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Article

Dietary Ethanolamine Plasmalogen from Ascidian Alleviates Chronic Hepatic Injury in Mice Treated with Continuous Acetaminophen

by
Ryosuke Sogame
1,2,
Yuki Tominaga
1,
Momoka Echigoya
1,
Kiyotaka Nakagawa
3,
Michihiro Fukushima
1,
Teruo Miyazawa
2,
Mikio Kinoshita
1 and
Shinji Yamashita
1,*
1
Department of Life and Food Sciences, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
2
Food and Biotechnology Platform Promoting Project, New Industry Creation Hatchery Center (NICHe), Tohoku University, Sendai 980-8579, Japan
3
Food Function Analysis Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(11), 5968; https://doi.org/10.3390/app15115968
Submission received: 23 April 2025 / Revised: 21 May 2025 / Accepted: 23 May 2025 / Published: 26 May 2025
(This article belongs to the Special Issue Marine-Derived Bioactive Compounds and Marine Biotechnology)
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Abstract

:
Background: Ethanolamine plasmalogen (PlsEtn) is a subclass of ethanolamine glycerophospholipids (EtnGpls) and is abundantly found in some marine invertebrates, including ascidian Halocynthia roretzi. PlsEtn is reported to exhibit physiological and nutritional hepatic functions; however, the effects of dietary PlsEtn on continuous acetaminophen (APAP)-induced hepatic injury, including oxidative stress and impaired lipid metabolism, remain unclear. Herein, we investigated the dietary effects of PlsEtn from ascidian on chronic hepatic injury in APAP-treated mice. Methods: Five-week-old male ICR mice were divided into four groups (n = 12), which were treated with the respective experimental diet for two weeks and then the respective APAP-containing diet for five weeks. The results obtained after administering the PlsEtn-rich diet were compared with those obtained after the administration of a phosphatidylethanolamine (PtdEtn)-rich diet, a major subclass of hepatic EtnGpls. Results: The PlsEtn-rich diet effectively suppressed the APAP-induced decrease in body and liver weights of mice; however, this suppressive effect was not observed in mice fed a PtdEtn-rich diet. APAP administration decreased the total fatty acid content in the liver, whereas a PlsEtn-rich diet alleviated this decrease and increased the hepatic content of docosahexaenoic acid (DHA), which exhibits various hepatic functions. Moreover, dietary EtnGpl rich in PlsEtn or PtdEtn suppressed APAP-induced lipid oxidation in the liver. The protein expression results revealed that dietary EtnGpls reduced the expression of certain apoptosis-related proteins in the livers of APAP-administered mice compared to that in the APAP group. This reduction was particularly more effective in mice fed the PlsEtn-rich diet than in those on the PtdEtn-rich diet. Conclusions: Dietary EtnGpls, particularly PlsEtn, alleviated the hepatic cellular stress caused by continuous APAP consumption. These beneficial effects may depend on the subclass and may be related to DHA metabolism in the liver. The results of this study contribute to the understanding of the role of PlsEtn in maintaining liver health.

