Figure 6.

YWPC alleviate DOX‐induced reactive oxygen species (ROS) and down‐regulation of transforming growth factor beta (TGF‐β)/smad3 pathway dependent on Nrf2 status, in vitro. (A) The intracellular ROS production of H9C2 cells under the stimulation of DOX (5 μmol/L for 24 h) with or without pre‐treatment of YWPC (50 mg/L for 24 h) was visualized under a fluorescence microscope with 2′, 7′‐dichlorodihydrofluorescein diacetate (DCFDA) probe. (B) The protein expression level of p‐smad3 and smad3 in H9C2 cells was determined by Western blot using phosphorylated smad3 and smad3 antibodies. (C) DOX treatment for 6 h induced the cytosol accumulation of Nrf2, and YWPC pre‐treatment enhanced the nuclear accumulation of Nrf2 activated by DOX; (D) Knockdown of Nrf2 in H9C2 cells was achieved by specific siRNA and the efficiency validated by Western blot analysis. RT‐qPCR was used to determine the expression levels of Nrf2 target genes, HO‐1 (E), NQO‐1 (F), and GCLM (G) in H9C2 cells managed with DOX and/or YWPC, CDDO‐EA Nrf2 siRNA. (H) The TGF‐β1 expression level in the culture supernatants was analysed by Enzyme‐linked immunosorbent assay (ELISA). *P < 0.05, vs. the control; ^# P < 0.05 vs. the DOX. DOX, doxorubicin; YWPC, Yellow Wine Polyphenolic Compounds