Dilong Prevents the High-KCl Cardioplegic Solution Administration-Induced Apoptosis in H9c2 Cardiomyoblast Cells Mediated by MEK

Abstract

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.

miR-19a protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis via PTEN/PI3K/p-Akt pathway

“…Signal transduction pathways involving PTEN, PI3K, and p-Akt are known to be associated with apoptosis and play an important role in the control of cardiomyocyte survival and function [ 39 ]. Activation of this pathway may be useful in promoting myocyte survival in the damaged heart [ 40 ]. The PI3K has the ability to phosphorylate PIP2 into the secondary messenger PIP3 and activate Akt.…”

Protocatechuic Acid from Alpinia oxyphylla Induces Schwann Cell Migration via ERK1/2, JNK and p38 Activation

“…Chinese herbal medicines have attracted a great deal of attention as alternative and complementary medicines (Zhang and Tang, 2006;Abdelfadil et al, 2013;Hu et al, 2013;Han et al, 2014;Lin et al, 2014). AOF, which is one of the most important traditional Chinese medicines, has been reported to be a potential neuro-protective agent (Yu et al, 2003;Koo et al, 2004;An et al, 2006;Shi et al, 2006;Shui et al, 2006).…”

The effect of earthworm extract on promoting skin wound healing

“…Extraction and use of biologically active compounds from earthworms have been traditionally practiced by indigenous people throughout the world, more particularly in Asia, including China, India, Myanmar, Korea, and Vietnam [ 8 ]. Previous earthworm studies have shown its antiulceral [ 9 ], antioxidative [ 10 ], hepatoprotective [ 11 ], anti-inflammatory [ 12 ], antimicrobial, anticancer [ 13 ], antiapoptosis [ 14 , 15 ], anticoagulation, and fibrinolytic activities [ 16 ]. All of these properties and action modalities may contribute to the acceleration of wound healing process.…”