Human il 6

Alternol (99.9% purity) is a kind gift from Strand Biotech Co. Shantou, China and its structural scheme is shown in Figure 1B. It was dissolved in dimethyl sulfoxide (DMSO) as a 10 mmol/l stock solution stored from light in aliquot package in −20°C. The working concentrations used for different experiments were prepared by diluting the stock solution with DMEM‐h. The antibodies used for western blot were as follows: rabbit anti‐actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐caspase‐3, anti‐caspase‐8, anti‐Bcl‐xl, anti‐PARP anti‐p27, anti‐p21, anti‐CyclinB1, anti‐CyclinA2, anti‐CyclinD1, anti‐CDc2, anti‐SAPK/JNK, anti‐phosph‐SAPK/JNK (Tyr183/185), anti‐p38MAPK, anti‐phosph‐p38MAPK (Tyr180/182), anti‐ERK1/2, anti‐phosph‐ERK1/2 (Tyr202/204), anti‐STAT3, anti‐phosph‐STAT3 (Tyr705), anti‐JAK2, anti‐phosph‐JAK2 (Tyr1007/1008), anti‐Src, anti‐phosph‐Src (Tyr416) (Cell Signaling Technology Inc., Danvers, MA, USA), caspase3 inhibitor Z‐VAD‐FMK, SAPK/JNK‐specific inhibitor SP600125, p38MAPK inhibitor SB203580 (Selleck, Selleckchemo Houston, TX, USA), ROS inhibitor antioxidant NAC (Beyotime, Shanghai, China), human IL‐6 (Sigma‐Aldrich, Inc., St. Louis, MO, USA).

The primary HCASMCs (ATCC® PCS-100-021™, American Type Culture Collection, Manassas, VA, USA) were cultured in smooth muscle cell growth medium 2 (#C-22062, PromoCell GmbH, Heidelberg, Germany), and all patient monocyte-derived macrophage cells were cultured in the RPMI-1640 culture media (Sigma-Aldrich Corporation, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 50 μg/mL streptomycin, and 50 U/mL penicillin in 5% CO₂ humidified atmosphere incubator at 37°C to 98%-100% confluence. Cells were subcultured and culture media changed every 48 h. Human CRP (#C1617, Sigma-Aldrich Corporation, St. Louis, MO, USA), human IL-6 (#407652, purity ≥ 95% by SDA-PAGE, Sigma-Aldrich Corporation, St. Louis, MO, USA), human low-density lipoprotein (LDL) (#LP2, purity ≥ 95% by SDA-PAGE, Sigma-Aldrich Corporation, St. Louis, MO, USA), methyllycaconitine (MLA), a selective and potent antagonist of the α7-nAChR, (351344-10-0, caymanchem, USA), and anti-IL-6 receptor antibody (tocilizumab) were also obtained from Sigma-Aldrich. Stock solutions of tocilizumab were prepared at a concentration of 10 mM in double-distilled water (ddH₂O) and stored at -20 °C until use. Methylergonovine (Methergine®) was obtained from Novartis (Novartis Pharmaceuticals Corp., Basel, Switzerland) and nitroglycerin from G. Pohl-Boskamp (Millisrol®; G. Pohl-Boskamp, Hohenlockstedt, Germany).

Murine C2C12 myoblasts and human Huh7 hepatoma cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 20% or 10% fetal bovine serum, respectively, non-essential amino acids, 100 U/mL penicillin and 100 μg/mL streptomycin. The medium was changed every 2–3 days, and the cells were passaged before reaching 80% confluence. For differentiation into myotubes, C2C12 cells were cultured for 2 weeks in media containing 2% fetal bovine serum. For differentiation into a cardiomyocyte-like lineage, C2C12 cells were cultured for 1 week in media containing 2% fetal bovine serum and supplemented with 500 nM retinoic acid. Where indicated, the cells were treated with the following recombinant proteins: 5 ng/mL human/mouse BMP6 and/or BMP2 (R&D Systems, Minneapolis, MN, USA), 20 ng/mL murine IL-6 (Cell Signaling, Danvers, MA, USA), or 10 ng/mL human IL-6 (Sigma-Aldrich, St. Louis, MI, USA).