第一部分 中草藥白英透過抑制血管增壓素-II所引起的心肌細胞肥大與凋亡以達到保護效果、第二部分 探討雌激素與雌激素接受體抑制ZAK所造成心肌細胞凋亡之訊息路徑

Abstract

Part 1 Angiotensin II (Ang II) is a very important inducer of cardiovascular disease and may cause cardiac apoptosis and hypertrophy. We used a valued Chinese traditional medicine, Solanum lyratum Thunb (SLT), to evaluate its therapeutic effect on Ang II-induced cardiac apoptosis and hypertrophy. SLT is well known for regulating immune system. It is also reported to exert the functions of anti-oxidation and anti-inflammation; some constituents of SLT have been treated in congestive heart failure and arrhythmia. However, the related mechanisms are not known yet. On this study, Ang II-treated H9c2 cells were administered with SLT at concentrations at 0, 0.5, 1, 2.5, 5 and 10μg/ml. Significant increases of TUNEL-positive cells, caspase-3, 8, 9 levels, pro-apoptotic protein, instability of mitochondria membrane potential, released cytochrome c, intracellular actin polymerization, NFAT3 translocation, and hypertrophic markers, ANP and BNP, were observed in Ang II-treated H9c2 cells. Interesting, these Ang II-enhanced effects were dose-dependently improved by the administration of SLT at all doses except 10 μg/ml, which caused cyto-toxicity and obvious cell damage, and the most effective concentration is at 5 μg/ml SLT. Based on above results, we make a conclusion that Solanum lyratum Thunb might have the potential to inhibit Ang II-induced apoptosis and hypertrophy in H9c2 cardiomyoblast cells. Part 2 Previous studies indicate a mixed lineage kinase, called leucine-zipper and sterile-alpha motif kinase (ZAK) could induce apoptosis through p-JNK activation, and northern blot analysis also reveal that ZAK in heart is the most abundantly level among other organs, implying it might cause cardiac cell death. 17β-Estradiol (E2) and Estrogen receptor-alpha (ERα) are researched widely and well known about the function of cardioprotection. Therefore, this study is to clarify the effect of E2/ERα on ZAK-induced cardiac apoptosis in H9c2 cells. We setup three different ZAK tet-on systems (wild type, constitute active and dominant negative) which exhibit different kinase activity to regulate downstream proteins. H9c2 cell apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling(TUNEL) assay, active-caspase-3, 8, 9, p-JNK, and calcineurin levels were evaluated by western blot analysis .In results, significant increases of TUNEL-positive cells and caspase-3, 8, 9 activities after the overexpression of wide type ZAK with tet-on systems were decreased following the treatment of E2/ERα. Inhibitory test (ICI, an inhibitor of E2/ERα) results showed E2/ERα might inhibit apoptosis through regulating p-JNK. According to the above data, we make a conclusion that E2/ERα may have the potential toinhibit ZAK- induced apoptosis in cardiomyoblast H9c2 cells.