Ethanol Extract of the Microalga Phaeodactylum tricornutum Shows Hepatoprotective Effects against Acetaminophen-Induced Acute Liver Injury in Mice

Abstract

Acute liver failure is an infrequent yet fatal condition marked by rapid liver function decline, leading to abnormalities in blood clotting and cognitive impairment among individuals without prior liver ailments. The primary reasons for liver failure are infection with hepatitis virus or overdose of certain medicines, such as acetaminophen. Phaeodactylum tricornutum (PT), a type of microalgae known as a diatom species, has been reported to contain an active ingredient with anti-inflammatory and anti-obesity effects. In this study, we evaluated the preventive and therapeutic activities of PT extract in acute liver failure. To achieve our purpose, we used two different acute liver failure models: acetaminophen- and D-GalN/LPS-induced acute liver failure. PT extract showed protective activity against acetaminophen-induced acute liver failure through attenuation of the inflammatory response. However, we failed to demonstrate the protective effects of PT against acute liver injury in the D-GalN/LPS model. Although the PT extract did not show protective activity against two different acute liver failure animal models, this study clearly demonstrates the importance of considering the differences among animal models when selecting an acute liver failure model for evaluation.

Keywords: D-galactosamine; Phaeodactylum tricornutum; acetaminophen; acute liver failure; hepatotoxicity.

Figure 1

The preventive (A,B) and therapeutic (C,D) effects of PT extract on acute liver injury after acetaminophen overdose. (A,B) C57BL/6J mice were administered SM, HD, and PT orally for 10 days, and then, the animals received 600 mg/kg of intraperitoneal acetaminophen after fasting overnight. After 24 h, the mice were sacrificed, and blood was collected for serum separation. Sera were separated from blood for AST (A) and ALT (B), where these acronyms refer to aspartate transaminase and alanine transaminase, respectively. (C,D) Mice received acetaminophen via intraperitoneal injection after fasting overnight and then administered PT orally for 3 days. The data from three independent experiments, each of which was performed in triplicate, are indicated as the mean ± SD. The significance of the differences between the value of the AP group and that of the SM, HD, and PT treatment groups after AP injection were determined through one-way ANOVA with Tukey’s comparisons test: * p < 0.05 and ** p < 0.01.

Figure 2

The preventive (A,B) and therapeutic (C,D) effects of PT extract on acute liver injury after D-GalN/LPS injection. (A,B) C57BL/6J mice were administered HD and PT orally for 3 days and then received intraperitoneal D-GalN (450 mg/kg) and LPS (10 μg/kg) after fasting overnight. After 6 h, the mice were sacrificed, and their blood was collected for serum separation. Sera were separated from blood for AST (A) and ALT (B). (C,D) The mice received D-GalN and LPS via intraperitoneal injection after fasting overnight and were then administered HD and PT orally. After 6 h, the mice were sacrificed, and their blood was collected for serum separation. Sera were separated from blood for AST (C) and ALT (D). The data from three independent experiments, each of which was performed in triplicate, are indicated as the mean ± SD. The significances of the differences between the value of the D-GalN/LPS group and that of the HD and PT treatment groups after D-GalN/LPS injection were determined through one-way ANOVA with Tukey’s comparisons test: ** p < 0.01.

Figure 3

Histological analysis of liver tissues. Liver tissue sections of normal and acetaminophen-injected mice stained with H&E are shown (magnification, 200× and 400×). (A) H&E staining images; (B) grayscale images converted with ImageJ; (C) percentage of area of extravasated RBCs in the image. The black circle in (A) indicates accumulated RBCs. The significance of the differences between the value of the AP group and that of the SM, HD, and PT treatment groups after AP injection was determined through one-way ANOVA with Tukey’s comparisons test: ** p < 0.01.

Figure 4

Histological analysis of liver tissues. Liver tissue sections of normal and D-GalN/LPS-injected mice stained with H&E are shown (magnification, 200× and 400×). (A) H&E staining images; (B) grayscale images converted with ImageJ; (C) percentage of area of extravasated RBCs in the image. The significance of the differences between the value of the D-GalN/LPS group and that of the HD and PT treatment groups after D-GalN/LPS injection were determined through one-way ANOVA with Tukey’s comparisons test: ** p < 0.01.

Figure 5

Effect of PT on the production of inflammatory cytokines in acute liver failure mouse models. Sera obtained from acetaminophen (A,B) and D-GalN/LPS (C,D) animal models were used in ELISA assays for TNF-α and IL-1β. The data from three independent experiments, each of which was performed in triplicate, are indicated as the mean ± SD. The significance of the differences between the value of AP or D-GalN/LPS groups and that of the SM, HD, and PT treatment groups after AP or D-GalN/LPS injection were determined through one-way ANOVA with Tukey’s comparisons test: * p < 0.05 and ** p < 0.01.

Figure 6

HPLC chromatogram of fucoxanthin standard (A) and PT extract (B) at 450 nm. The inset shows the absorption spectrum of the fucoxanthin peak. Red arrows indicate the fucoxanthin peak in each chromatogram.

Figure 7

Outline of the experimental design for the in vivo study of the preventive (A) and therapeutic (B) effects of PT extract on acetaminophen-induced acute liver injury and the preventive (C) and therapeutic (D) effects of PT extract on D-GalN/LPS-induced acute liver injury.