*2.4. YA Pre-Administration Attenuated Endoplasmic Reticulum (ER) Stress and Ferroptosis and Pyroptosis Signals in ALF Model*

ER stress is related to various cell death mechanisms. ER stress-related proteins such as GRP78, PERK, eIF2α, ATF4, ATF6, and CHOP were upregulated in the LPS/D-GalN group (Figure 4A). The upregulated ER stress markers were markedly decreased in the YA and silymarin pre-administered groups. SLC7A11, GPx4, and HO-1 suppression are linked to ferroptosis induction, while 4-HNE upregulation is related to lipid peroxidation during this process. SLC7A11, GPx4, and HO-1 protein expression levels were decreased in the LPS/D-GalN group, while the 4-HNE protein expression level was increased. The changes in the ferroptosis markers were significantly restored in the YA and silymarin pre-administered groups (Figure 4B, *p* < 0.05, n = 3).

Pyroptotic cell death was detected in the LPS/D-GalN group. The caspase-1 was cleaved, and the gasdermin D (GSDMD) was upregulated. In addition, the carboxyterminal gasdermin-C domain cleaved in gasdermin D (CL-C-terminal GSDMD) was detected in the LPS/D-GalN group (Figure 4C). IL-1β was highly secreted in the LPS/D-GalN group (see Figure 2D). The pyroptotic signals were significantly reduced in the YA+LPS/D-GalN and silymarin+LPS/D-GalN groups (Figure 4C, *p* < 0.05, n = 3).

The mechanisms involved in liver injury in LPS/D-GalN-injected mice are summarized in Figure 5.

**Figure 4.** Ferroptotic and pyroptotic signals decreased by YA pre-administration. (**A**) Changes in ER stress markers. (**B**) Changes in ferroptosis markers. (**C**) Changes in pyroptosis markers. Data were shown as the mean ± SD (n = 3 in each group). (**B**,**C**) share a label representing each experimental group. \* *p* < 0.05 compared to vehicle group. † *p* < 0.05 compared to the LPS/D-GalN group. The plus sign (+) means the combination of LPS/D-GalN and each substance.

**Figure 5.** Various mechanisms were observed in liver tissues obtained from the LPS/D-GalN-induced ALF model. Pre-administration of YA reduced LPS/D-GalN-induced liver damage factors. The mechanisms are interconnected and can exacerbate liver damage.

#### **3. Discussion**

This study analyzed the effect of YA on previously known mechanisms of hepatocellular damage in a mouse model capable of mimicking ALF symptoms. LPS/D-GalN-injected mice used as an ALF model in this study are also referred to as models for fulminant liver failure (FLF), acute liver injury (ALI), and acute hepatitis. Previous studies reported that liver damage in LPS/D-GalN-injected mouse models is induced by multiple complex mechanisms, such as inflammation, apoptosis, necrosis, autophagy, pyroptosis, necroptosis, and ferroptosis [14–19]. However, most previous studies using LPS/D-GalN-injected mouse models analyzed one or two of the mechanisms mentioned above and reported that many hepatoprotectants proposed in those studies modulate the analyzed mechanisms. Since various factors and mechanisms cause ALF, substances that can control several mechanisms at once will be more helpful in treating ALF. Many hepatoprotective natural

substances are more likely to exert their effects by regulating multiple mechanisms rather than specifically regulating a single mechanism. However, due to the lack of research, only some mechanisms of action of these substances are known. Even if the effects of these substances are excellent, it is challenging to develop new drugs or healthy functional foods if the mechanism of action is not sufficiently analyzed.

Here, we introduce a YA peptide that regulates many pathological mechanisms occurring in the LPS/D-GalN-induced ALF model. YA reduced inflammation, apoptosis, ER stress, ferroptosis, and pyroptosis in a LPS/D-GalN-injected mouse model, eventually reducing liver injury. Dipeptide YA used in this study has not been studied as much as other peptides. The mechanism of LPS/D-GalN-induced liver injury is also associated with increased autophagy [16]. However, in our study, autophagy-related signals did not show consistent results, so autophagy was excluded from the hepatoprotective mechanism of YA. Necrotic events were confirmed by H&E staining. Hepatocyte swelling along with shrinkage of the nucleus shown in the LPS/D-GalN-induced ALF model was reduced in the YA pre-administered group. Receptor-interacting protein kinase (RIPK) 1 and RIPK3, necroptosis markers, expression levels were also checked in the liver tissues obtained from the LPS/D-GalN-induced ALF model. There were no significant differences between the vehicle group and the LPS/D-GalN group. As a result, autophagy and necroptosis were excluded from YA-regulated mechanisms.

