP2, Ehhadh, and Acaa1. Those results enhanced our understanding of the prevention of dyslipidemia in mice with EPA−PL diet, which was obtained through molecular biology methods. It should be noted that genes under regulation of lipid metabolism are more complex.12 Further research should also be undertaken to clarify the specific mechanism. However, the mechanisms by which DHA−PL-diet intake exerts its effects are still unclear. Proteomics techniques are usually performed to explore a specific biological function or biochemistry via analysis of the © 2017 American Chemical Society
Received: Revised: Accepted: Published: 5107
February 2, 2017 April 5, 2017 April 24, 2017 April 25, 2017 DOI: 10.1021/acs.jafc.7b00478 J. Agric. Food Chem. 2017, 65, 5107−5113
Article
Journal of Agricultural and Food Chemistry
Table 1. Identification of Differentially Expressed Proteins in Mice Liver from Control, Hyperlipidemic, and DHA−PL Treated Groups gel spot
protein change
gene name
accession no.
mass
score
matches
sequences
calculated pI
fold change
701 1102 2102 2402 2404 2507 2603 2713 2714 3106 4001 4002 4003 4207 5003 5103
up up up up up up down up up up down down down down down down
Calr Pebp1 Apoa1 Bckdhb Eno1 Selenbp2 Krt8 Pdia3 Suox Prdx6 Fabp2 Ttr 54039306 Ech1 Hint1 Ca3
gi|6680836|NP_031617.1 gi|84794552|NP_061346.2 gi|160333304|NP_033822.2 gi|806776659|NP_001292864.1 gi|70794816|NP_001020559.1 gi|148840438|Q63836.2 gi|114145561|NP_112447.2 gi|200397|AAA39944.1 gi|74024924|NP_776094.2 gi|3219774|O08709.3 gi|6679737|NP_032006.1 gi|7305599|NP_038725.1 gi|54039306|P63324.2 gi|7949037|NP_058052.1 gi|33468857|NP_032274.1 gi|31982861|NP_031632.2
48136 20988 30597 43651 47453 53147 54531 57099 61231 24969 15117 15880 14853 36437 13882 29633
221 420 208 344 388 768 454 444 193 470 247 537 135 627 280 328
7 8 6 3 5 9 9 9 4 8 5 7 3 7 4 7
7 7 6 3 4 9 8 9 4 8 5 6 3 7 3 7
4.33 5.19 5.51 6.27 6.37 5.78 5.70 5.88 6.07 5.71 6.62 5.77 6.82 7.6 6.36 6.89
1.48 1.66 2.06 2.33 24.54 2.09 0.12 7.34 2.70 2.49 0.28 0.09 0.22 0.73 0.42 0.30
Protein Sample Preparation. After the 4 week feeding period, mice were euthanized. Their livers were collected and rinsed in cold phosphate-buffered saline three times in order to remove blood.19 Approximately 0.3 g of proteins was mixed with rehydration buffer [4% (w/v) CHAPS, 7 M urea, 65 mM 1,4-dithiothreitol (DTT), 2% IPG buffer, 2 M thiourea]. The homogenate was treated by a ultrasonication for 30 s. The supernatant was obtained by centrifugation at 120 000 × g for 15 min and analyzed for protein content using the Bradford method20 and stored at −80 °C until used. 2-DE. The 2-DE method was performed in accordance with the description by O’Farrell.21 Isoelectric focusing (IEF) was accomplished using an IPGphor IEF System (Amersham−Pharmacia Biotech) as the first dimension of protein separation. Approximately 1500 mg of protein from each mouse was mixed with rehydration buffer (450 μL total volume), before being loaded onto fixed pH gradient strips (24 cm, pH 3−10) rehydrated for 12 h at 20 °C and 30 V (constant), then separated at 20 °C in a stepwise manner: 200 V-1 h, 500 V-1 h, 1000 V-1 h, gradient 8000 V-0.5 h, and finally 8000 V until the total V h reached 42 000 V h. After IEF, all the strips were rinsed in balanced buffer I [50 mM Tris−HCl, 30% (v/v) glycerol, 1% DTT, 2% (w/v) sodium dodecyl sulfate (SDS), 6 M urea] for 15 min and then in balanced buffer II [50 mM Tris−HCl, 30% (v/v) glycerol, 2.5% iodoacetamide, 2% (w/v) SDS, 6 M urea] for 15 min with moderate shaking. After balance, strips were applied to 11% (w/v) polyacrylamide gels and sealed with an agarose overlay solution [0.5% (w/v) agarose] containing SDS buffer [192 mM glycine, 25 mM Tris, and 0.1% (w/v) SDS] and a small amount of bromphenol blue. As the second dimension of protein separation, SDS−polyacrylamide gel electrophoresis was carried out in an Ettan DALT-six System (Amersham Biosciences) at 18 °C, beginning at 10 mA gel (50 V maximum) for 10 h approximately. Then, gels were stained with CBBG250 and shaken for 2 h gently, and transferred to 1% (v/v) acetic acid destain with moderate shaking for about 12 h until the spots of interest were excised.22 Each gel was run in triplicate. Gel Scanning and Analysis. Stained gels were scanned at a resolution of 300 dpi with an Image Scanner (GE Healthcare, USA). All the gels were subjected to spot discovery, volumetric detection, and spot matching using PDQuest 8.0 software. Statistical analysis of spot intensities were conducted by Student’s t-test. Differences in protein content between groups were calculated as the fold ratio. The differentially expressed spots of interest were selected at a threshold of P ≤ 0.05 and fold change ≥2 or ≤0.5. These spots were identified and recorded in Table 1. Identification of Protein by Matrix-Assisted Laser Desorption/Ionization Tandem Time-of-Flight Mass Spectrometry (MALDI−TOF/TOF) and Database Search. The selected proteins were excised from gels and rinsed with Millipore pure water three
