| Part I Oxidative stress and apoptosisThe majority of the free radical within a body is reactive oxygen species (ROS). With the increasing of oxidative reaction and/or when anti-oxidative ability was damaged, the kinetic oxidation-reduction homeostasis would be lost, lead to change in ratio of reduced ion components to oxidized ion components of cells. In this condition, oxidative stress occurs. Active free radical can induce peroxidation injury of lipid, protein, DNA and RNA that conduce to cell physiological function and morphological damages.Apoptosis plays a crucial role in the proper development and function in neurological system. Several recent studies indicated that apoptosis could be induced by exogenous oxidants. ROS may play a central role in many apoptotic pathways. In addition, many types of apoptotic stimuli also result in generation of intracellular reactive oxygen species or are associated with evidence of oxidative stress. Apoptosis can be inhabitable by the addition of antioxidant. The active process of apoptosis, requiring energy, can be finely regulated by intracellular redox condition. However, how and when cellular redox changes regulate the apoptotic process as well as the specific redox-sensitive molecules involved in the regulation of this cellular pathway has not been known.In order to investigate the potential mechanisms between oxidative stress and apoptosis, explore the mechanisms of neuron damage inducing by oxidative stress and apoptosis, the following experiments were designed: if the addition of ROS can result in apoptosis; if apoptosis can be associated with the induction of ROS; if apoptosis can be blocked by the addition of compounds with antioxidant abilities; if the anti-apoptotic protein Bcl-2 can function as an antioxidant. Because the dosage of antioxidant was not explored in many researches, so we used different doses of GSHto study if antioxidative effect was on dose-depend.Twenty-four Wister rats were divided into four groups randomly: 1) OS group: six of them received 100nmol/l CumOOH 30uJ in left striatum.2) Control group.- six of them received the same volume of saline with the same regimen. 3) GSH group: six rats received GSH3,iv., 18h interval. CumOOH was given after last dose of GSH. This group was divided into two subgroups, some were injected GSH 120mg/kg; some 75mg/kg. All these rats were sacrificed at 2h after experiment.HPLC-ECD was used to detect DHBA and SA level in striatum and cortex; Bcl-2 positive ratio was examined by immunohistochemistry method; In situ cell death detection (TUNEL method) was applied to detected apoptotic cells.The following experimental results were obtained.1. Using the salicylate trapping method to determine the DHBA/SA level, measured by HPLC-ECD, to reflect hydroxyl radical ('OH) production, is more sensitive (10-15-10-12mol/L), and higher specific. Salicylic acid (SA) is nontoxic, available for animal experiments and clinic. This method which can examine' OH directly provides a credible mean to study free radical.2. Our present observations demonstrated that lOOumol/1 CumOOH, a kind of organic hydroperoxidative component, injected to striatum could induce lipid peroxidation rapidly, cause marked increasing of 'OH level: 211% of the control in rat cortex (P<0.05) and 241% of the control in striatum (P<0.05) measured by HPLC. We believed that CumOOH-treated rat might be a suitable oxidative stress model for our study.3. Examined by immunohistochemistry, the number of anti-apoptosis protein Bcl-2 positive neurons decreased after CumOOH-treated, only 43% of the control in rat cortex, 45% of the control in striatum (P<0.05).4. In situ cell death detection (TUNEL method) detected no apoptotic cells in control group. CumOOH, an increasing level of oxidative stress results in alteration of redox status, finally causes apoptosis.5. Pretreating rat with different dosage of reduced glutathione (GSH) three times, prior to CumOOH administration, could suppress the CumOOH-induced o...
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