Graphical Abstract

1. Introduction

Ethanolamine glycerophospholipids (EtnGpls) are the major phospholipids present in biological membranes and can be classified into three subclasses based on differences in linkages at the sn-1 position of the glycerol moiety. Ethanolamine plasmalogen (PlsEtn) and phosphatidylethanolamine (PtdEtn) are the alkenyl and acyl forms of the EtnGpls, respectively. The ratio of PlsEtn-to-EtnGpl in the body varies depending on the tissue or organ. For example, in the human brain and nervous system, PlsEtn represents more than 60% of the total EtnGpls, whereas in the liver, it represents only 2–5% [1]. Owing to its alkenyl bond (vinyl-ether bond), PlsEtn possesses antioxidant properties, and its sn-2 position predominantly consists of polyunsaturated fatty acids (PUFA), including docosahexaenoic acid (DHA; 22:6n-3). Neurodegenerative disorders, such as Alzheimer’s disease, result in decreased levels of PlsEtn, particularly PlsEtn-bearing DHA, in the brain and blood [2,3,4]. Endogenous plasmalogens, including choline plasmalogen, protect against hepatic steatosis and steatohepatitis in mice [5].
In some marine invertebrates, such as ascidian Halocynthia roretzi and pacific oyster Crassostrea gigas, the majority of EtnGpl is present as PlsEtn [6]. Marine-derived PlsEtn exhibits various nutritional functions, including improving cognitive performance and intestinal impairments [1]. The alkenyl bond in plasmalogens is acid-labile, but plasmalogens contained in the diet are hardly degraded under stomach-like conditions (pH 1.0 at 37 °C for 1 h) because of the buffering action of the diets [7,8,9]. Ingested PlsEtn is digested into lysoPlsEtn and free fatty acid in the small intestine, and PlsEtn digests are absorbed and are subsequently resynthesized into PlsEtn preferentially with PUFA. Some ingested PlsEtn and lysoPlsEtn reach the large intestine. Therefore, ingested PlsEtn can function in both the body and intestines. PlsEtn mitigates LPS-induced damage in intestinal epithelial cells in vitro [10]. Administration of PlsEtn or lysoPlsEtn suppresses intestinal inflammatory injury in a mouse model of colitis and increases the cecal levels of short-chain fatty acids, which are produced by microbiota and enhance the intestinal barrier [11,12]. PlsEtn protects HepG2 cells, which are used as an in vitro hepatic model, from lead-induced toxicity and subsequent oxidative stress [13]. Dietary PlsEtn enriched with eicosapentaenoic acid (EPA; 20:5n-3) has been shown to mitigate atherosclerosis and hepatic steatosis in rodents subjected to a high-fat diet, and these effects are more pronounced than those observed with dietary EPA-ethyl ester [14,15]. The proposed mechanism involves the enhancement of lipid and bile acid excretion in feces through the elevated hepatic expression of cytochrome P450 (CYP) 7A1. This process is achieved by the suppression of the farnesoid X receptor activation in the liver and the modification of microbiota abundance, which has bile salt hydrolase, thereby affecting secondary bile acid production. Although PlsEtn is present in the liver at low levels, it plays significant hepatic nutritional roles, including the enhancement of lipid metabolism and antioxidant activity. These effects may be exerted directly on the liver as well as through the gut–liver axis. Furthermore, the benefits conferred by dietary PlsEtn may mitigate liver impairment induced by excessive or prolonged drug dosage.
Acetaminophen (N-acetyl-p-aminophenol, APAP) is a frequently used analgesic and antipyretic, and its overdose causes acute liver failure (ALF), chronic active hepatitis, cholestasis, and jaundice [16,17]. Approximately 90% of the administered APAP is metabolized by UDP-glucuronosyltransferase and sulfotransferase into glucuronic acid and sulfate conjugates, respectively. Moreover, 5–9% of administered APAP is metabolized into the highly reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI) by the enzymatic reaction of CYP2E1 with APAP [18]. This metabolite is considered a pivotal molecule in APAP-induced hepatotoxicity and is conjugated by glutathione (GSH). Excessive NAPQI levels deplete GSH and covalently bind to cellular proteins, resulting in organelle dysfunction, such as mitochondria dysfunction. These impairments induce oxidative stress, cell malfunctions, and subsequently, cell death, such as ferroptosis and apoptosis [19,20]. The symptoms appear as loss of appetite and body weight and can develop into liver fibrosis and further disease.
Researchers have investigated the effects of various nutritional components on APAP-induced ALF and chronic liver injury. Rodents administered continuous APAP also serve as a model for chronic drug-induced liver disease, as they exhibit liver abnormalities similar to those observed with some lipid-lowering agents [16]. Continuous APAP consumption lasting more than four weeks causes liver injury in rodents through sustained oxidative stress and impaired lipid metabolism, including a decrease in the mRNA expression of fatty acid synthase [21,22], which are the primary toxicities of APAP overdose in the liver.
In this study, we investigated the effects of dietary PlsEtn from ascidian, in comparison with PtdEtn (a predominant subclass in hepatic EtnGpls), on continuous APAP-induced liver damage. Mice were treated with continuous APAP consumption to induce oxidative stress and impaired lipid metabolism in the liver, and the effects of diets supplemented with PlsEtn were evaluated based on the levels of malondialdehyde (MDA), a marker of lipid oxidation, fatty acid content, and expression of apoptosis-related proteins in the liver.

2. Materials and Methods

2.1. EtnGpl Preparation

Freeze-dried ascidian muscle (Halocynthia roretzi; AM) was provided by Yaizu Suisankagaku Industry Co., Ltd. (Shizuoka, Japan), and porcine liver (PL) was purchased from a meatpacker in Hokkaido (Japan). Fish oil from skipjack tuna (Katsuwonus pelamis) and mixed fish oil from skipjack tuna and tuna (Thunnus) were provided by Nissui Co., Ltd. (Tokyo, Japan). EtnGpls purified from AM and PL were prepared as described in a previous study [23]. Briefly, freeze-dried samples were treated with cold acetone to remove neutral lipids, and crude polar lipids were extracted from the treated samples using the Folch method. Subsequently, the EtnGpls were purified using two silica column chromatography systems. The first stepwise solution comprised chloroform/methanol, and the second solution comprised chloroform/methanol/28% ammonia solution. The purified EtnGpls were successfully one-spot detected via the ninhydrin-reagent and 50% sulfate using thin-layer chromatography. The lipids used in the experimental diets were treated with 10% acetyl chloride in methanol solution at 100 °C for 2 h to generate fatty acid-methyl esters and dimethylacetals from the acyl and alkenyl chains, respectively. The derivates extracted using n-hexane were identified using gas chromatography–mass spectrometry (GC-2030 and GCMS-QP2020NX instruments, Shimadzu, Kyoto, Japan) with a CP-Sil 88 GC column (50 m × i.d 0.20 mm, df 0.25 μm, Agilent, CA, USA) and were subsequently quantified using a gas chromatography–flame ionization detector (GC-2010, Shimadzu) with nonadecanoic acid as the internal standard [24,25]. The PlsEtn ratio in EtnGpl was obtained by doubling the ratio of the total alkenyl chain to the total fatty chain of EtnGpl.

