*4.8. Western Blot Analysis*

A Western blot analysis of total, cytoplasmic, and mitochondrial proteins was performed as described previously [25]. The total protein was isolated from liver tissue using the RIPA buffer (25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1% deoxycholate, 0.1% sodium dodecyl sulfate (SDS); Thermo Fisher Scientific, Carlsbad, CA, USA) containing a 1× protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). According to the manufacturer's protocol, mitochondrial and cytosolic fractions were isolated using a mitochondria isolation kit for the tissue (Thermo Fisher Scientific). Equal amounts (30 μg) of protein were analyzed among experimental groups. Equal volumes of the proteins and 2× SDS sample buffer were mixed, loaded on 10% SDS-polyacrylamide gel, and separated by electrophoresis for 120 min at 120 V. Then, the gel was transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) for 1 h at 100 V using a wet transfer system (Bio-Rad, Hercules, CA, USA). The membranes blocked with 5% (*w*/*v*) fat-free dry milk in TBS with tween-20 at room temperature for 60 min were incubated with anti-Bax (1:200 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Bcl-2 (1:200 dilution; Santa Cruz Biotechnology), anti-cytochrome C (1:1000; Cell Signaling, Danvers, MA, USA), anti-VDAC (1:1000; Cell Signaling, Danvers, MA, USA), anti-caspase-3 (1:1000; Cell Signaling), anti-GRP78 (1:1000 dilution, Abcam., Cambridge, UK), anti-pERK (1:200 dilution; Santa Cruz Biotechnology), anti-p-pERK (1:200 dilution; Santa Cruz Biotechnology), anti-elF2α (1:1000; Cell Signaling), anti-p-elF2α (1:1000; Cell Signaling), anti-ATF4 (1:200 dilution; Santa Cruz Biotechnology), anti-ATF6 (1:1000; Cell Signaling), anti-CHOP (1:200 dilution; Santa Cruz Biotechnology), anti-HO-1 (1:200 dilution; Santa Cruz Biotechnology), anti-SLC7A11/xCT (1:1000, arigo Biolaboratories Corp., Hsinchu City, Taiwan), anti-GPx4 (1:1000, arigo Biolaboratories Corp), anti-4-HNE (1:1000, arigo Biolaboratories Corp), anti-caspase 1 (1:1000, Adipogen Corporation, San Diego, CA, USA), anti-GSDMD (1:1000, Abcam), anti-cleaved C-terminal GSDMD (1:1000, Abcam), and anti-β-actin antibody (1:5000 dilution; Thermo Fisher Scientific) at 4 ◦C overnight. After the primary antibody incubation, it was incubated with a secondary HRP-conjugated anti-rabbit or anti-mouse antibody at 1:10,000 (Assay Designs, Ann Arbor, MI, USA). Immuno-positive bands were enhanced with chemiluminescence (EzWestLumi plus; ATTO Gentaur, Tokyo, Japan) and visualized using the iBright CL1500 imaging system (Thermo Scientific Fisher/ Life Technologies Holdings Pte Ltd., Singapore).

### *4.9. Measurement of IL-1β, IL-6, and TNF-α Concentrations in Liver Tissues*

According to the manufacturer's protocol, the concentrations of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the liver tissues were quantified using an ELISA kit (R&D system, Minneapolis, MN, USA). The protocol was previously described [25]. The absorbance of the plates at 450 nm was read with a microplate reader (Molecular Devices).

#### *4.10. Measurement of Total and Phospho-NF-kB p65 Protein*

The semi-quantitative measurement of NF-κB p65 (pS536) and total NF-κB p65 protein was performed using an ELISA kit (NF-κB p65 (pS536 + Total), Abcam) according to the manufacturer's protocol. The 100 mg of liver tissues were homogenized in cold 1× Extraction Buffer PTR (Abcam). The homogenates were incubated in ice for 20 min and subjected to centrifugation at 18,000× *g* for 20 min at 4 ◦C (Eppendorf Centrifuge 5424R, Eppendorf AG). After centrifugation, the supernatant was transferred to a clean tube and provided as tissue lysates. The protein concentration of tissue lysate was quantified using the BCA assay kit. Then, the tissue lysates were diluted to the desired concentration using 1× Extraction Buffer PTR. The diluted tissue lysates (50 μL), antibody cocktail (the mixture of Capture Antibody NF-κB p65 (pS536) or NF-κB p65 total with Detector Antibody) were added to a 96-well plate. The plates were covered with an adhesive strip, incubated for 1 h at room temperature, and washed three times with 1× wash buffer PT. Then, 100 μL of TMB substrate were added and incubated at room temperature for 15 min in the dark on a plate shaker set to 400 rpm. The reaction was quenched by adding 100 μL stop solution, and the absorbance of the plates was read at 450 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

#### *4.11. Data Analysis and Statistics*

The images of the Western blots and agarose gel were captured using an iBright CL1500 imaging system (Thermo Scientific Fisher/Life Technologies Holdings Pte Ltd.). The bands were quantified by ImageJ software (version 1.49, National Institutes of Health, Bethesda, MD, USA). Data are presented as the mean ± standard deviation (SD). The one-way ANOVA/Bonferroni test or the Kruskal–Wallis/Mann–Whitney test was selected after the normality test to analyze differences among groups (OriginPro2020, OriginLab Corp., Northampton, MA, USA). A *p* < 0.05 value was considered as the statistical significance criterion.

#### **5. Conclusions**

Our findings demonstrate that YA regulates a complex mechanism induced by LPS/D-GalN. YA has anti-inflammatory, anti-apoptotic, anti-pyroptotic, and anti-ferroptotic effects against LPS/D-GalN-induced hepatic inflammation and cell death. YA may be a potential marine anti-inflammatory and antioxidative agent for treating acute liver diseases such as ALF.

**Author Contributions:** Conceptualization, S.S.K., Y.J.C. and D.K.; Data curation, A.S.S., M.M.N. and D.K.; Formal analysis, A.S.S., M.M.N., E.-J.K. and D.K.; Funding acquisition, S.S.K., Y.J.C., D.R.K. and D.K.; Investigation, A.S.S., M.M.N., E.-J.K., M.S.W. and D.K.; Methodology, A.S.S., M.M.N.; Project administration, E.-J.K.; Resources, Y.J.C.; Supervision, S.-G.H., J.H.; Writing—original draft, A.S.S., S.B.C. and D.K.; Writing—review and editing, D.K.L., J.H., S.S.K., S.-G.H. and D.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Research Foundation of Korea (MSIT, NRF-2015R1A-5A-2008833 and 2021R1I1A3044128) and by the Ministry of Oceans and Fisheries (Korea, D11410119H480000110/#2014042).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the animal care and use committee of Gyeongsang National University (GNU-151208-M0068).

**Data Availability Statement:** The study did not report any data.

**Acknowledgments:** This work was from Adrian Syawaluddin Siregar's thesis for the degree of Doctor of Philosophy ("Hepatoprotective effect of dipeptide tyrosine-alanine in ethanol and lipopolysaccharide-induced liver injury models", 2021, Department of Convergence Medical Science, Graduate School, Gyeongsang National University).

**Conflicts of Interest:** The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**

