Molecular mechanisms of cytoprotective effects of tanshinones in H9c2 cells

Danshen is a traditional Chinese herbal medicine and has long been used for relieving cardiovascular disorders like angina and hypertension. Tanshinone IIA (TIIA) and cryptotanshinone (CT) are major bioactive compounds isolated from Danshen and have been reported with actions against inflammation, oxidative stress, and myocardial ischemia-reperfusion injury. However, the mechanisms of these compounds on chronic hypoxia induced apoptosis have not been fully understood. The objective of this study is to investigate cytoprotective effects of tanshinones and a related synthetic compound Com1 on molecular mechanisms involved against hypoxia injury. <br><br>An <em>in vitro</em> chronic hypoxia injury model was set out in H9c2 cell. Cells were subjected to a 8 hr hypoxia period and then various markers for cell apoptosis (cytochrome c, Bcl-2 proteins and membrane potential) and oxidative stress (O₂^-, H₂O₂, superoxide dismutase (SOD) and NO) were determined. H9c2 cells in hypoxia condition exhibited a significant decrease in cell viability. This was accompanied with significant increase in LDH enzyme release and HIF-1α expression. The chronic hypoxia induced cell death was characterized with late stage of apoptosis, increase in caspase 3 activity and cytochrome c release, hyperpolarization of mitochondria membrane potential and expression of pro-apoptosis proteins (Bax and Bak), which was also associated with increased mitochondrial superoxide and intracellular NO productions and inhibited mitochondrial SOD activity. <br><br>TIIA and CT significantly inhibited the late apoptosis rate compared to hypoxia control. These compounds also significantly inhibited caspase 3 activity, cytochrome c release, and hyperpolarization. The hypoxia-induced overexpression of Bax was significantly suppressed by TIIA and CT resulting a decreased ratio of Bax/Bcl-2. In addition, the hypoxia-induced increase in NO and mitochondrial superoxide production, but not hydrogen peroxide/peroxinitrite production, was inhibited by TIIA and CT. TIIA and CT markedly enhanced mitochondria SOD activity compared to hypoxia and normoxia group. TIIA also inhibited the increase in [Ca²⁺]_i and restored hypoxia induced ATP depletion. Similarly, Com1 inhibited the late apoptosis compared to the hypoxia control. It also markedly inhibited hyperpolarization, cytochrome c release and reduced Bax/Bcl-2 ratio. Com1 also inhibited hypoxia-induced productions of NO, mitochondrial superoxide, hydrogen peroxide/peroxinitrite. [Ca²⁺]_i increase vand ATP depletion induced by hypoxia was inhibited or restored by Com1. Com1 was more effective in increase in Bcl-2 protein expression than TIIA. <br><br>The results indicate that TIIA, CT and Com1 all protect against chronic hypoxia induced cell injury and apoptosis. These compounds acts on mitochondrial apoptosis signaling pathway by multiple mechanisms, by reducing mitochondria hyperpolarization, cytochrome c release and caspase 3 activity, and balancing anti- and pro-apoptotic proteins in Bcl-2 family proteins. They also have antioxidant effects as well as effects of regulating cellular ATP content and [Ca²⁺]_i level. These findings provide evidence for molecular mechanisms involved in the cardioprotective actions of tanshinones against hypoxia induced cell injury. As hypoxia is an important contributor of ischemia cardiac injury, preventing hypoxia induced cell damages may minimize ischemia-reperfusion injury. The findings help to develop new therapeutic agents to treat hypoxia/ischemia injury and heart disease. <br>