2.2. Animals

Four-week-old male ICR mice were obtained from CLEA Japan, Inc. (Tokyo, Japan). They were housed in pathogen-free conditions in microisolator cages at 22 ± 1 °C with a 12 h light/dark cycle. The mice had access to a CE-2 diet (CLEA Japan, Inc., Tokyo, Japan) and water ad libitum for one week. After one week of acclimatization, the mice were randomized into four experimental groups (n = 12): control (Ctrl), APAP, AM + APAP, and PL + APAP. For the experimental diets, the AIN-93G diet was slightly modified by reducing the soy oil content to 6% and supplementing with 1% fish oil (10 g/kg diet) to form the basal diet. Fish oil from skipjack tuna and mixed fish oil were used in the diets for the first four weeks and final three weeks, respectively, owing to changes in the product specifications. AM and PL diets were prepared by adding 0.1% purified AM- and PL-derived EtnGpls (1.0 g/kg diet), respectively, to the basal diet. In previous studies on phospholipid nutritional function in rodents, the range of phospholipids contained in the diets was 0.25–10 g/kg, and the most frequent amount used was 1.0 g/kg [1,14,26]. Subsequently, 1% fish oil was added to all three experimental diets to obtain similar n-3-to-n-6 ratios because the intake of fish oil with a high n-3 PUFA content enhances the impairment of chronic hepatic injury induced by the continuous oral administration of APAP [27]. The Ctrl group was fed a basal diet for seven weeks, and the APAP group was fed the basal and APAP-mixed basal (5 g/kg diet; Sigma-Aldrich Co., St. Louis, MO, USA) diets for two and five weeks, respectively. The AM + APAP (PL + APAP) group was fed the AM (PL) diet for two weeks and the APAP-mixed AM (PL) diet for five weeks. Finally, the mice were euthanized with a modified mixture of three anesthetic agents: domitor, dormicum, and vetorphale (0.3 mg, 4 mg, and 5 mg/kg body weight, respectively). The animal study protocol was approved by the Animal Care and Experiment Committee of the Obihiro University of Agriculture and Veterinary Medicine (License No.: 20-172).

2.3. Blood and Liver Preparation

Fresh blood samples were collected from the right ventricles of mouse specimens in tubes containing EDTA-2Na and subjected to centrifugal separation twice (15 min, 1000× g, 4 °C) for plasma preparation. Plasma was stored at −80 °C, and the liver was resected, washed, and stored at −80 °C. Frozen liver was homogenized with cold phosphate-buffered saline containing 1 mM EDTA using a digital homogenizer (AS ONE Corporation, Osaka, Japan) in an ice bath and stored at −80 °C.

2.4. Analysis of Liver Injury Parameters

Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured using the transaminase CII Test Wako kit (Fujifilm Wako Pure Chemical Corp., Osaka, Japan), according to the manufacturer’s protocol. Plasma TNF-α levels were quantified using mouse TNF-α enzyme-linked immunosorbent assay kits (FUJIFILM Wako Shibayagi Corp., Gunma, Japan) according to the manufacturer’s protocol. Hepatic MDA levels were measured using a thiobarbituric acid-reactive substance assay according to a previous report [28]. Hepatic GSH levels were analyzed using a GSSG/GSH Quantification kit (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer’s protocol. The hepatic fatty acid content was analyzed after lipid extraction using the Folch method, as described above. The protein content of the samples was measured using a DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA).

2.5. Apoptosis Array Assay

Oxidative stress in the liver induces apoptosis, which can lead to fibrosis [19]. We analyzed the expression of 21 apoptosis-related proteins in mouse livers. After freezing and thawing the liver homogenates, the levels of apoptosis-related proteins in the liver were determined using a mouse apoptosis array kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. The apoptosis-related proteins detected included B-cell lymphoma 2 (Bcl-2), Bcl/leukemia x (Bcl-x), catalase, claspin, MCL-1, p27 cyclin-dependent kinase 4 inhibitor 1B (p27/Kip1), X-linked inhibitor of apoptosis, Bcl-xL/Bcl-2-associated death promoter (Bad), cytochrome c (cyt c), second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (SMAC/Diablo), fibroblast-associated (Fas) receptor, TNF receptor 1 (TNF R1), TNF-related apoptosis-inducing ligand receptor 2 (TRAIL R2), cleaved caspase-3, p53, hypoxia-inducible transcription factor (HIF)-1α, heme oxygenase (HO)-1, HO-2, heat shock protein (HSP)27, HSP60, and HSP70.

2.6. Statistical Analysis

The Ctrl and APAP groups were analyzed using Student’s t-test. The three groups administered APAP were analyzed via the one-way analysis of variance using Tukey’s post hoc test. Differences were considered statistically significant at p < 0.05. All data were analyzed using BellCurve for Excel ver. 4.08 (Social Survey Research Information Co., Ltd., Tokyo, Japan) and are expressed as the mean ± standard error of the mean.

