Silibinin

Silibinin (S0417, Sigma-Aldrich, USA; purity ≥ 98%) was dissolved in dimethyl sulfoxide (DMSO) to form a 100 mM stock and then added to cells at the indicated concentrations. Silibinin and cisplatin (479306, Sigma-Aldrich, USA; purity ≥ 99%) were dissolved in DMSO, polyoxyethylated castor oil (C5135, Kolliphor EL, Sigma-Aldrich, USA), and phosphate-buffered saline (PBS) to be rendered available as a solution for administration in PDTX and xenograft models.

The mice model of Myocardial I/R was performed as described previously 17 (link). Male C57BL/6 mice (8-12 weeks old) were anesthetized by 2% isoflurane inhalation and ventilated mechanically. After left thoracotomy, the LAD was reversibly ligated with a slipknot (6-0 silk sutures). After 30 minutes of ischemia, reperfusion was achieved by releasing the slipknot for 24 hours. Sham-operated mice subjected to equivalent procedure with the absence of myocardial ischemia. Mice were randomized to four groups: sham+vehicle group, I/R+vehicle group, sham+Silibinin group and I/R+Silibinin group. Silibinin (Sigma, powder, S0417) (100mg/kg, dissolved in DMSO) or vehicle was consecutively administered by intraperitoneal injection for 7 days before I/R. To determine anti-fibrotic remodeling of Silibinin, mice were consecutively treated by intraperitoneal injection for 7 days before I/R, and continued for 2 weeks post reperfusion injury.

LO2 is a human foetal hepatocyte cell line that has been previously characterised [91 (link)]. TAMH was a kind gift from the late Prof. Nelson Fausto (University of Washington, Seattle, WA, USA); the isolation of TAMH was previously described [92 (link)]. LO2 was cultured in Dulbecco’s minimum essential medium (DMEM) (Sigma Aldrich, St. Louis, MO, USA) containing 10% v/v foetal bovine serum (FBS). TAMH was cultured in DMEM-F12 (Sigma Aldrich, St. Louis, MO, USA). Cells were incubated at 37 °C in a humidified incubator with 5% CO₂. Stocks of 100 mM silibinin (Sigma Aldrich, St. Louis, MO, USA), 10 mM sulphoraphane (SU) (Sigma Aldrich, St. Louis, MO, USA), 50 mM trans-cinnamaldehyde (CA) (Sigma Aldrich, St. Louis, MO, USA), and 5 M tert-butyl hydroperoxide (TBHP) (Sigma Aldrich, St. Louis, MO, USA) were prepared in dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Louis, MO, USA). Stocks were diluted with culture medium into different concentrations, ensuring that the final concentration of DMSO never exceeded 0.1% v/v.

Silibinin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-acetylcysteine (NAC), ascorbic acid (AA), 3-methyladenine (3-MA), bafilomycin-A1 (Baf-A1), mouse anti-human BNIP3 and rabbit anti-human light chain 3 (LC3) were all obtained from Sigma-Aldrich. Antibodies for rabbit anti-human Bcl-2, β-actin, rabbit anti-human Atg12 and rabbit anti-human Beclin-1 were all purchased from Cell Signaling Technology (Beverly, MA, USA). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazol ylcarbocyanine iodide (JC-1) and dihydroethidium (DHE) were obtained from Molecular Probes. BNIP3 siRNA was purchased from RiboBio Co. (guangdong, China).

The test substance was silibinin (also called silybin, Sigma-Aldrich); naringenin (Sigma-Aldrich) was used as internal standard for the analyses in human plasma and breast tissue, while naproxen (Sigma-Aldrich) was used as internal standard for human urine analysis. Different working solutions of silybin and of internal standards were prepared starting from the stock solutions 1 mg/mL. Stock solutions were prepared in methanol, working solutions were prepared in methanol-water (1:1, v:v). The ranges of the working solutions were 5 ng/mL–5000 ng/mL for human plasma, 5 ng/mL–5000 ng/mL for human urine, and 5 ng/mL–1250 ng/mL for pig muscle. Working solutions were stored at −25 ± 5 °C immediately after preparation.

Silibinin (purity >98%, as assessed by high-performance liquid chromatography) and N-acetyl-L-cysteine (NAC) were procured from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was used to dissolve Silibinin during in vitro experiments. Primary antibodies against Bcl-2 (#3498), cleaved PARP (#5625), Bax (#5023), cyclin D1 (#2922), cleaved caspase-3 (#9661), cyclin-dependent kinase4 (CDK4) (#12790), CDK6 (#3136), cyclin E1 (#4129), E-cadherin (#3195), N-cadherin (#4061), , p-AKT (#9271), AKT (#9272), p-ERK (#9101), ERK (#9102), p-p38 (#9216), p38 (#9212), cyclin D1 (#2922), p-c-Jun (#3270S), and c-Jun (#9165) were procured from Cell Signaling Technology (Danvers, MA, USA). And p53 (sc-1312), SOD1 (sc-11407), SOD2 (sc-18503), β-Actin (sc-47778) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Also, vimentin (ab92547) was obtained from abcam.