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Objective To evaluate the role of mitochondrial fission in hypoxia-reoxygenation (H/ R) injury to hippocampal neurons of rats.Methods Primarily cultured hippocampal neurons obtained from newborn Wistar rats were randomly divided into 5 groups (n =60 each) using a random number table: normal group (N group), vehicle group (V group), H/R group, H/R + vehicle group (H/R + V group),and mitochondrial division inhibitor group (group M).The cells were cultured in normal culture medium in group N.Dimethyl sulfoxide (DMSO) was added to the culture medium with the final concentration ＜ 0.1％, and the cells were incubated for 40 min in group V.The cells were subjected to 6 h hypoxia, followed by 20 h reoxygenation in H/R, H/R+V and M groups.DMSO was added to the culture medium with the final concentration ＜0.1％ at 40 min before hypoxia in group H/R+V.In group M, mitochondrial division inhibitor Mdivi-1 50 mmol/L (dissolved in DMSO, DMSO concentration ＜0.1％) was added to the culture medium at 40 min prior to hypoxia.Mito Tracker staining was used to examine mitochondrial morphology.Western blot was used to measure the expression of mitochondrial dynamin-related protein 1 (Drp1), mitofusin 2 (Mfn2) , peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α), nuclear respiratory factor-1 (NRF-1), and mitochondrial transcription factor A (TFAM).Multifunctional microplate reader and fluorescent microscope were used to detect the mitochondrial reactive oxygen species (ROS) level.The flow cytometer was used to detect the apoptosis in hippocampal neurons.Apoptosis rate was calculated.Results Compared with group N, the expression of Drp1, PGC-1α, NRF-1 and TFAM was significantly up-regulated, the ROS content and apoptosis rate were increased, and the expression of Mfn2 was down-regulated in group H/R (P＜0.05).Compared with group H/R, the expression of Drp1 was significantly down-regulated, the ROS content and apoptosis rate were decreased, and the expression of Mfn2, PGC-1α, NRF-1 and TFAM was up-regulated in group M (P＜0.05).Conclusion Mitochondrial fission is involved in H/R injury to hippocampal neurons of rats.
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Objective To investigate the effects of diazoxide pretreatment on the expression of hypoxia-inducible factor-1α (HIF-1α) mRNA and p53 mRNA in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation (H/R).Methods The SD rat myocardial microvascular endothelial cells were cultured. The cells were seeded in 96-well plates ( 100 μl/hole) or in 6 cm diameter dishes (2 ml/dish) with the density of 1 ×106/ml and randomly divided into 4 groups ( n = 24 each): normal control group (group C), H/R group, H/R +diazoxide pretreatment group (group DZ) and H/R + diazoxide pretreatment + mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate (5-HD) group (group DZ + 5-HD). The cells were exposed to 2 h hypoxia followed by 2 h reoxygenation. Diazoxide 100 μmol/L and diazoxide 100 μmol/L + 5-HD 100 μmol/L were added to the culture medium 2 h before hypoxia in DZ and DZ + 5-HD groups respectively. The cell vitality, apoptotic rate and expression of HIF-1α mRNA and p53 mRNA were detected at the end of reoxygenation. Results Compared with group C, the cell vitality was significantly decreased, apoptotic rate increased and the expression of HIF- 1α mRNA and p53 mRNA up-regulated in H/R group ( P ＜ 0.01). Compared with group H/R, the cell vitality was significantly increased, apoptotic rate decreased, the expression of HIF-1α mRNA up-regulated and the expression of p53 mRNA down-regulated in group DZ ( P ＜ 0.05 or 0.01 ). 5-HD could inhibit diazoxide pretreatment-induced changes mentioned above (P ＜ 0.05 ). Conclusion Diazoxide pretreatment can reduce H/R injury in rat myocardial microvascular endothelial cells through up-regulating the expression of HIF-1α and down-regulating the expression of p53, and the mechanism is related to activation of mitochondrial ATP-sensitive potassium channels.
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Objective To investigate the effects of diazoxide pretreatment on the expression of NF-κB mRNA and fractalkine (FKN) mRNA in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation (H/R). Methods The SD rat myocardial microvascular endothelial cells were cultured. The cells were seeded in 96-well plates (100μl/hole) or in 6 cm diameter dishes (2 ml/dish) with the density of 1 × 106/ml and randomly divided into4 groups (n = 24 each): Ⅰ normal control group (group C), Ⅱ H/R group, Ⅲ diazoxide pretreatment group (group DZ), Ⅳ diazoxide pretreatment + mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate (5-HD) group (group DZ + 5-HD). The cells were exposed to 2 h hypoxia followed by 2 h reoxygenation. Diazoxide 100 μmol/L and diazoxide 100 μmol/L + 5-HD 100μmol/L were added to the cultured medium 2 h before hypoxia in group DZ and DZ + 5-HD respectively. The cell vitality, apoptotic rate and expression of NF-κB mRNA and FKN mRNA were detected at end of reoxygenation. Results Compared with group C, the cell vitality was significantly decreased, apoptotic rate increased and the expression of NF-κB mRNA and FKN mRNA up-regulated in H/R group. Compared with group H/R, the cell vitality was significantly increased,apoptotic rate decreased and the expression of NF-κB mRNA and FKN mRNA down-regulated in group DZ. 5-HD could inhibit diazoxide pretreatment-induced changes mentioned above. Conclusion Diazoxide pretreatment can reduce H/R injury in rat myocardial microvascular endothelial cells through down-regulating the expression of NFκB and FKN, and the mechanism is related to activation of mitochondrial ATP-sensitive potassium channels.