**1. Introduction**

Acute liver failure (ALF) is the most common life-threatening disease in adults without pre-existing liver disease, and it mainly occurs in the 30s [1]. There are many causes of ALF that include hepatitis, acetaminophen overdose, toxins, autoimmune diseases, Wilson's disease, and unknown factors. Herbal supplements cannot be free from triggers of ALF [2]. Since there are few effective treatments for ALF other than liver transplantation, studies to find strategies for the treatment and prevention of ALF using experimental animal models are continuously being performed. In the early stages of ALF, the incidence of bacterial infection is high [3,4], which might aggravate the clinical condition and prognosis [5]. An

**Citation:** Siregar, A.S.; Nyiramana, M.M.; Kim, E.-J.; Cho, S.B.; Woo, M.S.; Lee, D.K.; Hong, S.-G.; Han, J.; Kang, S.S.; Kim, D.R.; et al. Oyster-Derived Tyr-Ala (YA) Peptide Prevents Lipopolysaccharide/D-Galactosamine-Induced Acute Liver Failure by Suppressing Inflammatory, Apoptotic, Ferroptotic, and Pyroptotic Signals. *Mar. Drugs* **2021**, *19*, 614. https:// doi.org/10.3390/md19110614

Academic Editors: Donatella Degl'Innocenti and Marzia Vasarri

Received: 6 October 2021 Accepted: 26 October 2021 Published: 28 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

uncontrolled inflammatory response not only impairs the liver's defenses but also causes massive cell death of hepatocytes, leading to acute liver damage and ultimately severe ALF [1,6].

A model made by the intraperitoneal injection of lipopolysaccharide (LPS) and Dgalactosamine (D-GalN) has been widely used to study the pathogenesis of human ALF and drug development [7] because it shows clinically similar symptoms to ALF [8]. LPS, the major pathogenic component of Gram-negative bacteria, induces the secretion of large amounts of pro-inflammatory cytokines and ultimately causes liver injury [9–11]. D-GalN, a selective hepatotoxin, induces depletion of the intracellular uridine moiety, which in turn disrupts the hepatocyte RNA metabolism and results in liver injury [12,13]. D-GalN increases the sensitiveness of LPS and causes hepatotoxicity within a few hours. The LPS/D-GalN model shows typical hepatocellular death manifested by necrosis, apoptosis, autophagy, and inflammatory responses [14–16]. Although many studies have not been conducted, recent studies reported that cell death mechanisms by ferroptosis, pyroptosis, and necroptosis are involved in liver injury in the LPS/D-GalN model [17–19]. Substances that regulate signals related to the hepatocyte death mechanism are expected to be helpful in the prevention and treatment of LPS/D-GalN-induced liver injury.

Conventional drugs used to treat liver diseases, such as corticosteroids, antiviral drugs, and immunosuppressants, can cause serious adverse effects and even liver damage with long-term use [20]. A common strategy for preventing liver damage includes using substances with antioxidant and anti-inflammatory activity [21]. Natural products with antioxidant and anti-inflammatory activities, such as silymarin, were developed as hepatoprotectants [22]. However, since silymarin interacts with CYP2C9 inhibitors, caution is required when taking drugs related to CYP2C9 inhibitors [23]. It is necessary to broaden the choice of natural medicines suitable for individual patients by developing natural hepatoprotectants and therapeutic agents with fewer side effects than silymarin.

In our previous studies, oyster-derived hydrolysate (OH) showed hepatoprotective effects in a single ethanol binge model and a LPS/D-GalN-induced liver injury model [24,25]. In particular, the Tyrosine-Alanine (YA) peptide, the main component of OH, enhanced the ethanol metabolism and protected the liver from ethanol-induced toxicity [25]. YA has antioxidant and anti-inflammatory activities. Bioactive peptides affect various biological functions, and peptides have been used as therapeutic agents for various diseases for a long time [26]. Arg-Gly-Asp (RGD) peptide attenuates LPS-induced pulmonary inflammation [27] and hepatic fibrosis [28]. Currently, there are few studies on the hepatoprotective mechanism of YA against the LPS/D-GalN-induced liver injury model. Since the YA peptide is a food-derived substance, the preventive effect was first investigated before the therapeutic effect. This study was performed to determine the hepatoprotective effect of the YA peptide in the LPS/D-GalN-induced ALF model. We also compared the effects of two different concentrations of YA (10 and 50 mg/kg).

## **2. Results**

#### *2.1. Generation of Acute Liver Failure (ALF) Mouse Model*

The method to produce an ALF mouse model and the experimental procedure to confirm the prophylactic effect of YA are summarized in Figure 1A. The ALF model was generated by the intraperitoneal injection of LPS (1 μg/kg) and D-galactosamine (400 mg/kg), and the mice were sacrificed 6 h after the LPS/D-GalN injection. The five experimental groups were divided into the vehicle, LPS/D-GalN, YA (10 or 50 mg/kg) + LPS/D-GalN, and silymarin (25 mg/kg) + LPS/D-GalN groups (each group with 10 mice). YA and silymarin were pre-administrated orally for 10 days. Saline was pre-administered instead of YA in the vehicle and LPS/D-GalN groups. Body weight was measured at the beginning and end of the experiment, and liver weight was measured immediately after sacrificing the mice. There was no significant change in body and liver weights among the experimental groups.

