Abstract
Abstract Mitotochondrial function in mammalian is generally presented as the central pathway for energy metabolism, but several other aspects of mitochondrial function have been described, such as key roles in apoptosis, Reactive oxygen species production, thermogenesis and calcium signaling.Reactive oxygen species (ROS), such as superoxide anion(.O2-), hydrogen peroxide(H2O2)and hydroxyl radical(.OH), are by-products of aerobic metabolism in cells. Upon neuronal injury stimulation and release immune modulators such as the potentially damaging nitric oxide (NO) and reactive oxygen species. In this study, major technology used Lascer scanning confocal microscopy observation astrocytes cells line had double staning of ROS generation detected by DCFH-DA （1μM）and △Ψm depolarization detected by TMRM（300 nM）；After exposure ionomycin （100 nM），the study results indicated that ionomycin-induced mROS generation soon reached to a peak at where △Ψm depolarized completely (<20 min) and resulted in severe plasma membrane blebing and apoptotic condensation of nuclei and cell death (<30 min). To confirm ionomycin-triggered MPT as well as to examine whether the MPT involved in the early phase of ionomycin-induced △Ψm depolarization, cells were treated with a classic MPT inhibitor, CsA. No cell death was observed within 30 min of ionomycin exposure. Interestingly, inhibition of MPT significantly reduced ionomycin-induced mROS formation by about 50 % CsA did not completely inhibit ionomycin-induced apoptosis suggesting ionomycin-induced Ca2+ stress together with a MPT-independent mROS formation acted synergistically to cause mitochondrial dysfunction and later apoptotic cell death. To remove ionomycin-induced mROS formation for an “oxidation free-Ca2+ stress” and associated MPT during ionomycin exposure, cells were treated with a classic antioxidant vitamin E to mask ionomycin-induced ROS formation. Ionomycin-induced mROS generation was completely neutralized by vitamin E for an exclusive condition of “oxidation free-Ca2+ stress”. Nevertheless, “oxidation free-Ca2+ stress” induced a depolarization of △Ψm whose time course was slighter slower than that observed in the absence of vitamin E. These results thus suggest that Ca2+ plays a major role in ionomycin-induced apoptosis and “oxidation free-Ca2+ stress” is sufficient to trigger MPT and apoptosis. Melatonin significantly prevented ionomycin-induced depolarization of △Ψm in a manner different from that seen upon CsA treatment. At the end of recording (30 min), ionomycin-induced △Ψm declined to around 40 % of the control level. Its associated apoptotic death of cells was significantly prevented long after ionomycin exposure (>40 min) in the presence of melatonin. These results thus suggest, in addition to anti-mROS formation, melatonin may target furthermore on mechanisms that regulate Ca2+ stress-induced depolarization of △Ψm and Ca2+ stress-mediated MPT. In the presence of vitamin E, melatonin significantly prevented ionomycin-induced apoptosis. Cells survival rate was greatly improved up to near 100 % and to 70% at 30 min and 50 min, respectively, after ionomycin exposure. Melatonin completely prevented “oxidation free-Ca2+ stress”-induced depolarization of △Ψm and associated MPT. When effects of melatonin on “oxidation free-Ca2+ stress”-mediated △Ψm depolarization and MPT was compared to that provided by CsA, melatonin seems provides a better protection than CsA. With the protection provided by CsA in the presence of vitamin E, ionomycin-induced △Ψm depolarization and MPT, although prevented for longer than 26 min, declined to close 20% of the control level at 28 min after ionomycin exposure. effect of melatonin plus CsA on ionomycin-induced △Ψm depolarization and MPT. Interestingly, synergistic protections provided by CsA and melatonin prevented completely ionomycin-induced plasma membrane blebing and apoptotic death long after ionomycin exposure (>70 min). Ionomycin-induced depolarization of △Ψm and MPT was preserved to 80% of the control level even after 70 min of ionomycin exposure As a summary, degrees of protection provided by sole effect of vitamin E, CsA, or melatonin, as well as combination of these compounds during ionomycin exposure are listed below: ionomycin < vitamin E < CsA < melatonin < vitamin E + CsA < vitamin E + melatonin < melatonin + CsA.