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
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
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DOI：
10.1111/jcmm.13973
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发表时间：
2019-02-01
影响因子：
5.3
通讯作者：
Yang, Hsin-Ling
中科院分区：
文献类型：
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作者：
Hseu, You-Cheng;Yang, Ting-Yu;Yang, Hsin-Ling
tgf - β 1在血管纤维化疾病的发病机制中起重要作用。据报道，查尔酮是富含水果和蔬菜的癌症化学预防食品成分。在本研究中，研究了番石榴提取物中天然存在的查尔酮黄卡文A （FKA, 2-30 μ M）在tgf - β 1刺激的血管平滑肌（A7r5）细胞中的抗纤维化和抗氧化特性，以及其潜在的分子作用机制。免疫荧光数据显示，在tgf - β 1刺激的A7r5细胞中，FKA处理可下调F-actin的表达。Western blotting结果显示，FKA处理抑制tgf - β 1刺激下α - sma和纤维连接蛋白的表达。伤口愈合和侵袭实验结果表明，FKA抑制tgf - β 1介导的迁移和侵袭。Western blotting结果显示，FKA可下调MMP-9和MMP-2的表达，上调TIMP-1的表达。进一步的证据表明，FKA降低了tgf - β 1介导的磷酸化和Smad3的转录活性。在A7r5细胞中，FKA可显著逆转tgf - β 1诱导的过量ROS生成，FKA或n -乙酰半胱氨酸（NAC）的抑制作用可显著降低tgf - β 1诱导的p-Smad3激活和伤口愈合迁移。有趣的是，fka介导的抗氧化特性与Nrf2核易位增加和抗氧化反应元件（ARE）荧光素酶活性升高有关。在fka处理的A7r5细胞中，Nrf2/ARE信号的激活伴随着HO-1、NQO-1和γ - gclc基因的诱导。值得注意的是，Nrf2的沉默（siRNA转染）显著降低了FKA介导的抗氧化作用，表明FKA可能通过抑制A7r5细胞中ROS的产生来抑制tgf - β 1诱导的纤维化。本研究结果提示，查尔酮黄蛋白A的抗纤维化和抗氧化活性可能有助于开发基于食品的纤维化疾病化学预防药物。
TGF-beta 1 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 mu M), a naturally occurring chalcone in kava extracts, was evaluated for its anti-fibrotic and antioxidant properties in TGF-beta 1-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-beta 1-stimulated A7r5 cells. Western blotting demonstrated that FKA treatment suppressed the expression of alpha-SMA and fibronectin proteins under TGF-beta 1 stimulation. Findings from wound-healing and invasion experiments showed that FKA inhibits TGF-beta 1-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-beta 1-mediated phosphorylation and the transcriptional activity of Smad3. TGF-beta 1-induced excessive ROS production was remarkably reversed by FKA treatment in A7r5 cells, and inhibition by FKA or N-acetylcysteine (NAC) substantially diminished TGF-beta 1-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 gamma-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-beta 1-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.