3. Results

3.1. Fatty Chain Composition in Experimental Diets

AM-derived EtnGpl comprised 84.2 mol% PlsEtn and 15.8 mol% PtdEtn in EtnGpl (Table 1). Conversely, PL-derived EtnGpl comprised 7.8 mol% PlsEtn and 92.2 mol% PtdEtn. The PlsEtn contents in the AM and PL diets were 108.7 μmol and 10.0 μmol/100 g diet, respectively, whereas the PlsEtn content in the basal diet was not detected. The n-3-to-n-6 ratios in the basal, AM, and PL diets were 0.24, 0.25, and 0.24, respectively, for the first four weeks using fish oil from skipjack tuna (Table 2). For the final three weeks using mixed fish oil, this ratio was 0.22, 0.23, and 0.22, respectively, in the basal, AM, and PL diets. The level of EPA in the AM diet was approximately 1.3 times higher than that in the other diets, and the level of DHA was similar among all diets.

3.2. Parameters of Hepatic Injury and Oxidative Stress in Mice Fed APAP and EtnGpl

Continuous APAP consumption significantly prevented body weight gain over five weeks, reduced liver weight, and non-significantly decreased splenic weight (p = 0.075) (Table 3). The AM diet, which was abundant in PlsEtn, improved the body and liver weights; however, these effects were not observed in the PM diet, which was abundant in PtdEtn. Continuous oral APAP administration showed a non-significant decrease in food intake during APAP treatment (p = 0.081), which was non-significantly suppressed by the AM diet (p = 0.066). Continuous oral APAP administration did not affect plasma AST and ALT activities, plasma TNF-α levels, which are markers of liver cell injury, or hepatic GSH levels. In contrast, continuous APAP consumption increased hepatic MDA levels, and dietary AM or PL suppressed APAP-induced oxidation in the liver. Spleen weight, which is affected by inflammation and hepatic cirrhosis, has no significant differences among groups.

3.3. Hepatic Fatty Acid Contents in Mice Fed APAP and EtnGpl

Continuous oral APAP administration reduced hepatic levels of fatty acids, including palmitic acid (16:0), oleic acid (18:1n-9), eicosatrienoic acid (20:3n-6), and total fatty acids (Table 4). Moreover, it non-significantly decreased the levels of linolenic acid (18:3n-3, p = 0.054) and arachidonic acid (ARA; 20:4n-6, p = 0.096). A PlsEtn- or PtdEtn-rich diet significantly alleviated the APAP-induced decrease in 16:0 levels. In addition, the PlsEtn-rich diet suppressed the decrease in total fatty acid levels and non-significantly alleviated decreased levels of 18:1n-9 (p = 0.097) and 20:3n-6 (p = 0.089). Notably, the PlsEtn-rich diet increased the hepatic levels of DHA and docosapentaenoic acid (22:5n-3) and the DHA/ARA ratio. In contrast, plasmalogen levels in the livers were extremely low (<0.5 nmol/mg protein) among all the groups.

3.4. Expression of Apoptosis-Related Proteins in the Livers of Mice Fed APAP and EtnGpl

Continuous oral APAP administration significantly decreased the expression of p53 and claspin, which play central roles as checkpoint proteins in the cell cycle in the liver (Figure 1). Additionally, it significantly increased the expression of HSP27. Dietary PlsEtn or PtdEtn significantly suppressed the expression of ten proteins, including antiapoptotic proteins (Bcl-2, Bcl-x, claspin, and MCL-1) and other proteins (HIF-1α, HO-1, and HO-2) as well as apoptosis-promoting proteins (cleaved caspase-3, an effector caspase of apoptosis, TRAIL-R2, and cyt c), compared to the APAP group. Dietary PlsEtn showed greater suppressive effects on the expression of Bcl-2, HIF-1a, and HSP27 than dietary PtdEtn. Moreover, dietary PlsEtn increased the expression level of XIAP and reduced the expression levels of Bad, p53, catalase, and p27/Kip1 compared to those in the APAP group.

