The natural compound Zerumbone (hereinafter referred to as ZER), a monocyclic sesquiterpenoid, has been reported to possess many pharmacological properties, including antioxidant and anti-inflammatory properties. This study aimed to investigate the underlying mechanism of ZER against acute liver injury (ALI) in CCl₄-induced mice models. ICR mice were pretreated intraperitoneally with ZER for five days, then received a CCl₄ injection two hours after the last ZER administration and were sacrificed 24 h later. Examination of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and the histopathological analysis confirmed the hepatoprotective effect of ZER. Biochemical assays revealed that ZER pretreatment recovered the activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), restored the glutathione (GSH) reservoir, and reduced the production of malondialdehyde (MDA), all in a dose-dependent manner. Furthermore, administration of ZER in vivo reduced the release amounts of pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) and inhibited the increased protein levels of Toll-like receptor 4 (TLR4), nuclear factor-kappaB (NF-κB) p-p65, and cyclooxygenase (COX-2). Further studies in lipopolysaccharide (LPS)-induced Raw264.7 inflammatory cellular models verified that ZER could inhibit inflammation via inactivating the TLR4/NF-κB/COX-2 pathway. Thus, our study indicated that ZER exhibited a hepatoprotective effect against ALI through its antioxidant and anti-inflammatory activities and the possible mechanism might be mediated by the TLR4/NF-κB/COX-2 pathway. Collectively, our studies indicate ZER could be a potential candidate for chemical liver injury treatment.

Heat stress will stimulate cells of living organisms to generate heat shock proteins (Hsps). In the mouse liver, impacts of heat stress on hepatocyte proliferation, apoptosis and metabolism have not been studied systematically at different temperatures. In this research, the test mice were heated to 40, 42, 44 and 46°C, respectively, for 20 min and recovered at room temperature for 8 h in normal feeding conditions; the control animals were kept at room temperature without heat stress. The expression levels of Hsp70, Pcna, Bax, Bcl2, cytochrome P450 1A2 (CYP1A2), CYP2E1 and analog of CYP3A4 (not reported in mouse before), the parameters reflecting stress strength, cell proliferation, apoptosis and metabolism, were detected by western blotting, immunohistochemistry and semi-quantitative RT-PCR in test and control mice. Haematoxylin–eosin (H&E) staining and TUNEL analysis were further used to study the impacts of heat stress at different temperatures on hepatocellular necrosis and apoptosis. Serum AST and ALT levels, the markers of liver injury, were measured after heat stress at different temperatures. The data show that Hsp70 expression was significantly increased when temperature increased (P < 0.05). At lower temperatures (40 or 42°C), expression of Pcna, CYP1A2 and analog of CYP3A4 were considerably increased (P < 0.05) while hepatocyte necrosis and apoptosis were not induced (P > 0.05). At higher temperatures (44 or 46°C), expression of Pcna was decreased while hepatocyte necrosis and apoptosis were induced (P < 0.05). Expressions of CYP1A2 and analog of CYP3A4 were decreased especially at 46°C (P < 0.05). Expression of CYP2E1 could not be detected to increase at 40°C but was at high levels at 42, 44 and 46°C (P < 0.05). Expressions of AST and ALT were not different between the test mice and control mice at 40°C while they were significantly higher in the test mice than those in the control mice at 42 (P < 0.05), 44 and 46°C (P < 0.01). In conclusion, heat stress at lower temperatures promotes hepatocyte proliferation and improves the metabolic efficiency in mouse liver while heat stress at higher temperatures inhibits hepatocyte proliferation, promotes hepatocyte apoptosis and induces hepatocyte necrosis. This may give a hint to understanding human liver injury in high temperatures. Moreover, it is the first time that the analog of CYP3A4 was detected in mouse hepatocellular cytoplasm. It is worthwhile to dissect its function in future work.