and reduce the risk of atherosclerosis. However, little information on the effect of DHA−PL has been reported. In the current study, our aim is to use proteomic analysis to understand the effect of DHA−PL from large yellow croaker roe on lipid metabolism in hyperlipidemic mice. Using a 2-D gel electrophoresis (2-DE)-based proteomics approach, the levels of differentially expressed liver proteins extracted from control, hyperlipidemic, and DHA−PL-fed mice were analyzed. Meanwhile, to further elucidate the role of 2-DE-identified proteins in regulation of lipid metabolism, we also used bioinformatics to identify affected metabolic pathways and constituents. The results would enhance our understanding of the mechanism involved in lipid metabolism, in hyperlipidemic mice, by DHA−PL extracted from large yellow croaker roe.
■
MATERIALS AND METHODS
Preparation of DHA−PL from P. crocea Roe. The DHA−PL was extracted from P. crocea roe by solvent extraction.16,17 Briefly, 10.0 g of homogenized roe was dissolved in 30 mL of cold acetone to remove neutral lipids then filtered through filter paper. The precipitated residue was then dissolved in 95% ethyl alcohol for 30 min, and the solvent phase was collected and evaporated at 65 °C in a rotary vacuum evaporator. Cold acetone was added to the dried residue and again subjected to vacuum drying for 12 h to obtain a purified mixture of PLs. All extracted PLs were analyzed and published in our previous work,18 which demonstrated PL-enrichment of DHA [31.0% ± 0.19% (w/w) of total PLs], and phosphatidylcholine (PC) as the dominant PL class [61.06% ± 0.02% (w/w) of total PLs]. In the following work, we also studied the regulation function of antihyperlipidemia with hyperlipidemic model mice. Meanwhile, we observed that DHA−PL had significant regulation ability in vivo experiment. Animals and Diets. Thirty healthy adult male ICR mice (5 weeks old) were purchased from Fujian Medical University (Fuzhou, China). Mice were kept under normal environment with humidity of 62% ± 5% and temperature of 22 °C ± 2 °C with an interval cycle of 12 h light and 12 h dark. After a 14 d adjustment period, ten of 30 mice were randomly segregated into 3 groups and fed a control group (AIN-96G with 5% corn oil), a high fat [20%, (w/w)], and high fructose [20%, (w/w)] diet to be the hyperlipidemic group, or a high fat diet supplemented with 3% DHA−PL (DHA−PL group). After 4 weeks of feeding, the mice were sacrificed after 12 h overnight. The study protocol was authorized by the Ethics Committee of Fujian Medical University, and special care was taken to minimize suffering of mice. 5108
DOI: 10.1021/acs.jafc.7b00478 J. Agric. Food Chem. 2017, 65, 5107−5113
Article
Journal of Agricultural and Food Chemistry times, destained two times with 50 mM NH4HCO3 in acetonitrile for CBB G250 stained spots, decreased with 10 mM DTT, alkylated with iodoacetamide in NH4HCO3, separately, dried two times with acetonitrile, and digested at 37 °C overnight with modified trypsin (Promega, Madison, WI, USA).23 Then peptides were extracted, pooled according to group, and lyophilized. MALDI-TOF-TOF MS analyses were carried out using a 4800 Plus MALDI-TOF-TOF analyzer (Applied Biosystems, Foster City, CA, USA). The protein spectra was submitted and searched against the NCBInr mouse database via MASCOT software (http://www.matrixscience.com). Data Analysis. Statistical analysis of data was subjected to the analysis of variance followed by the Student’s t-test. Differences with P < 0.05 were considered statistically significant among control, hyperlipidemic, and DHA−PL groups.
■
RESULTS
Differentially Expressed Protein. The results illustrated a consistent protein expression pattern on the selected gels. 2-DE profiles demonstrated that protein spots would be reproducibly resolved in the pH 3−10 scope and had relative molecular masses of 15−120 kDa. To analyze the quantitative image analysis of the biological, the replicates revealed a total of 67 spots displayed >2-fold, or 0.5, but the dotted lines mean