4. Discussion

PlsEtn has been reported to exhibit physiological and nutritional functions in the liver [5,13,15]. In this study, dietary PlsEtn from ascidian suppressed continuous APAP-induced hepatic injury, as evidenced by the prevention of body and liver weight loss, reduction in hepatic oxidative stress, and impairment of lipid metabolism. In contrast, dietary PtdEtn, another EtnGpl subclass, ameliorated APAP-induced lipid oxidation in the liver but did not suppress the decrease in hepatic total fatty acid content and the loss of liver weight. Additionally, we found that PlsEtn suppressed the expression of apoptosis-related proteins in the liver, and the extent of suppression by PlsEtn was greater than that by PtdEtn, as shown in Figure 1. In previous studies, dietary PtdEtn suppresses hepatic steatosis and inflammatory response in rodents fed a high-fat diet [29,30]. Liver levels of PtdEtn and its metabolites, including phosphatidylcholine (PtdCho) and phosphatidylserine, decrease to induce mitochondrial dysfunction in non-alcoholic fatty liver disease [31]. Thus, dietary PtdEtn may maintain their hepatic levels and protect the liver. Dietary PlsEtn also alleviates hepatic impairments in rodents fed a high-fat diet [15]; however, the concentration of PlsEtn in the liver is much lower compared to PtdEtn, and PlsEtn is not directly converted to PtdEtn due to the structural variation at the sn-1 position. Therefore, it is proposed that the hepatoprotective mechanism of dietary PlsEtn differs from that of PtdEtn.
Dietary PlsEtn increased the hepatic levels of DHA and the DHA/ARA ratio. Intracellular DHA suppresses oxidative and inflammatory stress, both by itself and via its metabolites [32], and the DHA/ARA ratio exhibits protective properties [33]. ARA is metabolized by oxidizing enzymes, resulting in ARA-derived eicosanoids that induce inflammation [34]. Conversely, DHA and EPA competitively inhibit ARA metabolism to suppress inflammation, and their metabolites, docosanoids and EPA-derived eicosanoids, exert anti-inflammatory effects. In the liver of mice expressing the gene encoding n-3 desaturase, an increase in DHA and EPA results in elevated levels of docosanoids and EPA-derived eicosanoids [35]. In this study, the basal diet contained nearly equivalent levels of DHA and EPA as the AM and PL diets. Glycerophospholipids are resynthesized preferentially with PUFAs following digestion [9,36]. Although PUFA absorption from glycerophospholipids in the small intestine is similar to that from triacylglycerols, PUFA accumulation in the liver is more efficient with glycerophospholipid intake than with triacylglycerol intake [37].
Dietary PlsEtn may not markedly influence the total plasmalogen level in the liver. The intracellular concentration of total EntGpl, comprising PlsEtn and PtdEtn, is meticulously regulated [38]. In mice with a hepatocyte-specific peroxisomal defect, which is crucial for plasmalogen biosynthesis, the hepatic PlsEtn levels are similar to those observed in the control group [39]. This suggests that the hepatic PlsEtn level is maintained through provision via plasma from/to other organs. Conversely, in mice subjected to diets deficient in n-3 PUFAs, the administration of PlsEtn or PtdCho enriched with EPA results in elevated hepatic DHA levels of both PlsEtn and PtdCho [40]. Furthermore, dietary PlsEtn significantly increases the hepatic concentration of DHA within PtdCho, a predominant phospholipid in the liver, compared to dietary PtdCho. When considered in conjunction with the previous paragraph, dietary PlsEtn can modify the fatty acid composition of phospholipids, including PlsEtn, in the liver. PlsEtn serves as a carrier of n-3 PUFAs when consumed alongside n-3 PUFA sources such as fish oil, and it exhibits hepatoprotective effects, potentially by enhancing membrane functions and generating bioactive metabolites.
In this study, the hepatic HSP27 expression was increased by APAP administration, and its increase was suppressed by the administration of EtnGpls, particularly PlsEtn. HSP27 is involved in mitochondrial damage and endoplasmic reticulum (ER) stress. The HSP family is induced by various inter- and intracellular stimuli, such as thermal and oxidative stress [41,42,43]. NAPQI can covalently bind to organellar proteins, and HSP27 expression increases with mitochondrial damage and ER stress [41]. Additionally, dietary PlsEtn decreased HO-1 expression and increased XIAP expression. HO-1 is a stress-inducible enzyme that functions to eliminate oxidative stress, and the administration of n-3 PUFA alleviates APAP-induced hepatic injury via the suppression of HO-1 expression in rats [44]. XIAP suppresses the caspase cascade pathway, and its upregulation alleviates APAP-induced liver injury [45]. Moreover, dietary PlsEtn suppressed the expression of TRAIL R2. TRAIL is a major mediator of APAP-induced hepatic injury in mice, and TRAIL R2 blocking alleviates impairments [46]. Moreover, EtnGpls, particularly PlsEtn, decreased the expression of antiapoptotic and apoptosis-promoting proteins. Thus, dietary PlsEtn may suppress the expression of apoptosis-related proteins by alleviating initial impairments, including oxidative stress and organelle dysfunction in the liver, caused by continuous APAP administration.
Owing to the alkenyl linkage, which exhibits antioxidant properties, PlsEtn was expected to markedly suppress hepatic lipid oxidation; however, its suppressive effect was the same extent as that by PtdEtn. PlsEtn may hardly exert antioxidant properties due to the alkenyl bond in the liver, which expresses high levels of lysoplasmalogenase [47]. Both PlsEtn and PtdEtn contain an ethanolamine base in their structures, and free ethanolamine and its metabolite choline exhibit to suppress lipid peroxidation [48]. Therefore, dietary PlsEtn and PtdEtn may be metabolized into free ethanolamine and its further metabolites, which may alleviate APAP-induced hepatic lipid oxidation.
In this study, continuous oral administration of APAP did not affect plasma AST and ALT activities, plasma TNF-α levels, which are markers of liver cell injury, or hepatic GSH levels. Fish oil, which is abundant in n-3 PUFA, was present in all the experimental diets to obtain similar n-3-to-n-6 ratios and has been reported to aggravate ALF and chronic liver injury via the oral administration of APAP in mice [27]. However, fish oil exhibits hepatic protection against fatty liver [49,50]. Additionally, fish oil and n-3 PUFA can also alleviate ALF induced by intraperitoneal injection, which is not via the intra-intestine, or oral administration in rodents [44,51]. Administration of excess PUFA along with oxidation promoters, such as heme iron, accelerates PUFA oxidation to impair the intestine [52], and PUFA oxides indirectly increase lipid peroxidation in the body [53]. Excess dietary fish oil and APAP may be oxidized in the intestine. In this study, the fish oil content in the diets was one-tenth of that in the Bad effect [27]; therefore, fish oil contained in the basal diet may suppress the increase in their markers of liver cell injury induced by continuous oral APAP administration. Given that repeated oral administration of APAP has also been documented to induce adaptation in the hepatic antioxidant system, including glutathione reductase [54], it is imperative to conduct further research to ascertain whether variations in dietary fish oil content influence liver injury resulting from oral APAP administration.
High feed intake may intensify the severity of liver injury due to the presence of APAP in the diet. However, in the AM + APAP group, which exhibited a higher intake, parameters of liver injury showed improvement compared to the APAP group, which had a lower intake. Although this difference in intake is not statistically significant, it metaphorically represents the protective effect of PlsEtn against APAP-induced injury. Furthermore, APAP impacts not only liver injury but also affects intestinal epithelial cells and microbiota. APAP impairs intestinal epithelial cells and decreases the permeability of small molecules (e.g., sugars) [55]. APAP has been reported to alter the gut microbiota, which aggravates liver injuries in mice with ALF [56], and alterations in the gut microbiota can aggravate or alleviate APAP-induced hepatotoxicity [57,58,59]. PlsEtn ameliorates intestinal impairments and alterations in microbiota caused by LPS-induced injury, colitis, and a high-fat diet [10,11,14]. Thus, dietary PlsEtn may also mitigate APAP-induced intestinal changes and prevent the reduction in nutritional absorption, appetite, and body weight through the gut–liver axis.
As mentioned above, the mitigation mechanism of dietary PlsEtn may involve intestinal protection and increased hepatic DHA levels. To validate these hypotheses, in vivo identification and quantification of PlsEtn species, DHA, and ARA metabolites—including PlsEtn oxides and fragments, DHA hydroxides and epoxides, and eicosanoids—in the liver, intestine, and blood should be conducted over time in the presence of APAP. Moreover, their in vitro effects on hepatic and intestinal cells should be examined using APAP and NAPQI.

