Impact of Higenamine on the Hepatic Stellate Cells Activation Stimulated by TGF-β1 in Vitro

Abstract

Objective: The current research aimed to explore the impact of higenamine on the hepatic stellate cells (HCC) activation Stimulated by transforming growth factor-β (TGF-β1) in vitro. Methods: This is a prospective study, we investigated the impact of higenamine on the hepatic stellate cells (HSCs) activation stimulated by TGF-β1 in vitro and Liver fibrosis (LF) stimulated by tetrachloromethane (CCl₄) in vivo. ‌Cell Counting Kit-8 (CCK-8) was adopted for detecting the proliferation of LX-2 cells, HSC stain. Reactive Oxygen Species (ROS) production was determined using fluoroprobe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA). The expression levels of ROS-producing enzymes (NOX₂ and NOX₄), as well as Smad2, p-Smad3, p-Smad2, and Smad3 were quantified via western blot (WB). The mRNA/protein expression of extracellular matrix (ECM) proteins (collagen I (Col I) and α-smooth muscle actin (α-SMA)) was detected via Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) and WB. Haematoxylin and eosin (H&E) dyeing was conducted using liver tissues to examine histopathological damage and fibrosis. The serum levels of fibrosis biochemical markers including hyaluronic acid (HA), PC-III, as well as Col IV were tested by Enzyme-Linked Immunosorbent Assay (ELISA). Results: Higenamine suppressed the proliferation and ROS production in TGF-β1-intervened LX-2 cells. The increased levels of NOX2/NOX4 and NOX activity in TGF-β1-intervened LX-2 cells were reduced by higenamine. Higenamine inhibited the mRNA/protein expression of α-SMA and Col I in TGF-β1-intervened LX-2 cells. Furthermore, the TGF-β1-intevened phosphorylation of Smad3 and Smad2 was attenuated by higenamine. Conclusion: To sum up, these findings showed that higenamine prevented HSCs activation via the TGF-β1/Smad pathway. Higenamine also attenuated CCl₄-caused hepatic damage and fibrosis in vivo. Thus, higenamine is one possible therapeutic agent for LF prevention.