Abstract: Objective To explore whether the inhibition of endogenous hydrogen sulfide, a novel and important gaseous transmitter generated in mammalian tissues mainly by Cystathionine β-synthase(CBS) or Cystathionine γ-lyase (CSE), induces apoptosis of the rat hepatic BRL cell line in physiological condition. Methods BRL cells were cultured, and divided randomly into several groups in different phases of the experiment. For example, negative-siRNA group, CBS siRNA groups and CSE siRNA groups were used to select the most efficient sequences of siRNAs at 48 or 24 hrs transfection. Solution group and (CBS+CSE) siRNA group were added to detect the variation of apotosis. The BRL cells line were observed and evaluated at 0, 4, 8, 12, and 24 hrs after siRNA transfection. When the mechanisms of apoptosis were detected, CBS/CSE siRNAs were transfected individually or jointly into BRL cells to examine the variation. The genic and protein expression of CBS/CSE were detected by RT-PCR and western blot. After transfection of CBS/CSE siRNA, BRL cells were detected apoptosis by Hoechst stain and Flow cytometry(FCM). The mitochondrial membrane potential(MMP) variation were observed by fluorescent staining. Western blot were used to examine the protein expression of cytochrome C and cleaved-caspase 3. Results CBS and CSE were observed in BRL cells. After transfection of CBS/CSE siRNA, endogenous H2S generation decreased and the apoptosis of BRL cells increased. Accordingly, the expression of intracytoplasm-Cyt C and cleaved-caspase 3 increased. Conclusion These results indicate that inhibition of endogenous H2S synthesis induces apoptosis of BRL cell under physiological condition. All these may involve in mitochondrial pathway of apoptosis.

Key words: Cystathionine β-synthase, Cystathionine γ-lyase, Hydrogen sulfide, siRNA, Apoptosis, BRL cells