5. Conclusions

In this study, we investigated the effects of PlsEtn from ascidian on chronic hepatic injury in APAP-treated mice, and to clarify the PlsEtn specificity, the effects were compared with those by dietary PtdEtn. The administration of PlsEtn or PtdEtn alleviated APAP-induced injuries via stress reduction, and the impacts of PlsEtn were more effective. One suggested mechanism for hepatic protection by dietary PlsEtn is related to increased hepatic DHA levels. These findings advance our understanding of the role of PlsEtn in maintaining hepatic health. Further studies are required to clarify the dynamics and detailed mechanisms of PlsEtn-related metabolites in rodents and humans during PlsEtn administration.

Author Contributions

Conceptualization, M.K. and S.Y.; methodology, M.F. and M.K.; formal analysis, investigation, and resources, Y.T. and M.E.; data curation, S.Y. and M.K.; writing—original draft preparation, R.S.; writing—review and editing, K.N. and S.Y.; visualization, S.Y.; supervision, T.M. and M.K.; project administration, M.K. and S.Y.; funding acquisition, K.N., T.M. and S.Y. All authors have read and agreed to the published version of the manuscript.

Funding

A Part of this research was supported by a grant from the Project of NARO Bio-oriented Technology Research Advancement Institution (R&D matching funds on the field for Knowledge Integration and Innovation) and JSPS KAKENHI grant number JP19K05892.

Institutional Review Board Statement

The animal study protocol was approved by the Animal Care and Experiment Committee of the Obihiro University of Agriculture and Veterinary Medicine (License No.: 20-172, approved on 10 July 2020).

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

We thank Yaizu Suisankagaku Industry Co., Ltd. and Nissui Co., Ltd. for providing ascidian muscle and fish oils, respectively.

Conflicts of Interest

The authors declare no competing financial interests.

