Activation of autophagy is required for Oroxylin A to alleviate carbon tetrachloride-induced liver fibrosis and hepatic stellate cell activation

Abstract

Liver fibrosis is a reversible pathophysiological process correlated with intense repair and cicatrization mechanisms, and its end-stage cirrhosis is responsible for high morbidity and mortality worldwide. Interestingly, the use of natural products as a realistic option for the treatment of liver fibrosis has broadly been accepted. Oroxylin A, a safe and natural product, shows a wide range of pharmacological activities such as anti-inflammatory, anti-oxidant, and anti-tumor properties. However, the effects of Oroxylin A on liver fibrosis remain poorly understood. In the present study, we sought to determine the effect of Oroxylin A on carbon tetrachloride (CCl₄)-induced liver fibrosis, and to further examine the molecular mechanisms. We found that treatment with Oroxylin A markedly decreased the level of liver injury markers, alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), in a dose dependent manner. Moreover, Oroxylin A treatment remarkably inhibited extracellular matrix (ECM) deposition, and significantly down-regulated the mRNA and protein expression of liver fibrosis markers including α1(I)collagen, fibronectin, alpha-smooth muscle actin (α-SMA), PDGF-βR, and TGF-βR1 in CCl₄-induced murine model of liver fibrosis. Furthermore, experimental results in vitro showed that Oroxylin A treatment reduced the mRNA and protein expression of HSC activation markers, α-SMA, desmin, α1 (I) collagen, fibronectin, TGF-β, and TNF-α, in a dose dependent manner. Attractively, Oroxylin A treatment also markedly up-regulated the expression of autophagy makers, LC3-B, Atg3, Atg4, Atg5, Beclin1/Atg6, Atg7, Atg9, ATG12, and Atg14, and apparently reduced the expression of autophagy substrate p62 in both CCl₄-induced murine model of liver fibrosis and PDGF-BB-treated HSCs. Importantly, inhibition of autophagy by specific inhibitor 3-methyladenine (3-MA) completely abolished Oroxylin A-induced anti-fibrosis effect, indicating that activation of autophagy was required for Oroxylin A to alleviate liver fibrosis. Overall, these results provide novel implications to reveal the molecular mechanism of Oroxylin A-induced anti-fibrosis properties, by which points to the possibility of using Oroxylin A for the treatment of liver fibrosis.

Keywords: Autophagy; Hepatic stellate cell; Liver fibrosis; Molecular mechanism; Oroxylin A.