Hepatoprotective Effect Of Dioscin Against APAP-induced Acute Liver Damage In Vitro And In Vivo

Objective: To investigate the hepatoprotective effect of dioscin againstAPAP-induced acute liver damage, and then to study the possible mechanism.Methods: In the in vitro tests, HepG2cells were pre-incubated with dioscin for6h and then APAP （10mM） was added for24h. Cell viability was evaluated by MTTassay. AST and GSH levels were detected. JC-1and TEM were used to evaluatemitochondrial membrane potential and mitochon-drial morphology. Cell apoptosis andnecrosis were evaluated by AO/EB, TUNEL, DAPI and SCGE assays. In the in vivoexperiments, dioscin （25,50and100mg/kg） was pretreated to mice for5days and thenthe animals were exposed to APAP. Silymarin （200mg/kg） was used as a positive drug.The levels of ALT, AST, MDA, GSH, GSSG and liver index were detected. H&Estaining, TUNEL and DAPI assays were deployed to evaluate liver necrosis andapoptosis. JC-1and TEM were used to evaluate mitochondrial membrane potential andmitochondrial morphology. The levels of Ca²⁺in mitochondria, the expression ofATP2A2and cardiolipin in mitochondrial membrane were also detected to investigatethe mitochondrial function. The possible mechanism was then investigated using2-DEcoupled with MALDI-TOF/TOF-MS/MS. Differentially expressed proteins werevalidated by western blot and ELISA assay. Then, the expression of some mitochon-drial related proteins including CYP2E1、p53、Bax、BAK、Bcl-2and Bid wereinvestigated.Results: The experimental results showed that dioscin inhibited the decreasementviability of HepG2cells induced by APAP and AST release. Mitochondrial membranepotential decrease, cell apoptosis and necrosis were significantly improved bypretreatment with dioscin.Pretreatment with dioscin prior to administration of APAP significan-tly inhibitedALT （2507±484vs.233±86U/L） and AST （702±184vs.106±57U/L） release, decreased hepatic MDA content, GSSG activity and liver index，increased GSH activityin liver homogenates. Dioscin stabilized mitochondrial membrane potential （76±11vs.102±6RFU）, decreased the levels of Ca²⁺in mitochondria （22.2±0.7vs.14.5±0.9RFU）, up-regulated the expression of ATP2A2with4.93times and increased thecontent of cardiolipin in mitochondrial membrane. The hepatoprotective effect ofdioscin at the dose of100mg/kg was even better than the effect produced by silymarinat the dose of200mg/kg. Fifteen differentially expressed proteins were found, and sixof them including sulfite oxidase （Soux）, peroxiredoxin-1（Prdx1）, regucalcin （Rgn）,cytoskeletal18（Krt18）, malate dehydrogenase （Mdh1） and purine nucleosidephosphorylase （Pnp） were validated. Compared with model group, the proteinexpressions of Suox, Prdx1and Rgn were up-regulated with1.65,20.2and1.46times,and the contents of Mdh1and Pnp were increased （p <0.01）, while the expression ofKrt18was down-regulated with1.53times. Deeply study indicated that dioscinmarkedly decreased the protein expressions of CYP2E1, p53, BAK and Bax, andincreased the protein expressions of Bcl-2and Bid.Conclusion: This is the first report on the hepatoprotective effect of dioscinagainst APAP-induced acute liver injury, and this protective effect is related to theregulation of mitochondrial function. The results provide useful information for furtherinvestigation of dioscin and also for prevention of liver diseases in future.