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Applsci 15 05968 g001
Figure 1. Expression of apoptosis-related proteins in the livers of mice fed diets containing APAP and EtnGpl. (A) Proapoptotic proteins. (B) Caspase and catalase. (C) Death receptors. (D) Antiapoptotic proteins. (E) Other apoptosis-related proteins. All values are presented as the mean ± standard error of the mean (n = 4; pooled mouse livers). Different letters indicate significant differences among groups treated with APAP at p < 0.05, as determined using a one-way analysis of variance by Tukey’s test. Asterisks indicate significant differences between the Ctrl and APAP groups at p < 0.05, as determined using the t-test. Please refer to Section 2.5 for abbreviations for apoptosis-related proteins. AM, ascidian muscle; APAP, acetaminophen; Ctrl, control; EtnGpl, ethanolamine glycerophospholipid; PL, porcine liver.
Figure 1. Expression of apoptosis-related proteins in the livers of mice fed diets containing APAP and EtnGpl. (A) Proapoptotic proteins. (B) Caspase and catalase. (C) Death receptors. (D) Antiapoptotic proteins. (E) Other apoptosis-related proteins. All values are presented as the mean ± standard error of the mean (n = 4; pooled mouse livers). Different letters indicate significant differences among groups treated with APAP at p < 0.05, as determined using a one-way analysis of variance by Tukey’s test. Asterisks indicate significant differences between the Ctrl and APAP groups at p < 0.05, as determined using the t-test. Please refer to Section 2.5 for abbreviations for apoptosis-related proteins. AM, ascidian muscle; APAP, acetaminophen; Ctrl, control; EtnGpl, ethanolamine glycerophospholipid; PL, porcine liver.
Applsci 15 05968 g001
Table 1. Fatty chain composition of EtnGpl from AM and PL.
Table 1. Fatty chain composition of EtnGpl from AM and PL.
AcylAM EtnGplPL EtnGplAlkenylAM EtnGplPL EtnGpl
mol% in Total Fatty Chainsmol% in Total Fatty Chains
16:01.37.416:0ol4.51.5
16:1n-71.60.118:0ol34.42.1
18:03.936.318:1ol3.20.3
18:1n-92.06.6Total42.1 3.9
18:2n-60.38.1
18:3n-30.80.0
20:4n-61.828.6
20:5n-332.90.5
22:4n-6nd1.3
22:5n-30.42.0
22:6n-39.43.7
Others0.80.9
All values are presented as the means of two analyses. AM, ascidian muscle; EtnGpl, ethanolamine glycerophospholipid; nd, not detected; PL, porcine liver.
Table 2. Fatty acid composition of each experimental diet.
Table 2. Fatty acid composition of each experimental diet.
(A) (B)
Acyl Basal DietAM DietPL DietAcyl Basal DietAM DietPL Diet
μmol/100 g Dietμmol/100 g Diet
14:0199.4200.6199.414:0209.5210.7209.5
16:03141.03146.13159.416:03352.83357.93371.3
16:1n-7254.9259.0254.916:1n-7216.2220.2216.2
18:0936.0948.51046.418:01086.31098.71196.6
18:1n-94853.54858.74866.818:1n-95053.55058.75066.8
18:1n-7374.2376.5375.818:1n-7384.4386.7386.0
18:2n-610,428.810,429.910,455.318:2n-610,464.510,465.610,490.9
18:3n-31220.01222.21220.318:3n-31271.51273.81271.8
20:4n-683.087.4145.420:4n-6134.3138.7196.7
20:5n-3243.3327.7244.220:5n-3255.7340.0256.5
22:4n-66.76.710.522:4n-6ndnd3.7
22:5n-336.737.842.522:5n-345.446.551.2
22:6n-31142.41167.01147.122:6n-3796.1820.7800.8
n-3/n-6 ratio0.250.260.25n-3/n-6 ratio0.220.230.22
(A) Diets containing fish oil from skipjack tuna for the first four weeks; (B) diets containing mixed fish oils from skipjack tuna and tuna for the final three weeks. All values are presented as the means of two analyses. AM, ascidian muscle; nd, not detected; PL, porcine liver.
Table 3. Changes in the parameters of hepatic injury and oxidative stress in mice fed APAP and EtnGpl.
Table 3. Changes in the parameters of hepatic injury and oxidative stress in mice fed APAP and EtnGpl.
CtrlAPAPAM + APAPPL + APAP
Initial body weight (g)24.4±0.424.3±0.4 a24.4±0.4 a24.3±0.3 a
Final body weight (g)44.7±1.540.5±0.8 b46.5±1.1 a41.0±1.1 b
Body weight gain9.4±0.8 *5.9±0.4 b9.7±0.6 a6.6±0.7 b
during APAP treatment (g)
Feed intake261.6±0.8243.9±7.6 a295.6±19.8 a269.5±6.8 a
during APAP treatment (g)
Plasma AST (IU/L)54.5±6.159.3±6.1 a48.4±5.5 a57.8±6.5 a
Plasma ALT (IU/L)14.4±3.110.5±1.0 a8.6±1.0 a11.6±1.7 a
Plasma TNF-α (pg/mL)19.3±0.620.8±0.6 a22.6±1.2 a21.0±1.4 a
Liver weight (g)2.2±0.2 *1.8±0.1 b2.2±0.1 a1.9±0.1 b
Liver GSH (µmol/mg protein)18.4±3.816.4±3.2 a19.9±2.0 a16.0±2.8 a
Liver MDA (nmol/mg protein)11.6±0.9 *16.2±1.5 a10.2±0.7 b10.6±1.1 b
Spleen weight (mg)140.0±10.7113.3±9.4 a120.8±6.2 a125.8±8.4 a
All values are presented as the mean ± standard error of the mean (n = 12 except for feed intake). Feed intake was expressed based on three data points and was calculated as the amount per mouse because each cage with one feed tray housed four mice. Different letters indicate significant differences among groups treated with APAP at p < 0.05, as determined using a one-way analysis of variance by Tukey’s test. Asterisks indicate significant differences between the Ctrl and APAP groups at p < 0.05, as determined using the t-test. ALT, alanine aminotransferase; AM, ascidian muscle; APAP, acetaminophen; AST, aspartate aminotransferase; Ctrl, control; EtnGpl, ethanolamine glycerophospholipid; GSH, glutathione; MDA, malondialdehyde; PL, porcine liver.
Table 4. Impacts of APAP and EtnGpl administration on fatty acid contents in mice livers.
Table 4. Impacts of APAP and EtnGpl administration on fatty acid contents in mice livers.
CtrlAPAPAM + APAPPL + APAP
nmol/mg protein
16:0111.9±11.0 *61.5±4.2 b81.3±2.1 a79.3±7.1 a
16:1n-79.4±1.3 *4.3±0.6 a5.9±0.6 a4.9±0.9 a
18:021.9±1.3 *17.9±1.2 a18.9±0.6 a18.9±0.8 a
18:1n-996.9±13.7 *38.5±4.5 a53.3±2.6 a40.1±5.1 a
18:1n-710.0±1.3 *3.8±0.4 ab5.1±0.5 a3.5±0.4 b
18:2n-661.9±3.9 *50.0±2.3 a59.1±2.6 a62.1±6.1 a
18:3n-33.5±0.42.6±0.2 a4.9±1.9 a3.0±0.4 a
20:3n-63.0±0.1 *2.3±0.1 ab2.7±0.2 a1.9±0.2 b
20:4n-611.7±0.310.7±0.5 a10.1±0.4 a10.9±0.6 a
20:5n-33.5±0.1 *2.7±0.3 a3.3±0.2 a2.9±0.4 a
22:5n-31.6±0.31.3±0.1 b2.1±0.2 a1.8±0.3 ab
22:6n-319.7±1.217.9±0.6 b22.6±0.7 a20.1±1.7 ab
Total354.7±29.3 *213.3±12.7 b269.2±7.8 a249.3±22.7 ab
DHA/ARA ratio1.7±0.11.7±0.1 b2.3±0.1 a1.8±0.1 b
All values are presented as mean ± standard error of the mean (n = 12). Different letters indicate significant differences among groups treated with APAP at p < 0.05, as determined using a one-way analysis of variance by Tukey’s test. Asterisks indicate significant differences between the Ctrl and APAP groups at p < 0.05, as determined using the t-test. AM, ascidian muscle; APAP, acetaminophen; ARA, arachidonic acid; Ctrl, control; DHA, docosahexaenoic acid; EtnGpl, ethanolamine glycerophospholipid; PL, porcine liver.
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MDPI and ACS Style

