AIF-mediated mitochondrial pathway is critical for the protective effect of diazoxide against SH-SY5Y cell apoptosis.

It has been well established that the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) opener, diazoxide, has protective effects on the heart and brain following ischemia/reperfusion injury. However, the mechanism of the neuroprotective effects of diazoxide remains unclear. This study highlights the anti-apoptotic effects of the drug, which are mediated by specific regulation of apoptosis-inducing factor (AIF) in the process of oxygen and glucose deprivation (OGD)-induced apoptosis in SH-SY5Y cells. Our data showed that pretreatment with diazoxide in SH-SY5Y cells following OGD concentration-dependently increased cell viability. Compared to cells induced by OGD alone, cells pretreated with diazoxide displayed reduced rates of apoptosis, increased mitochondrial transmembrane potential (ΔΨm), and reduced AIF translocation to the cell nucleus. The protective effects of preconditioning with diazoxide were attenuated by 5-hydroxydecanoic acid (5-HD), a selective mitoK(ATP) channel antagonist. Meanwhile, cell death was blocked in OGD-induced cells stably transfected with the AIF-shRNA plasmid, and down-regulation of AIF reduced the diazoxide-mediated prevention of cell apoptosis as well as the loss of ΔΨm induced by OGD. Taken together, our results demonstrate for the first time that the AIF-mediated mitochondrial pathway plays a critical role in the protective effect of diazoxide against SH-SY5Y cell apoptosis induced by OGD. Diazoxide treatment might represent a novel therapeutic target for the treatment of ischemic cerebrovascular diseases.

Effects of ATP sensitive potassium channel opener on the mRNA and protein expressions of caspase-12 after cerebral ischemia-reperfusion in rats.

OBJECTIVE: To investigate effects of K(ATP) opener on the expressions of caspase-12 mRNA and protein, and to explore the role of endoplasmic reticulum (ER) stress pathway in the mechanism of K(ATP) opener protecting against neuronal apoptosis after cerebral ischemia-reperfusion. METHODS: Two hundred rats were randomly divided into four groups: sham operation group, ischemia-reperfusion group, K(ATP) opener group, and K(ATP) blocker group. The middle cerebral artery occlusion (MCAO) model was established by intraluminal suture occlusion method; neuronal apoptosis was detected by TUNEL staining. The mRNA and protein expressions of caspase-12 were detected by semi-quantitative RT-PCR and immunohistochemical staining, respectively. RESULTS: In ischemia-reperfusion group, K(ATP) opener group and K(ATP) blocker group, the number of apoptotic cells and the mRNA and protein expressions of caspase-12 gradually increased following cerebral reperfusion, and reached the peak at 24 h. In K(ATP) opener group, the number of apoptotic cells was significantly less than that in ischemia-reperfusion group and K(ATP) blocker group at 12 h, 24 h, 48 h and 72 h (P< 0.05 or P< 0.01); while the mRNA and protein levels of caspase-12 were significantly less than those in ischemia-reperfusion group and K(ATP) blocker group at all times (P< 0.05 or P< 0.01). There were no differences between the ischemia-reperfusion group and K(ATP) blocker group at each time (P> 0.05). CONCLUSION: K(ATP) opener may protect neurons from apoptosis following the cerebral ischemia-reperfusion by inhibiting ER stress pathway.

Pinacidil reduces neuronal apoptosis following cerebral ischemia-reperfusion in rats through both mitochondrial and death-receptor signal pathways.

OBJECTIVE: To investigate effect of pinacidil, an ATP sensitive potassium channel (K(ATP)) opener, on the neuronal apoptosis and its signaling transduction mechanism following focal cerebral ischemia-reperfusion in rats. METHODS: One hundred male Wistar rats were randomly divided into four groups: A, sham-operated group; B, ischemia-reperfusion group; C, K(ATP) opener treatment group; and D, K(ATP) opener and blocker treatment group. The middle cerebral artery occlusion (MCAO) model was established by using the intraluminal suture occlusion method, neuronal apoptosis was determined by TUNEL staining, and expressions of caspase-8, caspase-9 and caspase-3 mRNA were detected by in situ hybridization. RESULTS: (1) The numbers of apoptotic neurons at 12 h, 24 h, 48 h, and 72 h were significantly less in group C than in groups B and D (P< 0.01 or P< 0.05); and there was no difference between groups B and D at all time points (P> 0.05). (2) The expressions of caspase-3 mRNA and caspase-8 mRNA at all times and the expressions of caspase-9 mRNA at 12 h, 24 h, 48 h, 72 h were significantly lower in group C than in groups B and D (P< 0.01 or P< 0.05); and there were no differences between groups B and D at all time points (P> 0.05). CONCLUSIONS: K(ATP) opener can significantly decrease the neuronal apoptosis and the expressions of caspase-3, caspase-8 and caspase-9 mRNAs following cerebral ischemia-reperfusion. The neuronal apoptosis may be decreased by the inhibition of both mitochondrial and death-receptor signal pathways.