Studies On The Protective Effect Of Silibinin Against Isoprterenol-induced Rat Cardiac Myocyte Injury And Apoptosis

Mechanism of silibinin against rat cardiac myocyte apoptosis in vitro was studied in this dissertation. Anti-apoptotic effects of silibinin on beta-adrenergic agonist isoproterenol-induced injury in cultured rat neonatal cardiac myocytes were demonstrated.In recent years, accumulating evidence has strongly suggested that dysregulation of apoptosis is the major cause of various diseases. It was first found in our study that silibinin, a plant flavonoid from seeds and fruits of milk thistle (Silybum marianum [L.] Gaertner) had protective effect against isoproterenol-induced rat cardiac myocyte apoptosis.Assays using the viability, activation of lactate dehydrogenase (LDH), and content of maleic dialdehyde (MDA) were chosen for measuring the degree of cardiac myocytes injury. As a result, silibinin protected isoproterenol-treated rat cardiac myocytes from death, and significantly decreased LDH release and MDA production. Silibinin increased superoxide dismutase (SOD) activity, decreased [Ca²⁺]_i, and increased mitochondrial membrane potential (ΔΨ). Furthermore, the release of pro-apoptotic cytochrome c from mitochondria was reduced by silibinin. Silibinin increased the expression of anti-apoptotic Bcl-2 family protein Bcl-2, and up-regulation of SIRT1 (a NAD⁺-dependent histone deacetylase) inhibited the translocation of Bax from cytoplasm to mitochondria, which caused mitochondrial dysfunction and cell injury. DNA damage, detected by the TUNEL and DNA fragmentation assay, was diminished after treatment of silibinin. Results of nitrite and Western blot assays showed that the amount of NO and the expression of iNOS were decreased after treatment with silibinin, while the expression of caspase-3 proenzyme and digestion of caspase-3 substrates, the inhibitor of caspase-activated DNase (ICAD) and poly-(ADP-ribose) polymerase (PARP), were increased simultaneously. The DNA damage was reversed by down-regulation of p53 phosphorylation after treatment with silibinin. Result of flow cytometric analysis showed that the cell cycle was not affected, and the expression of cell cycle regulatory protein p21 also had no change. Protective effect of silibinin was significantly suppressed by inhibitors, including genistein, manumysin A and GW5074 (inhibitors for protein tyrosine kinase (PTK), Ras and Raf-1 respectively). Exposure of rat cardiac myocyte to isoproterenol alone caused a decreased protein kinase C (PKC) activity, which was prevented by pretreatment with silibinin dose-dependently. Simultaneously, the increased expression of Ras and Raf-1 were blocked by the PKC inhibitor (stauroporine). Furthermore, Western blot analysis showed that an extracelluar signal-regulated kinase (ERK) phosphorylation was increased by silibinin, the expression of phosphorylated p38 MAPK was decreased and total ERK, p38, JNK and phosphorylated JNK MAPK did not change after treatment with both isoproterenol and silibinin. Furthermore, pretreatment of cardiac myocyte with PKC, Ras and Raf inhibitors significantly blocked ERK phosphorylation. Protective effect of silibinin was significantly suppressed by inhibitors, including wortmannin and rapamycin (inhibitors for phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt), respectively). Exposure of rat cardiac myocyte to isoproterenol alone caused a decreased Akt activity, which was reversed by pretreatment with silibinin. Translocation of a nuclear factor NF-κB/p65 to nuclear was increased after silibinin treatment. The effect of silibinin on p53 translocation was opposite to NF-κB/p65. The phosphorylation and degradation of the NF-κB cytosolic anchor IκBαwere consistentent with the changes of NF-κB/p65. Apoptitic-induced protein Caspase-9 activity, as well as FasL, was blocked by silibinin, whereas activated through rapamycin pretreatment.In conclusion, the present results show that silibinin appears to the same protective effect from isoproterenol-induced cell damage and apoptosis, which might broaden the potential therapeutic use of silibinin for the treatment of dysregulated apoptosis-related diseases...