## 4 Discussion
In this study, the hepatic HSP27 expression was increased by APAP administration, and its increase was suppressed by the administration of EtnGpls, particularly PIsEtn. 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 PIsEtn 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 PIsEtn 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 PIsEtn, decreased the expression of antiapoptotic and apoptosis-promoting proteins. Thus, dietary PIsEtn 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, PIsEtn was expected to markedly suppress hepatic lipid oxidation; however, its suppressive effect was the same extent as that by PtdEtn. PIsEtn may hardly exert antioxidant properties due to the alkenyl bond in the liver, which expresses high levels of lysoplasalogenase [47]. Both PIsEtn and PtdEtn contain an ethanolamine base in their structures, and free ethanolamine and its metabolite choline exhibit to suppress lipid peroxidation [48]. Therefore, dietary PIsEtn 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-\(\alpha\) 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 PIsEtn against APAP-induced injury.