Hmgb1

Tissues or cells were lysed by radioimmunoprecipitation assay lysis buffer (Solarbio). The proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated overnight using the following primary antibodies: ACSL4 (Santa Cruz, Dallas, USA), GPX4 (Santa Cruz), HMGB1 (Proteintech, Wuhan, China), HO-1 (Proteintech), α-SMA (Proteintech), GAPDH (Beyotime) and β-actin (Beyotime). The membranes were exposed using a ChemiDoc XRS + system (Bio-Rad, Hercules, CA, USA).

Total RNA was extracted from tissues or cells using Trizol reagent and reverse transcription into cDNA using a kit (Takara). RT-PCR was performed using suitable primers. Total proteins were extracted from tissues or cells for WB analysis. Antibodies used included SYVN1 (1:1000; Abcam), HMGB1 (1:1000; Proteintech) and GAPDH (1:5000; Proteintech). HRP-conjugated goat anti-rabbit (1:10000; Proteintech) antibodies were used as secondary antibodies.

For immunohistochemistry, fixed liver sections (5 μm thick) were deparaffinized, rehydrated, blocked with 0.3% nonspecific catalase, antigen retrieved with high pressure, and blocked with 10% nonspecific goat serum enzyme. Subsequently, the sections were incubated in polyclonal anti-rat antibody α-SMA (α-smooth muscle actin, diluted 1 : 200; Proteintech), Nrf2 (diluted 1 : 100; Proteintech), SOD2 (diluted 1 : 100; Proteintech), Hsp27 (diluted 1 : 100; Proteintech), and HMGB1 (diluted 1 : 200; Proteintech). After being washed in phosphate-buffered saline (PBS), the sections were incubated with the Polink-1-HRP-DAB Detection System to rabbit antibody for 1 h and visualized using DAB. Finally, the positive expressions of α-SMA, Nrf2, SOD2, Hsp27, and HMGB1 were observed at 400x under a light microscope (Olympus BX 53, Japan). Ten positive areas were randomly taken and analyzed using Image-Pro Plus 6.0 software.

E. ulmoides polysaccharide (EUP, content: 60%, batch number TR20180607, extracted from E. ulmoides leaves) was obtained from Xi'an Tianrui Bio-Tech Co., Ltd. (Xi'an, China). The lipid peroxidation malondialdehyde (MDA) assay kit, dihydroethidium (DHE), and CCK-8 reagent were acquired from Beyotime (Shanghai, China), and the superoxide dismutase (SOD) assay kit was acquired from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). Primary antibodies against Toll-like receptor 4 (TLR4), high-mobility group protein B1 (HMGB1), myeloid differentiation factor 88 (MyD88), NF-κB p65, IKB-α, interferon regulatory factor 1 (IRF-1), β-tubulin, β-actin, and horseradish peroxidase- (HRP-) conjugated secondary antibody were purchased from Proteintech (Wuhan, China). Primary antibodies against tumor necrosis factor-receptor-associated factor 6 (TRAF6), P-p65, and P-IKB-α were purchased from Affinity Biosciences (Cincinnati, OH, USA). HMGB1, TNF-α, and IL-1β ELISA kits were obtained from CUSABIO (Wuhan, China). Fetal bovine serum (FBS), RPMI-1640 medium, penicillin, and streptomycin were purchased from Gibco (Rockville, MD, USA). TLR-4 overexpression plasmid, empty plasmid, and polybrene were purchased from Hanbio Biotechnology Co., Ltd. (Shanghai, China).

Dulbecco’s modified eagle medium (DMEM)/Ham’s F12 medium supplemented with 2% fetal bovine serum (FBS) (Gibco BRL, Scotland, UK), tissue-culture dishes, and 96-well microtiter immunoplates (Nunc, Roskilde, Denmark) were purchased. Antibodies used in the study were for GLAST (1:2000, Chemicon, Temecula, CA, USA), β-actin (1:3000, Cell Signaling, Beverly, MA, USA), TLR4 (1:1000, Proteintech, Chicago, IL, USA), and HMGB1 (1:2000, Proteintech, Chicago, IL, USA). Eritoran and lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) were purchased from Calbiochem (San Diego, CA, USA). Recombinant HMGB1 (rHMGB1) was obtained from Sino Biological Inc. (Beijing, China).
Commercial kits, including HMGB1 ELISA (Elabscience Biotechnology Co., Shanghai, China), lactate dehydrogenase (LDH) release assay (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and Bio-Rad protein assay (Assay Kit II #5000002, Bio-Rad Laboratories, Feldkirchen, Germany), were used following the manufacturers’ instructions.
Immunoreactivities of HMGB1 siRNA and control siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were detected using a Western blot chemiluminescence reagent system (Perkin-Elmer, Boston, MA, USA).

Consecutive 4-μm sections were immunohistochemically stained using the immunoperoxidase technique described previously [38 (link)], with primary antibodies against c-Met (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), HMGB1 (Proteintech Group, Rosemont, IL, USA), RegIV (Biorbyt, St. Louis, MO, USA), and PCDHB8 established in our laboratory [13 (link)], and appropriate secondary antibodies (Medical and Biological Laboratories [39 (link)], Nagoya, Japan) (all 0.2 µg/mL). The tissue sections were then color-developed with diamine benzidine hydrochloride (DAKO, Glostrup, Denmark) and counterstained with Meyer’s hematoxylin (Sigma). For assessing the expression of c-Met, RegIV, and PCDHB9, cells that exhibited immunoreactivity at the cytoplasmic membrane were counted, and the staining intensity was scored between 0 to 1, (where a score of 0.3 was used to describe the expression level in a normal gastric foveolar epithelium). The staining positivity (0–100) was then calculated as the staining strength score multiplied by the staining area (%). The expression of HMGB1 was assessed by determining the percentage of nuclear immunoreactivity in 1000 examined nuclei.