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Psoralen has potential hepatotoxicity and has a certain promoting effect on the clinical liver injury of Psoralea corylifolia L (Fructus Psoraleae). This study investigated the underlying mechanisms of psoralen-induced hepatotoxicity in vitro . HepG2 cells were treated with psoralen for 6, 12, 24, or 48 h, and an endoplasmic reticulum (ER) stress-specific inhibitor, 4-PBA, was employed to investigate the mechanism of psoralen on ER stress and unfolded protein response (UPR). Cell viability was tested by MTT assay, ATP assay, and cell death by LDH. The apoptosis was reflected by the flow cytometry, caspase-8, and caspase-3 activates. The expression of ER stress-related markers was determined by RT-PCR and western blot. We found that psoralen significantly decreased cell viability, increased activities of caspase-8 and caspase-3, and upregulated expression of CHOP and BAX in a time- and dose-dependent manner. Moreover, psoralen significantly increased the expression and transcription levels of ER stress-related markers, including Grp78, PERK, eIF2α, ATF4, and ATF6, while IRE1α was not significantly affected. And 4-PBA could effectively inhibit psoralen-induced cell death and apoptosis along with the inhibition of ER stress responses. These results suggested that psoralen causes liver injury due to the induction of the ER stress-mediated apoptosis via PERK-eIF2α-ATF4-CHOP and ATF6-CHOP related pathways.

Background & aims Leucine-rich repeat G-protein-coupled receptor 4 (LGR4) and its ligands R-spondin1-4 (Rspos) have been vastly investigated in embryonic development. The biological functions of Rspos-LGR4 system in liver remains largely unknown. Here, we explored whether it protects hepatocytes against hypoxia/reoxygenation (H/R) induced damage. Methods H/R injury was induced by dimethyloxalylglycine (DMOG) in AML12 cells and the effects of Rspo3 on cell proliferation and apoptosis were assessed. Specific shRNAs were used to interfere LGR4 or β-catenin. Results DMOG caused hepatocytes damage evidenced by increase in HIF-1α, cell death and apoptosis genes p27 and Bax , with concurrent decrease of cell proliferation genes PCNA and CyclinD1 . Of all the Rspos, Rspo3 is predominantly expressed in AML12 hepatocytes. Importantly, Rspo3 demonstrated an alteration in a manner similar to proliferation-related genes during H/R injury. Rspo3 pretreatment rendered hepatocytes less vulnerable to DMOG induced H/R injury. Ablation of LGR4 using shRNA attenuated the protective effects of Rspo3. Wnt3a also protected AML12 cells from damages caused by H/R, showing enhanced proliferation activity. Notably, knockdown of β-catenin in hepatocytes completely abolished the effect of Rspo3 pretreatment on the expression levels of PCNA and CyclinD1. Conclusion Rspo3-LGR4 axis protects hepatocytes from H/R injury via activating β-catenin. [ABSTRACT FROM AUTHOR]