Protective Effects Of Inonotus Obliquus On Tacrine-induced Hepatotoxicity And Its Potential Mechanism

Hepatotoxicity was the most side effects in drug toxicity achieving10%to15%ofthe total number of drug toxicity in clinical using. With the development of newchemicals, more and more hepatotoxic drugs were used, and the incidence ofdrug-induced liver disease was more and more. The hepatotoxicity restricts the clinicalapplication of many drugs. It is also the main reason of many drugs withdrawn from themarket. Tacrine was the first drug licensed in the USA and Europe for the treatment ofmild to moderate dementia from Alzheimer’s disease. However, tacrine treatment forAlzheimer’s disease results in reversible hepatotoxicity in30—50%of patients. After4to12weeks of treatment, serum alanine aminotransferase (ALT) levels increase, whichseriously limits its clinical use,There is a blind spot in clinical treatment to the liver injury. First, the possibilityof liver injury from drugs can not be predicted in advance. Secondly, there is no preciseand feasible drug against liver damage, and finally the damage mechanism is not clear.Therefore it is important to search for the hepatoprotective agents from natural sourcesand supply through dietary intake.Inonotus obliquus, a white rot fungus, belongs to the hymenochaetaceae family ofBasidomycetes. It grows on birch trees in colder northern climates. There is a richresource of Inonotus obliquus in Heilongjiang and Jilin provinces in China. Inonotusobliquus is often used as a deep color of food coloring raw materials, and also be usedas beverages, condiments, biscuits and other supplements. Inonotus obliquus has beenused as a folk medicine for the prevention and treatment of gastrointestinal,cardiovascular, and cancer and other diseases. A large body of research indicates thatInonotus obliquus possesses antitumor, immunomodulating, hepatoprotective,antibacterial, antiangiogenic and antioxidant activity. However, its molecular mechanisms have not been well documented.Objective: Using HepG2cells were treated with tacrine as a model, the protectiveeffects of Inonotus obliquus polysaccharides (IOP) on tacrine-induced hepatotoxicityand its potential mechanisms will be explored.Methods: In our study, using HepG2cells treated with tacrine as a in vitro model,HepG2cells were pretreated with IOP for1h. The production of reactive oxygen species(ROS) was measured using the2,7-dichlorofluorescein diacetate (DCFH-DA) method.Rhodamine123was used to measure the changes of mitochondrial membrane potential.An immunoperoxidase method using a monoclonal8-OHdG antibody has beendeveloped for detection and quantitation of oxidative damage in mitochondrial DNA.Flow cytometry with AnnexinV-FITC conjugated with propidium iodide staining wasused to study the apoptosis in HepG2cells. The release of cytochrome C and theactivity of Caspase-3were measured using immunolfluorence staining method. Resultsare expressed as means and SDs. Statistical analyses were performed using SPSS17.0.Differences were considered statistically significant when p <0.05.Results: The results demonstrated that HepG2cells challenged with tacrinshowed a significant decrease of the mitochondrial membrane potential, followed by asignificant increase of ROS and8-OHdG formation in mitochondrial DNA in adose-dependent manner (P<0.05). Apoptosis was detected after24h treatment. Therelease of cytochrome c into the cytosol and increase of caspase-3activity were alsodetected after24h-treatments. With the pretreatment of IOP, the results showed that IOPat the concentrations of0.0625mg/ml and0.125mg/ml significantly reducedtacrine-induced ROS production, decrease of the mitochondrial membrane potential,8-OHdG formation in mitochondrial and subsequently the release of cytochrome c intothe cytosol and increase of caspase-3activity, and the apoptosis in HepG2cells.Conclusion: These data suggest that mitochondrial depolarization and ROSformation is a prerequisite for tacrine-induced apoptosis and that the mitochondrialpathway leads to apoptosis in HepG2cells. IOP could attenuate the apoptosis inducedby tacrine in HepG2cells. The protection is probably mediated by an antioxidantprotective mechanism. Consumption of IOP may be a plausible way to preventtacrine-mediated hepatotoxicity.