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

Abstract

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 CCl4 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 CCl4-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.

Blockade of glycolysis-dependent contraction by oroxylin a via inhibition of lactate dehydrogenase-a in hepatic stellate cells

“…Collagen gel contraction assays were performed as we previously described [18]. Percentages of original gel area were quantified using the Image J software (Media Cybernetics, Rockville, MD, USA).…”

“…Cytoskeleton was visualized using FITC-conjugated phalloidin (Beyotime Biotechnology, Haimen, China) according to our previously described methods [18]. The nuclei of cells were stained with diamidino-phenyl-indole (DAPI).…”

Efficacy and Mechanism of a Chinese Classic Prescription of Yueju in Treating Nonalcoholic Steatohepatitis and Protecting Hepatocytes from Apoptosis

“…For Yueju, several active components from this medicine were reported to be effective in regulating these targets. Ligustrazine, which is an active component from Rhizoma Chuanxiong, could directly stimulate PPAR γ by ligand activation to suppress the function of hepatic stellate cells in liver fibrosis [ 35 ]. Geniposide, an active component of Fructus Gardeniae, could enhance leptin signaling to attenuate the level of A β 1–42 in Alzheimer's disease and activate PPAR γ to reduce the blood glucose level in type 2 diabetes [ 36 , 37 ].…”

Targeting Hepatic Stellate Cells for the Treatment of Liver Fibrosis by Natural Products: Is It the Dawning of a New Era?

“…The antifibrotic action of ligustrazine was reported to be suppression of HSCs via SMRT-mediated transrepression of HIF-1a. It was shown to attenuate the HSC activities by inhibition of proangiogenic cytokines, suppression of migration and adhesion, restriction of contraction, and lowering the pericyte functions of HSCs (Zhang et al, 2018). The suppression of angiogenic properties of HSCs was also found to be related to the inhibition of canonical hedgehog signaling.…”