Apoptosis Induced By Natural Products On Hepatic Stellate Cell

Objective: The aim of our study was to clarify the apoptosis pathway induced by aloe emodin. an hydroxyanthraquinone present in aloe vent leaves. and cryptotanshinone present in Salvia miltiorrhiza standard fraction in rat hepatic stellate cells transformed by simian virus 40 (t-HSC/Cl-6). which retain the features of activated rat stellate cells.Methods: Apoptosis was determined by DNA fragmentation. caspase activity assay and western blotting analysis. And Salvia miltiorrhiza standard fraction was introduced orally to rats, which were induced acute or subacute liver injury by carbon tetrachloride in vivo.Results: (1) Treatment of t-HSC/Cl-6 cells with these two compounds (12.5-50 μM aloe emodin or 2.5-10 uM cryptotanshinone) both inhibited t-HSC/Cl-6 cell viability in a dose- and time-dependent manner. (2) The induction of apoptosis by the both compounds was confirmed by typical DNA ladder formation and annexin v-propidium iodide flow-cytometric analysis. (3) Both two compounds treatment on t-HSC/Cl-6 cells caused the activation of caspase-3 and caspase-9. detected with a caspase activity assay, although no change was observed in caspase-8 activity. Western blotting showed caspase-3 and caspase-9 active forms and the subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. Both two compounds induced mitochondrial membrane depolarization; our data also show that cytochrome c increased in the cytosol butdecreased in the mitochondria in a time-dependent manner. After aloe emodin treatment increased Bax and unchanged Bcl-2 levels resulted in an increased Bax/Bcl-2 ratio. The result of ervptotanshinone suggests that full-length Bax resided only in the mitochondrial fraction and it is cleaved into the pl8/Bax. but it is not translocated from cytosolic Bax into the mitochondria: Bcl-2 protein was only found in the mitochondrial fraction, and the mitochondrial Bcl-2 levels were no change. (4) Salvia miltiorrhiza standard fraction reduced serum biomarkers (GOT and GPT) and suppressed the synthesis of a-smooth muscle actin.Conclusions: Thus, our research provides elementary evidence that AE-induced apoptosis involves a mitochondria-associated apoptosis pathway. Cryptotanshinone-induced apoptosis is coupled with release of cytochrome c. Bax confonnational change and activation of caspase-3 in crvptotanshinone-induced apoptosis in vitro, and mitochondria pathway may exist in crvptotanshinone-induced apoptosis.These findings suggest that these compounds are potent apoptosis inducers of hepatic stellate cells.