Salvianolic Acid B Inhibits Activation of Human Primary Hepatic Stellate Cells Through Downregulation of the Myocyte Enhancer Factor 2 Signaling Pathway.

Wenwei Zhang,Jian Ping,Gaofeng Chen,Lieming Xu,Yang Zhou

doi:10.3389/fphar.2019.00322

Abstract

Various isoforms of myocyte enhancer factor 2 (MEF2) have been shown to play a role in the activation of rat hepatic stellate cells (HSCs) in culture. The signals that regulate MEF2 in HSCs are unknown. In addition, whether MEF2s regulate the activation of human HSCs (H-HSCs) is unclear. Here, we studied the expression and function of MEF2s in H-HSCs. Our data showed that the levels of MEF2A, C, and D proteins were high in liver tissues from patients with cirrhosis and increased during culture-induced activation of primary H-HSCs. Exposure of H-HSCs to transforming growth factor beta 1 (TGF-β1) led to a significant increase in MEF2A and C protein levels and enhanced MEF2 activity. Interestingly, TGF-β1 did not further enhance MEF2D levels. Furthermore, TGF-β1 activated p38 mitogen-activated protein kinase (MAPK) and led to increased phosphorylation of MEF2C at its p38 recognition site. Inhibition of p38 MAPK inhibited both TGF-β1- and culture-induced activation of MEF2. The activity of collagen I reporter in H-HSCs was significantly reduced when MEF2A and MEF2C were blocked with overexpression of dominant negative MEF2 mutants. Salvianolic-acid B (SA-B), a water-soluble element of Salvia miltiorrhiza known to have anti-fibrosis effects, attenuated both basal and TGF-β1-induced increased levels of MEF2A and C mRNA and protein. In addition, SA-B inhibited MEF2 activity, which correlated with reduced expression of the HSC activation markers, α-smooth muscle actin (α-SMA), and collagen I. Administration of SA-B reduced MEF2A in vivo, which was accompanied by reduced levels of α-SMA in a model of dimethylnitrosamine-induced rat liver fibrosis. We concluded that the MEF2 transcription factor was stimulated by TGF-β1 in H-HSCs. Antagonizing TGF-β1-induced activation of the MEF2 signaling pathway may account in part for the anti-fibrosis effects of SA-B.

Highlights

Hepatic stellate cells (HSCs) are the main cells responsible for liver fibrosis
Polyclonal antibodies for MEF2A and monoclonal antibodies for p-38, p-ERK1/2, and ERK1 were purchased from Santa Cruz Biotechnology (TX, United States); polyclonal antibodies for MEF2C and p-p38, from Cell Signaling; polyclonal antibodies for collagen I, from Calbiochem (CA, United States); monoclonal antibody for MEF2D, from BD Biosciences (MD, United States); and monoclonal antibodies for β-actin and α-smooth muscle actin (α-SMA) from Sigma-Aldrich (St Louis, MO, United States)
Passage 1 human hepatic stellate cells (HSCs) (H-HSCs) was proliferated at a faster rate, and these cells developed a fibroblastic morphology and prominent contractile filaments, similar to those observed in rat HSCs (Figure 1A)

Summary

Introduction

Hepatic stellate cells (HSCs) are the main cells responsible for liver fibrosis. Activation of HSCs, which involves the transition of quiescent cells into highly proliferative, fibrogenic, and contractile myofibroblasts, represents the final common pathway of the hepatic response to liver injury induced by various factors. Many studies suggest that HSCs isolated from rats and humans, and myofibroblasts, their activated counterparts, in particular, express a number of neuronal and muscle cell markers, such as neurotrophins, neurotrophin receptors, brain-derived nerve growth factor, MyoD, and α-smooth muscle actin (α-SMA) (Trim et al, 2000; Geerts, 2001; Vincent et al, 2001; Cassiman et al, 2002) This suggests that factors involved in the regulation of neuronal and muscle cells may play a role in HSCs. our previous studies have shown that the transcription factor, myocyte enhancer factor 2 (MEF2), represents a class of proteins known to play key roles in neurons and muscle cells and participates in the activation of rat HSCs in culture (Wang et al, 2004). Whether and how MEF2 is regulated by TGF-β1 during liver fibrosis is not entirely clear

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