Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Jiangsu Key Laboratory of Therapeutic Material of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Theranostics. 2018 Jan 1;8(3):610-626. doi: 10.7150/thno.22237. eCollection 2018.
Hepatic stellate cells (HSCs) are liver-specific pericytes regulating vascular remodeling during hepatic fibrosis. Here, we investigated how ligustrazine affects HSC pericyte functions. Rat HSC-T6 and human HSC-LX2 cells were cultured, and multiple molecular experiments including real-time PCR, Western blot, flow cytometry, immunofluorescence, electrophoretic mobility shift assay and co-immunoprecipitation were used to elucidate the underlying mechanisms. Molecular simulation and site-directed mutagenesis were performed to uncover the target molecule of ligustrazine. Rats were intoxicated with CCl for evaluating ligustrazine's effects . Ligustrazine inhibited angiogenic cytokine production, migration, adhesion and contraction in HSCs, and activated PPARγ. Selective PPARγ inhibitor GW9662 potently abrogated ligustrazine suppression of HSC pericyte functions. Additionally, HIF-1α inhibitor PX-478 repressed HSC pericyte functions, and ligustrazine inhibited the transcription of HIF-1α, which was diminished by GW9662. Moreover, ligustrazine downregulation of HIF-1α was rescued by knockdown of SMRT, and ligustrazine increased PPARγ physical interaction with SMRT, which was abolished by GW9662. These findings collectively indicated that activation of PPARγ by ligustrazine led to transrepression of HIF-1α via a SMRT-dependent mechanism. Furthermore, molecular docking evidence revealed that ligustrazine bound to PPARγ in a unique double-molecule manner via hydrogen bonding with the residues Ser289 and Ser342. Site-directed mutation of Ser289 and/or Ser342 resulted in the loss of ligustrazine transrepression of HIF-1α in HSCs, indicating that interactions with both the residues were indispensable for ligustrazine effects. Finally, ligustrazine improved hepatic injury, angiogenesis and vascular remodeling in CCl-induced liver fibrosis in rats. We discovered a novel ligand activation pattern for PPARγ transrepression of the target gene with therapeutic implications in HSC pericyte biology and liver fibrosis.
肝星状细胞 (HSCs) 是肝脏特异性周细胞，在肝纤维化过程中调节血管重塑。在这里，我们研究了川芎嗪如何影响 HSC 周细胞功能。培养大鼠 HSC-T6 和人 HSC-LX2 细胞，并进行了包括实时 PCR、Western blot、流式细胞术、免疫荧光、电泳迁移率变动分析和共免疫沉淀在内的多种分子实验，以阐明潜在的机制。进行分子模拟和定点突变以揭示川芎嗪的靶分子。用 CCl 对大鼠进行中毒以评估川芎嗪的作用。川芎嗪抑制 HSCs 中血管生成细胞因子的产生、迁移、黏附和收缩，并激活 PPARγ。选择性 PPARγ 抑制剂 GW9662 可有效阻断川芎嗪对 HSC 周细胞功能的抑制作用。此外，HIF-1α 抑制剂 PX-478 抑制 HSC 周细胞功能，而川芎嗪抑制 HIF-1α 的转录，GW9662 可减少 HIF-1α 的转录。此外，SMRT 敲低可挽救川芎嗪对 HIF-1α 的下调，并且川芎嗪增加了 PPARγ 与 SMRT 的物理相互作用，GW9662 可消除这种相互作用。这些发现共同表明，川芎嗪通过一种依赖 SMRT 的机制激活 PPARγ 导致 HIF-1α 的反式抑制。此外，分子对接证据表明，川芎嗪通过与残基 Ser289 和 Ser342 形成氢键，以独特的双分子方式与 PPARγ 结合。Ser289 和/或 Ser342 的定点突变导致川芎嗪在 HSCs 中对 HIF-1α 的反式抑制丧失，表明与这两个残基的相互作用对于川芎嗪的作用都是必不可少的。最后，川芎嗪改善了 CCl 诱导的大鼠肝纤维化中的肝损伤、血管生成和血管重塑。我们发现了一种新的配体激活模式，用于 PPARγ 对靶基因的反式抑制，这对 HSC 周细胞生物学和肝纤维化具有治疗意义。
