Hispidin, a phenolic compound from Phellinus linteus (a medicinal mushroom), has been shown to possess strong anti-oxidant, anti-cancer, anti-diabetic, and anti-dementia properties. However, the cardioprotective efficacy of hispidin has not yet been investigated. In the present study, we investigated the protective effect of hispidin against oxidative stress-induced apoptosis in H9c2 cardiomyoblast cells and neonatal rat ventricular myocytes. While the treatment of H9c2 cardiomyoblast cells with hydrogen peroxide caused a loss of cell viability and an increase in the number of apoptotic cells, hispidin significantly protected the cells against hydrogen peroxide-induced cell death without any cytotoxicity as determined by XTT assay, LDH release assay, Hoechst 33342 assay, and Western blotting of apoptosis proteins such as caspase-3, Bax, and Bcl-2. Our data also shows that hispidin significantly scavenged intracellular ROS, and markedly enhanced the expression of antioxidant enzymes such as heme oxygenase-1 and catalase, which was accompanied by the concomitant activation of Akt/GSK-3β and ERK1/2 phosphorylation in H9c2 cardiomyoblast cells. The effects of hispidin on Akt and ERK phosphorylation were abrogated by LY294002 (a PI3K/Akt inhibitor) and U0126 (an ERK1/2 inhibitor). The effect of hispidin on GSK-3b activities was also blocked by LY294002. Furthermore, inhibiting the Akt/GSK-3β and ERK1/2 pathway by these inhibitors significantly reversed the hispidin-induced Bax and Bcl-2 expression, apoptosis induction, and ROS production. These findings indicate that hispidin protects against apoptosis in H9c2 cardiomyoblast cells exposed to hydrogen peroxide through reducing intracellular ROS production, regulating apoptosis-related proteins, and the activation of the Akt/GSK-3β and ERK1/2 signaling pathways.
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In addition, a group of survival protein kinases which include Akt and ERK1/2 that confer powerful cytoprotection provides an amenable pharmacological target for cardioprotection  . Therefore we speculate that the anti-apoptotic mechanism of hispidin may be responsible for the activation of Akt and ERK1/2.Akt, a serine/threonine kinase, is activated subsequently to the production of PIP3 by phosphatidylinositol-3-kinase (PI3K), and mediates several functions through the phosphorylation and inactivation of the pro-apoptotic kinase, glycogen synthase kinase-3 (GSK-3α/β)  . Akt directly phosphorylates GSK-3β at Ser9, which negatively regulates its kinase activity. Thereafter, phosphorylated GSK-3β inhibits the opening of mitochondrial permeability transition pores  . A number of studies have shown that GSK-3β is the phosphorylation target of PI3K/Akt [34,35] . Persistent inhibition of GSK-3β induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility  . Ischemic preconditioning, an endogenously triggered cardioprotective stimulus, has also been shown to enhance the phosphorylation of Akt, which further leads to phosphorylation and the subsequent inactivation of GSK-3β  . So far, three major MAPK signaling pathways – ERK1/2, p38 MAPK, and JNK – have been identified. They are activated in response to myocardial I/R, and the activation of ERK1/2 (beneficial effect) and p38 MAPK–JNK (a deleterious effect) exerts opposite effects on myocardial apoptosis and cardiac function recovery  . ERK is known to be a signal of the cardiac reperfusion injury salvage kinase (RISK) pathway  . When activated in the setting of cardiac I/R, the ERK1/2 cascade plays an important role in preventing cellular apoptosis  . The activation of either the PI3K/Akt or the ERK1/2 pathway inhibits the conformational change in Bax required for its translocation to the mitochondria, therefore preventing apoptosis [40,41] . Furthermore the up-regulation of ERK1/2 and Akt inactivates the caspase cascade, a proposition which is supported by inhibiting caspase-3 and caspase-9 activation [42,43] . In the present study hispidin increased the phosphorylation of Akt/GSK-3β and ERK1/2 but not p38 MAPK and JNK, which are reduced by H 2 O 2 ( Fig. 4 ). We clearly demonstrated that LY294002, a specific inhibitor of PI3K, can inhibit the functions of downstream target kinases of PI3K, and U0126, a specific inhibitor of ERK1/2, significantly reversed the anti-apoptotic effect of hispidin ( Figs. 4 and 5 ). These findings suggest an essential role of the Akt/GSK-3β and ERK1/2 pathways in hispidin anti-apoptotic activity.
This work was supported by Kyungbok University , National Research Foundation of Korea 322 (NRF) grant funded by the Korea government (MSIP; 2009-0092964 , 2009-0083522 , 2010-323 0017984 , and 2012M3A9D1054520 ), Center for Food and Drug Materials of Agriculture Science and Technology Development 324 ( PJ0079772012 ), Rural Development Administration 325 , the National R&D Program, Ministry of Health and Welfare ( 0620360-1 ), and the Brain Korea 21 Plus Project, Republic of Korea .
All Science Journal Classification (ASJC) codes
- Cell Biology