Infusion of high-KCl
cardioplegic solution (High-KCS) is the most common method used to induce
asystole before cardiac surgery. However, our previous study showed the High-KCS can cause the apoptosis of cardiomyocytes in 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
complementary medicine to reduce this damage. Dilong (earthworm) 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 to reduce myocardial cell damage from High-KCS infusion and further investigated the mechanisms. H9c2 cardiomyoblast cells were cultured in serum-free medium for 4 h and then treated with dilong at 31.25, 62.5, 125, and 250 mg/mL for 24 h, which was 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 that High-KCS-induced cardiomyocyte apoptosis, enhanced the
protein level of pro-apoptotic Bad, released
cytochrome c, and activated
caspase-3 in H9c2 cells. 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 up-regulated, but the Akt for cardiomyocyte survival was greatly deactivated in postcardioplegic H9c2 cardiomyoblast cells. However, dilong was highly protective and totally reversed 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 which is the mediator for dilong attenuated High-KCS stimulated
caspase 3 activation.
MEK (
U0126) inhibitor completely blocked dilong inhibited
caspase 3 activation in High-KCS treated H9c2 cells. The
MEK siRNA was further applied to knockdown
MEK to confirm our finding. We found dilong worked through
MEK to inhibit
caspase 3 activity induced by High-KCS in H9c2 cells. Furthermore, we used the pure component of dilong,
Lumbrokinase, to block the High-KCS effect. Using the microscope to observe the cell viability, we found
Lumbrokinase could reverse the High-KCS effect.
Lumbrokinase could also reduce the
protein levels of
caspase 8,
caspase 9, and
caspase 3, and enhance the survival related
proteins PI3K/Akt and Bcl2. These results demonstrate that dilong could be used as a potential agent to block the side effects caused by High-KCS in CABG surgery patients.