Pifithrin μ

Male Sprague-Dawley rats were obtained from the Animal Center of the Third Military Medical University (Chongqing, China). An experimental HPS rat model was successfully established by CBDL as previously described^{28 (link),29 (link)}. Rats were randomly divided into different groups (no blinding was done). Under isoflurane inhalation anesthesia, the HPS group underwent CBDL, while the control group underwent common bile duct exposure but no ligation. The SMase inhibitor GW4869 (15 mg/kg) (Sigma, USA), p53 inhibitor pifithrin-μ (15 mg/kg) (Sigma, USA), or miR-194 inhibitor anta-miR-194 (2 mg/kg) (GeneChem, China) was administered every 5 days for 5 weeks by intravenous injection following CBDL. Control group rats were injected with saline (0.9% NaCl) containing no drug. Four rats were excluded owing to complications during surgery. Specimens were collected at 5 weeks postoperatively. All rats were housed under standard laboratory living conditions (22–24 °C, 12 h light/12 h dark cycle) and were fed a standard laboratory diet (Altromin, Germany). All procedures performed on the animals were conducted according to the guidelines from the National Institutes of Health. In addition, all experimental protocols were approved by the ethical committee of Third Military Medical University.

CMs from ventricles were dissociated as described in a previous study (Sander et al., 2013 (link)) and cultured at 28.5°C for 24 h in L-15 media with 10% fetal bovine serum (Invitrogen) before being exposed to drugs. Shz-1 (a compound for upregulating Nkx2.5), RA (a compound for inhibiting Calr) and pifithrin-μ (a specific inhibitor of p53) were purchased from Sigma–Aldrich (Shanghai, China) and dissolved in DMSO for storage. Drug treatment conditions included 20 mM glucose for 24 h, 2.5 μM Shz-1 for 72 h, 1 μM RA for 96 h and 10 μM pifithrin-μ for 24 h. The final DMSO concentration was kept at 0.1%.

BALB/c mice were injected intraperitoneally with 10 μg LPS (Sigma-Aldrich) in 1 ml sterile saline. Peritoneal macrophages were harvested 3 days post LPS injection by peritoneal lavage with 6 mL cold PBS and collected by centrifugation. Macrophages were enriched by adherence to plastic, recovered overnight and re-stimulated in vitro with 100 ng/mL LPS and 25 U IFN-γ (R&D systems) at 37°C or 39.5°C for indicated time points. Supernatant was collected for the presence of TNF-, IL-1 (Biolegend), IL-6 (BD Biosciences) and HSP70 (R&D systems) by ELISA. In some experiments, macrophages were treated with HSP70 inhibitors (KNK437, EMD chemicals; Pifithrin-μ, Sigma-Aldrich) together with LPS/IFN-γ.