The Effects of Different Fluorescent Indicators in Observing the Changes of the Mitochondrial Membrane Potential during Oxidative Stress-Induced Mitochondrial Injury of Cardiac H9c2 Cells.

We evaluated the ability of different fluorescent indicators by various analytical instruments, including a laser scanning confocal microscope (LSCM), fluorescence plate reader, and flow cytometer (FCM), to measure the mitochondrial membrane potential (ΔΨm) of cardiac H9c2 cells during oxidative stress-induced mitochondrial injury. The mitochondrial oxygen consumption rate and a transmission electron microscope were used to detect changes in mitochondrial functions and morphology, respectively. Cardiac H9c2 cells were exposed to H2O2 (500, 750, 1000, and 1250 µM) to induce mitochondrial oxidative stress injury, and fluorescent indicators including tetramethyl rhodamine ethyl ester (TMRE), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1), and rhodamine 123 (R123) were used to detect changes in ΔΨm using an LSCM, fluorescence plate reader, and FCM. The decrease in ΔΨm caused by H2O2 was determined by endpoint and dynamic analyses after staining with JC-1 or TMRE. With the R123 probe, the LSCM could only detect the change in ΔΨm caused by 1000 µM H2O2. Moreover, R123 was less effective than JC-1 and TMRE for measurement of ΔΨm by the LSCM. Our data indicated that an LSCM is the most suitable instrument to detect dynamic changes in ΔΨm, whereas all three instruments can detect ΔΨm at the endpoint.

[Role of mitochondrial permeability transition pore in mediating the inhibitory effect of gastrodin on oxidative stress in cardiac myocytes in vitro].

OBJECTIVE: To explore the role of mitochondrial permeability transition pore (mPTP) in mediating the protective effect of gastrodin against oxidative stress damage in H9c2 cardiac myocytes. METHODS: H9c2 cardiac myocytes were treated with H2O2, gastrodin, gastrodin+H2O2, cyclosporin A (CsA), or CsA+gas+H2O2 group. MTT assay was used to detect the survival ratio of H9c2 cells, and flow cytometry with Annexin V-FITC/PI double staining was used to analyze the early apoptosis rate after the treatments. The concentration of ATP and level of reactive oxygen species (ROS) in the cells were detected using commercial kits. The mitochondrial membrane potential of the cells was detected with laser confocal microscopy. The expression of cytochrome C was detected with Western blotting, and the activity of caspase-3 was also assessed in the cells. RESULTS: Gastrodin pretreatment could prevent oxidative stress-induced reduction of mitochondrial membrane potential, and this effect was inhibited by the application of CsA. Gastrodin significantly lowered the levels of ROS and apoptosis-related factors in H2O2-exposed cells, and such effects were reversed by CsA. CsA significantly antagonized the protective effect of gastrodin against apoptosis in H2O2-exposed cells. CONCLUSIONS: Gastrodin prevents oxidative stress-induced injury in H9c2 cells by inhibiting mPTP opening to reduce the cell apoptosis.