Dilong and Lumbrokinase prevent the High-KCL Cardioplegic Solution Administration Induced Apoptosis and Fibrosis in H9c2 cardiomyoblast cells

• Main Authors: Feng-Yueh Lo, 羅鋒岳
• Other Authors: 黃志揚
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/33773851462909667862

碩士 === 中國醫藥大學 === 中醫學系 === 98 === Infusion of high KCl cardioplegia solution (High-KCS) is the most common method for inducing asystole before cardiac surgery. However, our previous study showed the High-KCS can cause cardiomyocytes apoptosis and might lead to cardiac fibrosis in cardiomyocytes and patients who were administered High-KCS prior to undergoing coronary artery bypass graft (CABG) to treat coronary artery disease (CAD).Therefore, it is urgent today to find a compliment medicine to smoothe this damage. Dilong（earthwarm）has been used as a traditional medicine in China for several thousand years, and extract from the Dilong has been empirically used in Asia for the treatment of vascular disorders.In this study, we applied Dilong extract and its pure component Lumbrokinase to reduce the myocardial cell damage by high KCl cardioplegia solution Infusion and further investigate the mechanisms. H9c2 cardiomyoblast cells were cultured in serum-free medium for 4 h then treated Dilong at 31.25, 62.5, 125 and 250 mg/ml for 24h , and then followed by High-KCS treatment for 3 h to detect the protective mechanisms of Dilong behind cardiomyocyte apoptosis and cardiac fibrosis. Cells were harvested for MTT assay, TUNEL assay, and western blot analysis. We found the High-KCS induced cardiomyocyte apoptosis (TUNEL assay) and enhanced the protein level of pro-apoptotic Bad, cytochrome c released and active caspase-3 in H9c2 cells when exposed to High-KCS. The IGF-I/IGF-IR/ERK pathway involved in non-cardiomyocyte proliferation, and the expression/activation of uPA, Sp-1 and CTGF, which are implicated in the development of cardiac fibrosis were upregulated, but the Akt for cardiomyocyte survival was greatly deactivated in postcardioplegic H9c2 cardiomyblast cells. However, Dilong highly protective and totally reverse the apoptosis and cardiac fibrosis effects induced by High-KCS. Chemical inhibitors P38（SB203580）, JNK（SP600125）, MEK（U0126）, IGF-1（AG1024）and PI3K（LY294002）were applied to investigate who is the mediator for Dilong attenuated high KCl cardioplegia solution (High-KCS) stimulated caspase 3 actvation. MEK（U0126）inhibitor totally block Dilong inhibited caspase 3 activation in high KCl cardioplegia solution (High-KCS) treated H9c2 cells.The MEK siRNA was further applied to knockdown MEK to confirm our finding.We found Dilong mediated through MEK to inhibit caspase3 activity, induced by high KCl cardioplegia solution (High-KCS) in H9c2 cells. Further more, we used the pure component of Dilong, Lumbrokinase, to block the High-KCS effect. Using microscope to observe the cell vibility, we found Lumbrokinase could reverse the High-KCS effect. Lumrokinase could also reduced the protein levels of caspase 8, caspase 9, caspase 3, and enhanced the survival related proteins PI3K/Akt and Bcl2. Taken together, our studies indicated that Dilong and Lumrokinase could be used as potential agents to block the side effects caused by High KCl cardioplegia solution in CABG surgery patients.