The Endocannabinoid 2-Arachidonoyl Glycerol Induces Apoptosis in Primary Hepatic Stellate Cells and Is Upregulated During Hepatic Fibrogenesis

Aims: The endogenous cannabinoid 2-arachidonoyl glycerol (2-AG) is a lipid mediator that blocks proliferation and induces cell death in many cell types. AIM: To determine 2-AG levels during hepatic fibrogenesis and evaluate its effect on primary hepatic stellate cells (HSCs) and hepatocytes. Methods: Primary rat hepatocytes and rat and human HSCs were isolated from healthy human or rat liver by collagenase perfusion. 2-AG-induced cell death was analyzed by LDH assay and western blot for caspase 3- and PARP-cleavage. Reactive oxygen species (ROS) formation was monitored by DCFDA fluorescence and confocal microscopy. Expression of the cannabinoid receptors CB1 and CB2 was measured by real time PCR. 2-AG was measured by liquid chromatography/mass spectrometry in liver tissue from mice 3 weeks after bile duct ligation (BDL, n=4) or sham operation (n=4). Results: Hepatic 2-AG levels were elevated 2.5-fold three weeks after BDL (p<0.05). Treatment of primary activated human and rat HSCs with 2-AG dose-dependently induced cell death (70% after 24h at 10 μM) and was accompanied by PARP- and caspase 3-cleavage. In contrast, 2-AG did not induce cell death in primary rat hepatocytes at concentrations of up to 100 μM. Rat HSCs expressed 10-fold lower levels of GSH than rat hepatocytes and 2-AG induced an extremely strong increase in ROS production in rat HSCs. Confocal fluorescence microscopy revealed mitochondria as the site of excessive ROS production. Pretreatment with the antioxidant Trolox reduced 2-AG-mediated cell death in HSCs, whereas GSH depletion sensitized HSCs even further to 2-AG. GSH depletion also rendered hepatocytes sensitive to 2-AG induced cell death (32% at 25 μM 2-AG after 24h). CB1 and CB2 were expressed in HSCs, but absent in rat hepatocytes. The CB1 and CB2 receptor antagonists SR 141716 and SR 144628 failed to block 2-AG-induced cell death whereas membrane cholesterol depletion with methyl-β-cyclodextrin efficiently blocked 2-AG-mediated ROS formation and cell death in HSCs. Conclusion: 2-AG is elevated in hepatic fibrosis and induces cell death in HSCs at concentrations found in fibrotic liver tissue. 2-AG induces cell death in HSCs in an ROS- and membrane cholesterol-dependent manner whereas hepatocytes are largely resistant to 2-AG-induced cell death unless depleted of GSH. 2-AG may act as an endogenous antifibrogenic mediator in vivo and may serve as a candidate for antifibrotic therapy.

Key words

2-Arachidonoyl Glycerol - Anti-Fibrotic Therapy - Cell Death - Endocannabinoids - Hepatic Stellate Cells - Hepatocytes