Anisomycin

The high glucose cell model was constructed as described [22 (link),23 (link)]. Briefly, HRMECs were cultured in conditioned medium with 5 mM (serving as the normal glucose (NG) group) or 30 mM (HG group) d-glucose (Sigma, Darmstadt, Germany) and incubated at 37°C with 5% CO₂, to control osmotic pressure, 25 mM mannose was added to the medium. Then, the HG group was treated with or without 10 μM p38 inhibitor SB203580 (MedChemExpress, New Jersey, U.S.A.) or 50 ng/ml p38 agonist Anisomycin (MedChemExpress, New Jersey, U.S.A.) for 24 h. Each inhibitor or agonist was dissolved in dimethyl sulfoxide (DMSO) to a 50 mM concentration for use as stock solutions that were diluted to the required concentrations for in vitro studies.
In another experiment, the HRMECs were treated with or without p38 agonist for 24 h, and then with or without RUNX1 adenovirus (sh- RUNX1) or sh-NC for 12 h. Then, the cells were used in Western blotting and tube formation experiments.

Cepharanthine was bought from Aladdin Chemistry Co., Ltd (C102706-5q), solubilized in DMSO, and stored at −20°C for later use in experiments. DMEM/F12 medium, FBS, pancreatic enzyme, and streptomycin/penicillin were acquired from Gibco (NY, United States). Collagenase II and DMSO were bought from Sigma-Aldrich (Merck KGaA, MO, United States). 17β-oestradiol (E2) was obtained from Selleck (Shanghai, China). Recombinant IL-1β and TNF-α were obtained from R&D Systems (Abingdon, United Kingdom). The radio-immuno precipitation assay (RIPA) lysis buffer and the bicinchoninic acid (BCA) assay kit were purchased from Hangzhou Fude Biological Technology Co., Ltd (Hangzhou, China). Also, 3-methyladenine (3-MA) and anisomycin were obtained from MedChemExpress (Monmouth Junction, NJ).

Male C57BL/6 mice (18–22 g, 5–7 weeks old) used in this research were obtained from GemPharmatech Co., Ltd. (Nanjing, China). The animals were housed five per cage under standard laboratory conditions, with free access to a regular commercial diet and water. The experiment was carried out in the institutional animal care and use committee of the First Affiliated Hospital of Zhejiang University. APAP (HY-66005), NAC (HY-B0215) and Anisomycin (HY-18982) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). All animals were fasted overnight before APAP administration and were administered a single dose of 500 mg/kg APAP by intraperitoneal (i.p.) injection to establish a mouse model of APAP-induced liver injury. NAC (300 mg/kg via i.p. injection) and AMSCs (2 × 10⁵ via tail vein) were treated immediately after APAP administration. A JNK activator, Anisomycin (20 mg/kg), was administered via i.p. injection 30 min prior to APAP injection to further investigate the role of the JNK pathway in mediating the protective effect of AMSCs. For AMSCs delayed treatment assay, AMSCs were injected 1 h, 2 h, 4 h and 6 h after APAP overdosing. Mice were killed 6 and 24 h after receiving APAP treatment. Serum samples and liver tissues were harvested. Liver tissues were flash frozen in liquid nitrogen and stored at −80 °C for further use.