Iso hydrochloride

All animal experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and the National Standard of the People’s Republic of China for Laboratory animal Guidelines for ethical review of animal welfare. Male Sprague-Dawley rats (9-10 weeks, SPF, Guangdong Medical Experimental Animal Center) were housed at 20 ± 3°C and 55% ± 10% humidity, under a 12-h–12-h light/dark cycle. ISO hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) was dissolved and injected subcutaneously at different doses once daily for two days. Choose the best dose for the animal study. The rats were divided into three groups, including control (CTL), ISO injection (ISO), and ISO injection with pirfenidone (PFD) treatment (ISO+PFD) groups. PFD is a broad-spectrum antifibrotic drug that has shown potential in numbers of animal models of fibrosis and clinical trials (Lopez-de La Mora et al., 2015 (link)). One week after injection, PFD (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in water and gavaged for 4 weeks at a dose of 300 mg/kg in the ISO+PFD group. Meanwhile, equal volume of water was gavaged to the control and ISO groups for 4 weeks.

H9C2 rat cardiomyoblast cells obtained from the Korean Cell Line Bank (Seoul, Korea) were maintained in a humidified atmosphere at 37 °C and 5% CO₂ in Dulbecco’s modified Eagle’s medium containing 1 g/L glucose (L-DMEM; Lonza, Switzerland) supplemented with 10% fetal bovine serum (FBS; Serena, Germany) and 1% penicillin/streptomycin. Cells were differentiated over 7 days by incubation in media containing 1% FBS that was changed daily. Experiments were performed 24 to 48 h after seeding of differentiated cells by adding compounds to culture medium. N-Acetyl-L-cysteine (NAC) and curcumin was added 1 h prior to initiation of ISO treatment.
ISO hydrochloride, NAC, and curcumin were from Sigma Aldrich (St. Louis, MO). MitoSOX Red, Lipofectamine RNAi MAX reagent, and GIBCO Opti-MEM media were from Invitrogen Life Technologies (Carlsbad, CA). Antibodies against phosphorylated extracellular-signal-regulated kinase-1/2 (ERK1/2; #9101S), total ERK2 (#9108S) and p53 (#9282S), and anti-rabbit and anti-mouse secondary antibodies were from Cell Signaling Technology (Danvers, MA). Non-targeted small interfering RNA (siRNA), siRNA against p53, and anti-β-actin antibody (#sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, USA).

ISO hydrochloride and wortmannin were procured from Sigma–Aldrich Company, United States. RUP was obtained from ATCO Pharma, Egypt. All other chemicals and reagents, unless specified, were obtained from Sigma-Aldrich, United States.