Kaempferol attenuates carbon tetrachloride (CCl4)-induced hepatic fibrosis by promoting ASIC1a degradation and suppression of the ASIC1a-mediated ERS

Abstract

Background: Kaempferol is a flavonoid derived from the herb, Kaempferia galanga L., in addition to exhibiting a wide range of pharmacological properties, kaempferol is also an anti-inflammatory, anti-lipid metabolizing, and anti-oxidative stress agent. The underlying molecular mechanisms of its effects on vascular endothelial growth factor (VEGF) secretion and activation of hepatic stellate cells (HSCs) are yet unknown. Activated HSCs induces VEGF release and extracellular matrix (ECM) accumulation which are important factors in hepatic fibrosis.

Purpose: Our aim is to explore how kaempferol may affect hepatic fibrosis and the mechanisms behind its effects.

Methods: The in vivo model was Sprague-Dawley rats induced with carbon tetrachloride (CCl₄). Histological staining was used to observe histological features of the liver. The levels of (alanine aminotransferase) ALT and (aspartate aminotransferase) AST were detected by the corresponding kits. Platelet-derived growth factor (PDGF) was used to stimulate the HSC-T6 rat hepatic stellate cells. The mechanisms underlying this process were investigated using a variety of molecular approaches, including immunofluorescence, RT-qPCR, and western blotting. Moreover, intracellular Ca²⁺ were observed by laser confocal microscope.

Results: It was found that kaempferol significantly reduced the expression of ASIC1a, VEGF, α-SMA and Collagen-I proteins in a model of CCl₄-induced hepatic fibrosis in rats. In HSC-T6, kaempferol inhibits activation of HSCs by decreasing expression of ASIC1a, eIF2α, p-eIF2α and ATF-4. Laser confocal fluorescence showed that kaempferol inhibited Ca²⁺ influx and reduced Ca²⁺ concentration around the endoplasmic reticulum. Molecular docking and cellular thermal shift assay (CETSA) results further indicated that kaempferol interacted with ASIC1a. We found that kaempferol may promote the degradation of ASIC1a and inhibited ASIC1a- mediated upregulation of ERS.

Conclusion: The data from our in vivo experiments demonstrate that kaempferol effectively attenuates hepatic fibrosis. In vitro studies we further propose a novel mechanism of kaempferol against hepatic fibrosis which can interact with ASIC1a and promote ASIC1a degradation while inhibiting the activation and VEGF release of HSCs by suppressing the ASIC1a-eIF2α-ATF-4 signaling pathway.

Keywords: ASIC1a; ERS; Hepatic fibrosis; Hepatic stellate cell; Kaempferol; VEGF.