Alleviating Effects of Inonotus obliquus Extract on Liver Injury Induced by AFB1 in Mice
[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 AFBi group(P＜0.01).The contents of TNF-a,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.
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