Tanshinone IIA and Cryptotanshinone Prevent Mitochondrial Dysfunction in Hypoxia-Induced H9c2 Cells: Association to Mitochondrial ROS, Intracellular Nitric Oxide, and Calcium Levels

Abstract

The protective actions of tanshinones on hypoxia-induced cell damages have been reported, although the mechanisms have not been fully elucidated. Given the importance of nitric oxide (NO) and reactive oxygen species (ROS) in regulation of cell functions, the present study investigated the effects of two major tanshinones, Tanshinone IIA (TIIA) and cryptotanshinone (CT), on hypoxia-induced myocardial cell injury and its relationships with intracellular NO and ROS, calcium, and ATP levels in H9c2 cells. Chronic hypoxia significantly reduced cell viability which accompanied with LDH release, increase in mitochondrial ROS, intracellular NO and calcium levels, decrease in superoxide dismutase (SOD) activity, and cellular ATP contents. TIIA and CT significantly prevented cell injury by increasing cell viability and decreasing LDH release. The protective effects of tanshinones were associated with reduced mitochondrial superoxide production and enhanced mitochondrial SOD activity. Tanshinones significantly reduced intracellular NO and Ca(2+) levels. ATP levels were also restored by TIIA. These findings suggest that the cytoprotective actions of tanshinones may involve regulation of intracellular NO, Ca(2+), ATP productions, mitochondrial superoxide production, and SOD activity, which contribute to their actions against hypoxia injuries.

Figure 1

Effects of TIIA and CT on hypoxia-induced H9c2 cell injury. (a) Cell viability by MTT assay. The cell viability of normoxia was adjusted to 100% (n = 5), ^## P < 0.01  versus hypoxia, ***P < 0.001  versus normoxia. (b) LDH release. The LDH release of normoxia was adjusted to 100% (n = 3), ΔP < 0.05  versus CT,   ^## P < 0.01  versus hypoxia, and ^### P < 0.001  versus hypoxia, ***P < 0.001 versus normoxia. (c) Cellular ATP contents. The cellular ATP contents of normoxia was adjusted to 1 (n = 6), *P < 0.05  versus normoxia, *P < 0.05  versus normoxia, ^# P < 0.05  versus hypoxia, and  ^Δ P < 0.05  versus CT.

Figure 2

Effects of TIIA and CT on hypoxia-induced decrease in intracellular superoxide level and NADPH oxidase activity. (a) Images of cells labelled with DHE in normoxia and hypoxia groups. (b) The quantified value of DHE fluorescence intensity. Represented data are mean value of 500 each cells with 4 independent experiments. (c) Quantitative value of NADPH oxidase activity. Data shown are representative of 4 independent experiments.  **P < 0.01  versus normoxia,  ***P < 0.001  versus normoxia.

Figure 3

Effects of TIIA and CT on hypoxia-induced increase in H₂O₂/ONOO⁻ production. Quantitative value of DCFH-DA fluorescence intensity (n = 4). ^# P < 0.05  versus hypoxia, ^## P < 0.01  versus hypoxia, and **P < 0.01 versus normoxia.

Figure 4

Effects of TIIA and CT on hypoxia-induced increase in mitochondrial superoxide production. (a) Cell images illustrate MitoSOX (red) and DAPI (blue) in each group. (b) The quantified value of MitoSOX fluorescence intensity. Represented data are mean value of 500 each cells with 5 independent experiments. ^# P < 0.05  versus hypoxia, ^## P < 0.01  versus hypoxia, ^### P < 0.001  versus hypoxia, and ***P < 0.001 versus normoxia.

Figure 5

Effects of TIIA and CT on SOD enzyme activity. (a) Cytosolic SOD activity. (b) Mitochondrial SOD activity. Data shown are representative of four independent experiments. **P < 0.01  versus normoxia, ^## P < 0.01  versus hypoxia, and ^### P < 0.001  versus hypoxia.

Figure 6

Effects of TIIA and CT on hypoxia-induced increase in intracellular nitric oxide production. (a) Cell images illustrate DAF-2 (green) and DAPI (blue) in each group. (b) Quantitative DAF-2 fluorescence intensity. Represented data are mean value of 500 each cells with 3 independent experiments (n = 3). ^## P < 0.01  versus hypoxia,  ***P < 0.001  versus normoxia.

Figure 7

Effects of TIIA and CT on hypoxia-induced increase in intracellular calcium level. Data shown are representative of four independent experiments. *P < 0.05  versus normoxia, ^## P < 0.01  versus hypoxia, and ***P < 0.001  versus normoxia.