Abstract: 【Objective】 The objective of this study was to investigate the protective effect and mechanism of Inonotus obliquus extract (IOE) on liver injury in aflatoxin B₁ (AFB₁) exposed mice.【Method】 Thirty 6-week-old SPF male C57BL/6 mice with similar body weight were randomly divided into 5 groups.The control group was continuously gavage with distilled water for 10 days.AFB₁ group received continuous intragastric administration of distilled water from day 1 to day 7, and 2 mg/kg AFB₁ from day 8 to 10.Groups with different concentrations of IOE were given continuous intragastric administration of 25, 50 and 100 mg/kg IOE on days 1 to 7, and 2 mg/kg AFB₁ on days 8 to 10, respectively.The intragastric dose was 0.2 mL, once a day.After the experiment, the mice were weighed, blood and liver tissues were collected, and liver index was calculated.The pathological changes of liver were observed by hematoxylin-eosin (HE) staining.The apoptosis of liver cells was analyzed by flow cytometry.The contentss of inflammatory factors interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNF-α) in serum and cells were detected by ELISA.The mRNA expression of TNF-α, IL-6, IL-1β, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) genes in liver and AML12 cells were detected by Real-time quantitative PCR.The expression of NLRP3, Bax and Bcl-2 proteins in liver and AML12 cells were detected by Western blotting.【Result】 Compared with control group, hepatocyte arrangement was disordered, cell swelling and apoptosis rate were extremely significantly increased in AFB₁ group (P＜0.01).The contents of TNF-α, IL-6 and IL-1β in serum were extremely significantly increased (P＜0.01).The expressions of TNF-α, IL-6, IL-1β, NLRP3 and Bax genes in liver were extremely significantly increased, and the expression of Bcl-2 gene was extremely significantly decreased (P＜0.01).The protein levels of NLRP3 and Bax were extremely significantly increased, and the level of Bcl-2 protein was extremely significantly decreased (P＜0.01).Compared with AFB₁ group, inflammatory cell infiltration was decreased, cells were arranged neatly, and cell apoptosis rate was extremely significantly decreased in IOE groups (P＜0.01).Serum TNF-α, IL-6 and IL-1β contents were extremely significantly decreased (P＜0.01).The mRNA expressions of TNF-α, IL-6, IL-1β, NLRP3 and Bax genes in liver were extremely significantly decreased, and the expression of Bcl-2 gene was extremely significantly increased (P＜0.01).NLRP3 and Bax proteins levels were extremely significantly decreased, while Bcl-2 protein levels was extremely significantly increased (P＜0.01).The results of cytotoxicity test showed that the optimal condition for AFB₁-induced AML12 cell damage was 10 μg/mL AFB₁ for 24 h, and the concentration gradients of IOE for prevention and treatment of AFB₁-induced AML12 cell damage were 1.5, 3 and 6 μg/mL, respectively.The results of in vitro test showed that the secretion of inflammatory factors, apoptosis-related genes and protein expression of AML12 cells were consistent with the results of in vivo test.【Conclusion】 IOE could inhibit the secretion of inflammatory factors and reduce apoptosis by regulating the expression of NLRP3, Bax and Bcl-2, thus alleviating the liver injury induced by AFB₁ in mice.

Key words: Inonotus obliquus extract (IOE); aflatoxin B₁ (AFB₁); liver; apoptosis; inflammation