| Objective In this study, rats models of ischemia/reperfusion(I/R) injury and models of noninvasive delayed limb ischemic preconditioning(NDLIP) were enrolled to investigate the role of mitochondria ATP-sensitive potassium(mitoKATP) channels and mitochondrial permeability transition pore(m PTP) respectively in the neuroprotective effects of NDLIP against I/R injury and explore the mitochondria-related mechanisms.Methods Healthy male Wistar rats were divided randomly into 9 groups.(1)Sham group: Left common carotid artery was dissociated, while was not occluded.(2) I/R group: rats were subjected to 1h occlusion of middle cerebreal artery followed by 24 h of reperfusion.(3) NDLIP group: rats were subjected to 3 cycles of 5 min of ischemia and reperfusion on the left hind limb for 3 days to induce NDLIP. On the forth day, 1h of ischemia and 24 h of reperfusion was performed.(4) DIA group: rats were tail-introvenously injected with 0.6ml of DIA at 5mg/kg and 1h of ischemia and 24 h of reperfusion was performed 30 min later.(5) 5-HD group: rats were subjected to 3cycles of 5 min of ischemia and reperfusion on the left hind limb for 3 days to induce NDLIP. On the forth day, rats were tail-introvenously injected with 0.6ml of 5-HD at10mg/kg and 1h of ischemia and 24 h of reperfusion was performed 30 min later.(6)NaOH group: rats were tail-introvenously injected with 0.6ml of NaOH(0.1M) and1 h of ischemia and 24 h of reperfusion was performed 30 min later.(7) Atr group: rats were subjected to 3 cycles of 5 min of ischemia and reperfusion on the left hind limb for 3 days to induce NDLIP. On the forth day, rats were intracerebroventricularly injected with 10 ul of Atr(3mM) and 1h of ischemia and 24 h of reperfusion was performed 30 min later.(8) CsA group: rats were intracerebroventricularly injected with 10 ul of CsA(3uM) and 1h of ischemia and 24 h of reperfusion was performed 30 min later.(9) NS group: rats were intracerebroventricularly injected with 10 ul of NS and 1h of ischemia and 24 h of reperfusion was performed 30 min later. Nuerological scores were assessed 24 h after reperfusion. Cerebral infarct size was determinedbased on 2, 3, 5-triphenyltetrazolium chloride staining. expressions of apoptosis-associated proteins Bax, Bcl-2 and Caspase 3 were measured using Western blot method. Mitochondrial membrane potential were assessed with JC-1. Activity of Mitochondrial respiratory chain complexes I, II and IV were detected by spectrophotometer.Results1. Compared with I/R group, the nuerological scores of NDLIP group, DIA group and CsA group decreased(P<0.01). And the scores of 5-HD group, NaOH group, Atr group and NS group increased(P<0.01) compared to NDLIP group.2. Compared with I/R group, cerebral infarct size were diminished(P<0.01) in NDLIP group, DIA group and CsA group. And cerebral infarct size were enlarged(P<0.01) in5-HD group, NaOH group, Atr group and NS group compared to NDLIP group.3. Compared with Sham group, expressions of Bax, Caspase 3 in cortex of Ischemic hemisphere of other groups increased(P<0.01),while expressions of Bcl-2decreased(P<0.01). Compared with I/R group, expressions of Bax, Caspase 3decreased(P<0.01)in NDLIP group, DIA group and CsA group, while expressions of Bcl-2 increased(P<0.01).Compared with NDLIP group, expressions of Bax, Caspase3 increased(P<0.01), while expressions of Bcl-2 decreased(P<0.01).4. Compared with Sham group, Mitochondrial membrane potential of other groups decreased(P<0.01). Compared with I/R group, Mitochondrial membrane potential increased(P<0.01)in NDLIP group, DIA group and CsA group. And mitochondrial membrane potentialdecreased(P<0.01) in 5-HD group, NaOH group, Atr group and NS group compared to NDLIP group.5. Compared with Sham group, activities of mitochondrial respiratory chain complexes in other groups decreased(P<0.01). Compared with I/R group, activities of mitochondrial respiratory chain complexes increased(P<0.01) in NDLIP group, DIA group and CsA group. And the complexes activity decreased(P<0.01) in 5-HD group,NaOH group, Atr group and NS group compared to NDLIP group.Conclusion1. NDLIP can activate the mitoKATP, inhibiting the mPTP, reduce the cerebral infarctarea and alleviate neurological dysfunction after I/R injury, which plays an important role in brain protection.2. After cerebral I/R injury, Bcl-2 expression was inhibited In the injuried hemisphere of brain tissue, Bax and apoptosis protease caspases 3 increased.content decreased in Bcl-2/Bax showed that mitochondria related apoptosis pathway which is regulated by Bcl-2 proteins family has been activated after cerebral I/R injury. NDLIP can significantly inhibit the apoptosis signal pathway,Maybe its mechanism is that it can promoting of the opening of mitoKATP channels and inhibiting the opening of mPTP, which can resist apoptosis.3. After cerebral I/R injury, NDLIP is able to maintain Mitochondrial membrane potential, improve the activity of cerebral cortex mitochondrial oxidative respiratory chain complex I, complex II and complex IV in the injury area. Maybe its mechanism is that it can promoting of the opening of mitoKATP channels and inhibiting the opening of mPTP. Thus it can enhance the ability to resist oxidative damage to mitochondrial oxidative respiratory chain in a directly or indirectly way. |
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