title: Protective mechanisms of N-acetyl-cysteine against pyrrolizidine alkaloid clivorine-induced hepatotoxicity.
abstract: Pyrrolizidine alkaloid (PA) clivorine, isolated from traditional Chinese medicinal plant Ligularia hodgsonii Hook, has been shown to induce apoptosis in hepatocytes via mitochondrial-mediated apoptotic pathway in our previous research. The present study was designed to observe the protection of N-acetyl-cysteine (NAC) on clivorine-induced hepatocytes apoptosis. Our results showed that 5 mM NAC significantly reversed clivorine-induced cytotoxicity via MTT and Trypan Blue staining assay. DNA apoptotic fragmentation analysis and Western-blot results showed that NAC decreased clivorine-induced apoptotic DNA ladder and caspase-3 activation. Further results showed that NAC inhibited clivorine-induced Bcl-xL decrease, mitochondrial cytochrome c release and caspase-9 activation. Intracellular glutathione (GSH) is an important ubiquitous redox-active reducing sulfhydryl (--SH) tripeptide, and our results showed that clivorine (50 microM) decreased cellular GSH amounts and the ratio of GSH/GSSG in the time-dependent manner, while 5 mM NAC obviously reversed this depletion. Further results showed that GSH synthesis inhibitor BSO augmented clivorine-induced cytotoxicity, while exogenous GSH reversed its cytotoxicity on hepatocytes. Clivorine (50 microM) significantly induced cellular reactive oxygen species (ROS) generation. Further results showed that 50 microM Clivorine decreased glutathione peroxidase (GPx) activity and increased glutathione S transferase (GST) activity, which are both GSH-related antioxidant enzymes. Thioredoxin-1 (Trx) is also a ubiquitous redox-active reducing (--SH) protein, and clivorine (50 microM) decreased cellular expression of Trx in a time-dependent manner, while 5 mM NAC reversed this decrease. Taken together, our results demonstrate that the protection of NAC is major via maintaining cellular reduced environment and thus prevents clivorine-induced mitochondrial-mediated hepatocytes apoptosis.
authors.affiliation: Key Laboratory of Standardization of Chinese Medicines of Ministry of Education, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, PR China.
authors.forename: Lili
authors.initials: L
authors.lastname: Ji
authors.forename: Tianyu
authors.initials: T
authors.lastname: Liu
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.forename: Zhengtao
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury / Q000517:prevention & control*; D045304:Cytochromes c / Q000378:metabolism; D003603:Cytotoxins / Q000302:isolation & purification / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000378:metabolism; D005981:Glutathione Synthase / Q000037:antagonists & inhibitors; D005982:Glutathione Transferase / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects*; D006801:Humans; D011763:Pyrrolizidine Alkaloids / Q000302:isolation & purification / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism
chemical_list: C494811:BCL2L1 protein, human; D003603:Cytotoxins; D011763:Pyrrolizidine Alkaloids; D017382:Reactive Oxygen Species; D051020:bcl-X Protein; D013879:Thioredoxins; D045304:Cytochromes c; D005979:Glutathione Peroxidase; D005982:Glutathione Transferase; D053148:Caspase 3; D053453:Caspase 9; D005981:Glutathione Synthase; D005978:Glutathione; C028677:clivorine; D000111:Acetylcysteine