Sogame, R.; Tominaga, Y.; Echigoya, M.; Nakagawa, K.; Fukushima, M.; Miyazawa, T.; Kinoshita, M.; Yamashita, S. Dietary Ethanolamine Plasmalogen from Ascidian Alleviates Chronic Hepatic Injury in Mice Treated with Continuous Acetaminophen. Appl. Sci. 2025, 15, 5968. https://doi.org/10.3390/app15115968

AMA Style

Sogame R, Tominaga Y, Echigoya M, Nakagawa K, Fukushima M, Miyazawa T, Kinoshita M, Yamashita S. Dietary Ethanolamine Plasmalogen from Ascidian Alleviates Chronic Hepatic Injury in Mice Treated with Continuous Acetaminophen. Applied Sciences. 2025; 15(11):5968. https://doi.org/10.3390/app15115968

Chicago/Turabian Style

Sogame, Ryosuke, Yuki Tominaga, Momoka Echigoya, Kiyotaka Nakagawa, Michihiro Fukushima, Teruo Miyazawa, Mikio Kinoshita, and Shinji Yamashita. 2025. "Dietary Ethanolamine Plasmalogen from Ascidian Alleviates Chronic Hepatic Injury in Mice Treated with Continuous Acetaminophen" Applied Sciences 15, no. 11: 5968. https://doi.org/10.3390/app15115968

APA Style

Sogame, R., Tominaga, Y., Echigoya, M., Nakagawa, K., Fukushima, M., Miyazawa, T., Kinoshita, M., & Yamashita, S. (2025). Dietary Ethanolamine Plasmalogen from Ascidian Alleviates Chronic Hepatic Injury in Mice Treated with Continuous Acetaminophen. Applied Sciences, 15(11), 5968. https://doi.org/10.3390/app15115968

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