Nrf2 knockdown attenuates the ameliorative effects of ligustrazine on hepatic fibrosis by targeting hepatic stellate cell transdifferentiation

Abstract

Hepatic fibrosis is a frequent reparative process in response to chronic liver injury and inflammation, which is mainly attributed to hepatic stellate cell (HSC) activation. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was recently highlighted for its negative regulation of HSC behaviors. Our previous studies have revealed the potent antifibrotic effects of ligutrazine without elaborating potential molecular mechanisms. In this work, our in vitro results showed that ligustrazine significantly enhanced Nrf2 expression and nuclear translocation in HSC. Mechanistic investigations using RNAi technology demonstrated that Nrf2 knockdown abolished the ameliorative effects of ligustrazine on serum enzyme activities, hepatic histological architecture, levels of proinflammatory cytokines in serum and liver, intrahepatic inflammatory cell infiltration. Nrf2 shRNA also abrogated the antifibrotic effects of ligustrazine evidenced by increased serum fibrotic biomarkers, hepatic hydroxyproline, profibrogenetic factors in serum and liver, and intrahepatic collagen deposition. Ligustrazine inhibited the induction of CCl4 on β-catenin in HSC, which was cancelled by Nrf2 shRNA lentivirus. In vitro experiments also showed that Nrf2 siRNA abrogated the inhibition of ligustrazine on β-catenin expression. Nrf2 siRNA and IWR-1-endo (a specific antagonist of β-catenin) were applied to investigate the correlation between Nrf2 and β-catenin in mediating the effects of ligustrazine. Results suggested that ligustrazine not only suppressed the viability, contraction, and migration of human HSC but also alleviated lipid droplet loss and extracellular matrix production. Nrf2 siRNA yet weakened the inhibitory action of ligustrazine on HSC behaviors while IWR-1-endo further impaired the suppression of Nrf2 siRNA and restored the capacity of ligustrazine. Collectively, we drew a conclusion that the favorable antifibrotic effects of ligustrazine were attributed to its negative modulation on HSC behaviors by interrupting Nrf2/β-catenin pathway. The findings broaden the width and depth of molecular mechanisms involved in the ligustrazine action, facilitating the development of ligustrazine in antifibrotic therapies.

Keywords: Hepatic fibrosis; Hepatic stellate cell; Ligustrazine; Nuclear factor (erythroid-derived 2)-like 2; β-catenin.