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Abstract
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.
Highlights
Fibrosis can be described as a non‐physiological scarring process in response to chronic diseases, wherein excessive extracellular matrix (ECM) deposition contributes to irreversible tissue damage and the malfunction of vital organs, including the liver, heart, lung, kidney and skin.[1,2] Various chemokines and growth factors are primarily involved in the onset and progression of fibrotic pathology; the cytokine transforming growth factor‐β (TGF‐β) is a crucial regulator of all types of fibrosis.[3]
We found that 24‐ hour TGF‐β1 treatment induced significant migration of A7r5 cells (>3‐fold), which was effectively inhibited by Flavokawain A (FKA) treatment (2 h), particularily at 7.5 and 30 μM doses (Figure 4A and B)
We found that TGF‐β1‐induced substantial increase in A7r5 cell migration (>3‐fold), which was significantly inhibited by FKA pretreatment, at 7.5 and 30 μM doses (Figure 4A and B)
Summary
Fibrosis can be described as a non‐physiological scarring process in response to chronic diseases, wherein excessive extracellular matrix (ECM) deposition contributes to irreversible tissue damage and the malfunction of vital organs, including the liver, heart, lung, kidney and skin.[1,2] Various chemokines and growth factors are primarily involved in the onset and progression of fibrotic pathology; the cytokine transforming growth factor‐β (TGF‐β) is a crucial regulator of all types of fibrosis.[3]. Myofibroblasts, which express α‐smooth muscle actin (α‐SMA) and show an enormous capacity for producing ECM and collagen (types I and III), inhibit the activity of matrix metalloproteinases (MMPs) and are known to be the main effector cells responsible for cardiac fibrosis.[11,12]. In VSMCs, TGF‐β1 increases the phosphorylation of receptor‐associated Smads (Smad[2] and Smad3), which form heterodimers with Smad[4] This complex translocates into the nucleus and triggers the transcription of genes, including fibronectin and type 1 collagen, that are involved in vascular fibrosis.[13,14]. The fibrotic events associated with TGF‐β1 are coincident with the induction of ROS‐producing enzymes and/or reduction of ROS scavenging enzymes.[18,19] In these circumstances, the redox‐sensitive protein nuclear factor E2‐related factor 2 (Nrf2) is reported to be involved in the dynamics of fibrogenesis.[18]. We determined the key molecular proteins demonstrating anti‐ fibrotic effects in FKA and revealed the underlying induction of antioxidant Nrf2/ARE signaling pathways
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