FTO 的抑制作用减弱了益母草碱在大鼠心肌成纤维细胞中的抗纤维化作用。
The inhibition of FTO attenuates the antifibrotic effect of leonurine in rat cardiac fibroblasts.
机构信息
Department of Pharmacy, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China; Institute of Life Science, Chongqing Medical University, Chongqing, 400016, China.
Department of Breast and Thyroid Surgery, Daping Hospital, Army Medical University (Third Military Medical University), Chongqing, 400042, China.
出版信息
Biochem Biophys Res Commun. 2024 Jan 22;693:149375. doi: 10.1016/j.bbrc.2023.149375. Epub 2023 Dec 12.
BACKGROUND
Myocardial fibrosis (MF) is a common pathological condition in cardiovascular diseases that often causes severe cardiac dysfunction. MF is characterized by changes in cardiomyocytes, cardiac fibroblasts (CFs), levels of collagen (Col) -1, -3, and overdeposition of the extracellular matrix. Our previous research showed that leonurine (LE) effectively inhibits collagen synthesis and differentiation of CFs, but the mechanism is not fully elucidated. Recent evidence indicates that fat mass and obesity-associated proteins (FTO) regulates the occurrence and development of MF. This study aimed to explore the role of FTO in the antifibrotic effects of LE.
METHODS
Neonatal rat CFs were isolated, and induced using angiotensin II (Ang II) to establish a cell model of MF. Cell viability, wound healing and transwell assays were used to detect cell activity and migration ability. The protein and mRNA levels of MF-related factors were measured following stimulation with Ang II and LE under normal conditions or after FTO knockdown. The RNA methylation level was measured by dot blot assay.
RESULTS
The results showed that LE (20, 40 μM) was not toxic to normal CFs. LE reduced the proliferation, migration and collagen synthesis of Ang II-induced CFs. Further investigation showed that FTO was downregulated by Ang II stimulation, whereas LE reversed this effect. FTO knockdown facilitated the migration of CFs, upregulated the protein levels of Col-3, α-SMA and Col-1 in Ang II and LE-stimulated CFs, and enhanced the fluorescence intensity of α-SMA. Furthermore, LE reduced N-methyladenosine (mA) RNA methylation, which was partially blocked by FTO knockdown. FTO knockdown also reduced the expression levels of p53 protein in Ang II and LE-stimulated CFs.
CONCLUSIONS
Our findings suggest that the inhibition of FTO may attenuate the antifibrotic effect of LE in CFs, suggesting that FTO may serve as a key protein for anti-MF of LE.
背景
心肌纤维化（MF）是心血管疾病中常见的病理状态，常导致严重的心脏功能障碍。MF 的特征是心肌细胞、心脏成纤维细胞（CFs）、胶原（Col）-1、-3 水平的变化，以及细胞外基质的过度沉积。我们之前的研究表明，益母草碱（LE）能有效抑制胶原合成和 CFs 的分化，但机制尚未完全阐明。最近的证据表明，脂肪量和肥胖相关蛋白（FTO）调节 MF 的发生和发展。本研究旨在探讨 FTO 在 LE 抗纤维化作用中的作用。
方法
分离新生大鼠 CFs，用血管紧张素 II（Ang II）诱导建立 MF 细胞模型。采用细胞活力、划痕愈合和 Transwell 实验检测细胞活性和迁移能力。在正常条件下或 FTO 敲低后，用 Ang II 和 LE 刺激，检测 MF 相关因子的蛋白和 mRNA 水平。采用斑点印迹法检测 RNA 甲基化水平。
结果
结果表明，LE（20、40μM）对正常 CFs 无毒性。LE 降低了 Ang II 诱导的 CFs 的增殖、迁移和胶原合成。进一步研究表明，Ang II 刺激可下调 FTO 的表达，而 LE 可逆转这一作用。FTO 敲低促进了 CFs 的迁移，上调了 Ang II 和 LE 刺激的 CFs 中 Col-3、α-SMA 和 Col-1 的蛋白水平，并增强了 α-SMA 的荧光强度。此外，LE 降低了 N-甲基腺苷（mA）RNA 甲基化，这一作用部分被 FTO 敲低所阻断。FTO 敲低还降低了 Ang II 和 LE 刺激的 CFs 中 p53 蛋白的表达水平。
结论
我们的研究结果表明，抑制 FTO 可能会减弱 LE 在 CFs 中的抗纤维化作用，提示 FTO 可能是 LE 抗 MF 的关键蛋白。
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