Ligand Activation of PPARγ by Ligustrazine Suppresses Pericyte Functions of Hepatic Stellate Cells via SMRT-Mediated Transrepression of HIF-1α

Rationale: Hepatic stellate cells (HSCs) are liver-specific pericytes regulating vascular remodeling during hepatic fibrosis. Here, we investigated how ligustrazine affects HSC pericyte functions. Methods: 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(4) for evaluating ligustrazine's effects in vivo. Results: 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(4)-induced liver fibrosis in rats. Conclusions: We discovered a novel ligand activation pattern for PPARγ transrepression of the target gene with therapeutic implications in HSC pericyte biology and liver fibrosis.