YA exerts an anti-inflammatory effect by reducing MAPK/NF-κB activity. Apoptosis plays a role in normal liver development. However, the over-activation of apoptosis may lead to hepatocellular damage [32,33]. YA exerts anti-apoptotic effects by decreasing the Bax/Bcl2 ratio, PARP and caspase 3 cleavages, and cytochrome C translocation from the mitochondria to the cytoplasm. ER stress plays a role during LPS/D-GalN-induced apoptosis in the ALF model [34–36]. YA decreased most of ER stress protein expression. ER stress is related to apoptosis, inflammation, and pyroptosis [37,38]. Ferroptosis agents cause ER stress responses, which play an essential role in the cross-talk between ferroptosis and other types of cell death [39]. ER stress appears to mediate many kinds of cell death. Dysregulation of ferroptosis has also been associated with various liver diseases [40]. Ferroptosis occurs mainly due to downregulated system xc activity, inhibited glutathione peroxidase 4 (GPX4), and increased lipid ROS [41]. Functional subunit solute carrier family member 11 of system xc (SLC7A11), GPx4, and HO-1 protein expression are reduced in the LPS/D-GalNinduced liver injury model [19,42]. YA pre-administration reversed ferroptotic signals in the LPS/D-GalN-induced ALF model. Apoptosis, necroptosis, and pyroptosis can be switched by some molecules. GSDMD is a pore-forming protein that promotes pyroptosis and the release of pro-inflammatory cytokines [43]. GSDMD-mediated hepatocyte pyroptosis extends the inflammatory response to ALF by upregulating monocyte chemotactic protein 1/CC chemokine receptor-2 to recruit macrophages [17,18]. YA pre-administration also decreased the upregulation of GSDMD, caspase 1 activation, C-terminal of GSDMD cleavage in the LPS/D-GalN induced ALF model. These signals are intricately intertwined in the ALF model and will act in complex ways. In addition, the analyzed mechanism may not be perfect. Other mechanisms will work. YA regulates various mechanisms, which can occur in the LPS/D-GalN-induced ALF model. Involvement in multiple mechanisms can be either an advantage or a disadvantage. The advantage is that it can be effective because it can block numerous pathways that can act as mechanisms of liver damage in the ALF model at once. The disadvantage is that since it blocks several pathways, the probability of side effects can be high, and YA may not work specifically for the ALF model. However, in terms of side effects, since YA is a peptide derived from natural products, it is considered that the possibility of side effects is low.

YA was used as a standard material for OH. Although several peptides have been suggested as standard materials in the OH, YA has advantages over other peptides. YA is readily available to be used because YA is synthesized and sold by several companies, including Sigma-Aldrich (#T5128). Short peptides produced from proteins that have biological activity beyond their nutritional value are known as bioactive peptides. To achieve

their "bioactive" roles, these peptides must be released by proteolysis (in vivo digestion, in vitro enzymatic hydrolysis, or bacterial fermentation) [44]. YA was released from oysters by enzymatic hydrolysis. Our previous studies demonstrated that OH produced by in vitro enzymatic hydrolysis of oysters contained various bioactive peptides such as TAY, VK, KY, FYN, and YA and displayed antihypertensive, anti-inflammatory, antidiabetic, antioxidative, and hepatoprotective effects in in vitro and in vivo tests [24,25,45–47]. YA can be a bioactive peptide.