**Figure 1.** LPS/D-GalN-induced acute liver failure (ALF) mouse model. (**A**) Experimental design to determine the protective effect of YA in the ALF mouse model. Saline, YA, or silymarin was pre-administered daily for ten days by oral gavage before intraperitoneal injection of LPS/D-GalN. (**B**) LPS/D-GalN-induced pathological alterations in liver tissue attenuated by YA. The morphological changes were identified by H&E staining. The dotted rectangle representing the hemorrhage area is expanded to show. Blue arrowheads indicate nuclear fragmentation. Scale bar, 100 μm. (**C**) Effect of YA pre-administration on plasma ALT and AST levels in the LPS/D-GalN group. Data are shown as the mean ± SD (n = 10 in each group). \* *p* < 0.05 compared to vehicle group. † *p* < 0.05 compared to the LPS/D-GalN group.

The morphological changes of the liver observed in the experimental groups were evaluated by hematoxylin and eosin (H&E) staining. The LPS/D-GalN group showed a remarkable increase in hemorrhage and nuclear fragmentation (dotted rectangle, Figure 1B). The morphological features of cell damage were reduced in the YA+LPS/D-GalN and silymarin+LPS/D-GalN groups. Comparing the effects of two different concentrations of YA, the cell damage was decreased more in the 50 mg/kg YA pre-administered group than in the 10 mg/kg YA pre-administered group. Silymarin (25 mg/kg), a positive control showing hepatoprotective effects, reduced the LPS/D-GalN-induced morphological features of cell damage (n = 3, Figure 1B). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the LPS/D-GalN group were significantly increased compared to the vehicle group (*p* < 0.05). In contrast, they were significantly decreased in the YA and silymarin pre-administered groups (Figure 1C, n = 10, *p* < 0.05).

#### *2.2. YA Pre-Administration Attenuated Inflammatory Signals in ALF Model*

YA significantly decreased the activity of the biosynthesis enzymes cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO), which are involved in the inflammatory process. The effect was dose-dependent (n = 4, *p* < 0.05, Figure 2A). The nuclear factor kappa-lightchain-enhancer of activated B cells (NF-κB), a key transcription factor for pro-inflammatory gene induction, was significantly activated in liver tissues obtained from the LPS/D-GalN groups compared to the vehicle group (Figure 2B, n = 4, *p* < 0.05). The NF-κB activation was significantly decreased in the 50 mg/kg YA + LPS/D-GalN and silymarin + LPS/D-GalN groups (Figure 2B, n = 4, *p* < 0.05). In the NF-κB activity, the 10 mg/kg YA + LPS/D-GalN group showed no significant difference from the vehicle and LPS/D-GalN groups.

Mitogen-activated protein kinase (MAPK) activation is related to LPS-induced inflammation [29]. Extracellular signal-regulated kinase 1/2 (ERK), c-Jun N-terminal kinases (JNK), and p38 MAPKs were significantly activated in the LPS/D-GalN group compared to the vehicle group (Figure 2B, *p* < 0.05, n = 4). ERK and JNK activation was significantly decreased in the 50 mg/kg YA + LPS/D-GalN group (*p* < 0.05), whereas p38 activation was significantly reduced in the 10 mg/kg and 50 mg/kg YA and silymarin pre-administered groups (Figure 2C, n = 4, *p* < 0.05), indicating that YA and silymarin may act through different mechanisms. Activation of NF-κB and MAPK is associated with the secretion of pro-inflammatory cytokines such as interleukin (IL)-1 β, IL-6, and tumor necrosis factor (TNF)-α [30,31]. High concentrations of IL-1β, IL-6, and TNF-α in the LPS/D-GalN

group were significantly reduced in the YA + LPS/D-GalN group (Figure 2D, n = 4, *p* < 0.05). The secretion of IL-1β, IL-6, and TNF-α was more decreased in the 50 mg/kg YA pre-administered group than in the 10 mg/kg YA pre-administered group. The mRNA expression levels of IL-1β, IL-6, and TNF-α were also decreased in the YA + LPS/D-GalN groups (Figure 2D).

**Figure 2.** Anti-inflammatory effect of YA in LPS/D-GalN-induced ALF model. (**A**) Inhibition of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) activity by YA. \* *p* < 0.05 compared to 25 μg/mL YA. (**B**) Changes in NF-κB activation. The NF-κB activity was measured using a phospho-NF-κB p65 (S536) ELISA kit. (**C**) Suppression of MAPK activation by YA. (**D**) Decrease in pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) by YA. Data are shown as the mean ± SD (n = 4 in each group). \* *p* < 0.05 compared to vehicle group. † *p* < 0.05 compared to the LPS/D-GalN group. ‡ *p* < 0.05 compared to YA (10 mg/kg) + LPS/D-GalN group. The plus (+) sign, such as in YA10+, YA50+, and silymarin+, represents a combination of LPS/D-GalN and each substance.
