Chalcone flavokawain A attenuates TGF‐β1‐induced fibrotic pathology via inhibition of ROS/Smad3 signaling pathways and induction of Nrf2/ARE‐mediated antioxidant genes in vascular smooth muscle cells

TGF‐β1 plays a crucial role in the pathogenesis of vascular fibrotic diseases. Chalcones are reportedly cancer chemo‐preventive food components that are rich in fruits and vegetables. In this study, flavokawain A (FKA, 2‐30 μM), a naturally occurring chalcone in kava extracts, was evaluated for its anti‐fibrotic and antioxidant properties in TGF‐β1‐stimulated vascular smooth muscle (A7r5) cells, as well as its underlying molecular mechanism of action. Immunofluorescence data showed down‐regulated F‐actin expression with FKA treatment in TGF‐β1‐stimulated A7r5 cells. Western blotting demonstrated that FKA treatment suppressed the expression of α‐SMA and fibronectin proteins under TGF‐β1 stimulation. Findings from wound‐healing and invasion experiments showed that FKA inhibits TGF‐β1‐mediated migration and invasion. Western blotting demonstrated that treatment with FKA down‐regulated MMP‐9 and MMP‐2 and up‐regulated TIMP‐1 expression. Further evidence showed that FKA decreased TGF‐β1‐mediated phosphorylation and the transcriptional activity of Smad3. TGF‐β1‐induced excessive ROS production was remarkably reversed by FKA treatment in A7r5 cells, and inhibition by FKA or N‐acetylcysteine (NAC) substantially diminished TGF‐β1‐induced p‐Smad3 activation and wound‐healing migration. Interestingly, FKA‐mediated antioxidant properties were associated with increased nuclear translocation of Nrf2 and elevated antioxidant response element (ARE) luciferase activity. Activation of Nrf2/ARE signaling was accompanied by the induction of HO‐1, NQO‐1 and γ‐GCLC genes in FKA‐treated A7r5 cells. Notably, silencing of Nrf2 (siRNA transfection) significantly diminished the FKA‐mediated antioxidant effects, indicating that FKA may inhibit TGF‐β1‐induced fibrosis through suppressing ROS generation in A7r5 cells. Our results suggested that anti‐fibrotic and antioxidant activities of the chalcone flavokawain A may contribute to the development of food‐based chemo‐preventive drugs for fibrotic diseases.