Department of Respiratory and Critical Care Medicine, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China.
Department of Neurosurgery, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China.
Acta Biochim Biophys Sin (Shanghai). 2020 Aug 5;52(8):810-820. doi: 10.1093/abbs/gmaa067.
Isoliquiritigenin (ISL), a natural flavonoid derived from the root of liquorice, has been reported to possess anti-inflammatory and antioxidant activities. Previous studies have found that ISL plays a crucial role in anti-fibrosis of adipose tissue and renal tissue; however, its effect on pulmonary fibrogenesis has not been demonstrated. In this study, we aimed to explore the roles and the underlying mechanisms of ISL in TGF-β1-induced fibrogenesis using human lung fibroblast-derived MRC-5 cells. Cell proliferation and migration were determined by MTT and wound healing assay, respectively. The expression levels of alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 (COLIA1) and fibronectin (FN), microtubule-associated protein light chain 3 (LC3) and related signaling molecules were detected by quantitative real-time PCR, western blot and immunofluorescence assay, correspondingly. EGFP-LC3 transfection was used for autophagy analysis. The results showed that ISL inhibited the TGF-β1-induced proliferation and migration, and down-regulated the expressions of α-SMA, COLIA1 and FN. ISL treatment led to up-regulation of LC3 in TGF-β1-treated MRC-5 cells, accompanied by significant decrease in the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In addition, the inhibitory effects of ISL on TGF-β1-induced fibrogenic features in MRC-5 cells were enhanced by pretreatment with autophagy activator Rapmycin and PI3K/AKT inhibitor LY294002 and reversed by autophagy inhibitor 3-methyladenine and PI3K/AKT activator IGF-1. Taken together, our results demonstrated that ISL could attenuate the fibrogenesis of TGF-β1-treated MRC-5 cells by activating autophagy via suppressing the PI3K/AKT/mTOR pathway. Therefore, ISL holds a great potential to be developed as a novel therapeutic agent for the treatment of pulmonary fibrosis.
异甘草素（ISL）是一种从甘草根中提取的天然类黄酮，具有抗炎和抗氧化活性。先前的研究发现，ISL 在脂肪组织和肾组织的抗纤维化中起关键作用；然而，其对肺纤维化形成的影响尚未得到证实。在这项研究中，我们旨在使用人肺成纤维细胞衍生的 MRC-5 细胞探索 ISL 在 TGF-β1 诱导的纤维化中的作用及其潜在机制。通过 MTT 和划痕愈合试验分别测定细胞增殖和迁移。通过定量实时 PCR、western blot 和免疫荧光试验分别检测α-平滑肌肌动蛋白（α-SMA）、I 型胶原α1（COLIA1）和纤维连接蛋白（FN）、微管相关蛋白轻链 3（LC3）和相关信号分子的表达水平。EGFP-LC3 转染用于自噬分析。结果表明，ISL 抑制 TGF-β1 诱导的增殖和迁移，并下调 α-SMA、COLIA1 和 FN 的表达。ISL 处理导致 TGF-β1 处理的 MRC-5 细胞中 LC3 的上调，同时伴随着磷酸化水平的显著降低。磷脂酰肌醇 3-激酶（PI3K）、蛋白激酶 B（AKT）和雷帕霉素靶蛋白（mTOR）。此外，自噬激活剂 Rapmycin 和 PI3K/AKT 抑制剂 LY294002 预处理增强了 ISL 对 MRC-5 细胞 TGF-β1 诱导的纤维生成特征的抑制作用，而自噬抑制剂 3-甲基腺嘌呤和 PI3K/AKT 激活剂 IGF-1 则逆转了这种抑制作用。总之，我们的研究结果表明，ISL 通过抑制 PI3K/AKT/mTOR 通路激活自噬，可减轻 TGF-β1 处理的 MRC-5 细胞的纤维化。因此，ISL 具有开发为治疗肺纤维化的新型治疗剂的巨大潜力。