In the case of peptides, when administered orally, they are broken down into amino acids in the gastrointestinal tract, which may weaken their effectiveness. When comparing the effects of oral and intraperitoneal administrations in a preliminary study, the YA effect was slightly higher when injected intraperitoneally, indicating that the peptide may be digested into amino acids without being wholly absorbed when YA was administered orally. However, it is thought that this disadvantage can be overcome by intramuscular, subcutaneous, or intravenous injection. If YA is catabolized, tyrosine and alanine will be produced. Tyrosine and alanine are non-essential amino acids. Alanine is the most common amino acid catabolized by the liver in mammals, and it contributes the most to the gluconeogenesis of the 15 glucogenic amino acids [48,49]. To clear the N metabolites generated by amino acid catabolism, peripheral tissues such as skeletal muscle produce alanine and glutamine as nitrogen carriers in the blood, which are then taken up by the liver and gut and safely disposed of ureagenesis, resulting in glucose production from alanine [48,49]. Furthermore, the ALT expressed in the liver is responsible for the alanine-pyruvate interconversion [50]. When hepatocytes are damaged, ALT is released into the bloodstream, increasing serum ALT activity [51]. In an ALF rat model treated with D-GalN, alanine administration was found to lower plasma levels of ALT and total bilirubin dramatically [52]. In a CCl4-induced hepatocyte necrotic rat model, alanine administration was shown to reduce the ALT rise and histological liver damage [53]. In addition, alanine treatment dramatically reduced lactate dehydrogenase levels in D-GalNtreated rat hepatocytes [53]. When dietary tyrosine levels are low, the liver can produce tyrosine by hydroxylating phenylalanine. Tyrosine can become an essential amino acid in conditions where the liver fails. Tyrosine shortage can cause net protein catabolism and muscle wasting, so it is important to get enough [54]. Furthermore, tyrosine that is overused is oxidized. Tyrosine is a ketogenic and glucogenic amino acid. Both glucose and fatty acids can be produced by tyrosine [54]. Blood tyrosine levels are supposed to rise as a result of all-cause liver disease [55]. However, the exact mechanism is not known, and there is little evidence that tyrosine has a direct influence on liver disease. The YA concentration in the gastrointestinal system did not vary significantly in the simulated digestion experiment, suggesting that YA can be absorbed into the blood without significant loss [45].

In addition, when comparing the hepatoprotective effect of YA between the group pre-administered with YA once a day for 10 days (10 days YA group) and the group preadministered with YA once a day (1 day YA group), the hepatoprotective effect of YA was slightly lower in the 1 day YA group than the 10 day YA group, with a reduction in liver damage. At 10 and 50 mg/kg concentrations of YA, both concentrations effectively reduced liver damage in the LPS/D-GalN-induced ALF model, except for effects on ERK and JNK activation. The hepatoprotective effect was higher in the 50 mg/kg YA pre-administered group than in the 10 mg/kg YA pre-administered group. In addition, significant activation of ERK and JNK in the 50 mg/kg YA pre-administered group could act as a signaling pathway distinct from the silymarin pre-administered group. A single ethanol binge model with 50 mg/kg YA demonstrated a hepatoprotective effect [25]. Therefore, we compared the effect of low-dose (10 mg/kg) and high-dose (50 mg/kg) YA in the LPS/D-GalNinduced ALF model. It can become a more effective functional food and is more likely to be used as a pharmaceutical if it has an effect at a low concentration. Although less effective than 50 mg/kg YA, 10 mg/kg YA had a hepatoprotective effect, suggesting that it could be developed as a medication. YA did not cause hepatotoxicity at 50 mg/kg, and it may affect other mechanisms that were not fully explored in this study.

Food-derived bioactive peptides and peptide-rich protein hydrolysates could provide a safe alternative to synthetic pharmaceuticals for the prevention and treatment of acute and chronic diseases with fewer side effects. The positive effect of YA was confirmed in the acute inflammation models, but its effect should also be analyzed in the chronic models. The substances that modulate multiple mechanisms may be effective because they can control complex mechanisms that can coexist in a single disease. However, it will be necessary to investigate continuously the side effects of the substance on normal tissues. Our findings suggest that YA can be a hepatoprotectant in acute liver injury, such as ALF, FLF, and acute hepatitis as a bioactive peptide.
