ABSTRACT
Objective To evaluate the role of cyclic adenosine monophosphate-protein kinase A-cAMP response element-binding protein (cAMP-PKA-CREB) signaling pathway in hypoxic preconditioninginduced reduction of propofol-induced central neurotoxicity in the developing rats.Methods A total of 70 SPF male Sprague-Dawley rats,aged 7 days,weighing 10-15 g,were divided into 7 groups (n=10 each) using a random number table method:normal saline group (N group),propofol group (P group),hypoxic preconditioning plus propofol group (HP group),hypoxic preconditioning plus propofol plus PKA inhibitor H89 group (HPH group),propofol plus PKA agonist SP-CAMP group (PS group),normal saline injected via the lateral cerebral ventricle group (NI group),and 5％ dimethyl sulfoxide (DMSO) injected via the lateral cerebral ventricle group (DI group).In P group,propofol 50 mg/kg was intraperitoneally injected,and an increment of propofol 50 mg/kg was given after recovery of righting reflex.The equal volume of normal saline was given instead in N group.In HP group,hypoxic preconditioning (rats were subjected to 5 cycles of 10-min hypoxia of 8％ O2 and 1O-min normoxia of 21％ O2) was performed,and propofol was intraperitoneally injected at 2 h after the end of hypoxic preconditioning and the method was similar to those previously described in P group.In HPH group,H89 5 μmol/5 μl was injected via the lateral cerebral ventricle,and 30 min later the other treatment was similar to those previously described in HP group.In PS group,SP-CAMP 20 nmol/5 μl was injected via the lateral cerebral ventricle,and 30 min later propofol was injected using the method previously described in P group.In NI and DI groups,5 μl normal saline and 5％ DMSO were injected via the lateral cerebral ventricle,respectively.Rats were immediately sacrificed after the righting reflex was recovered,brains were removed and hippocampi were isolated and cut into sections which were stained with haematoxylin and eosin for determination of PKAc and p-CREB positive cells (by i mmuno-histochemistry) and expression of cleaved caspase-3,Bcl-2,Bax,PKAc and posphorylated (p-CREB) protein (by Western blot).Results Compared with N group,the expression of cleaved caspase-3 and Bax was significantly up-regulated,the expression of Bcl-2,PKAc and p-CREB was downregulated,and the percentage of PKAc and p-CREB positive cells was decreased (P＜0.05),hippocampal cells had irregular arrangement,and cells was atrophied in P group.Compared with P group,the expression of cleaved caspase-3 was significantly down-regulated,the expression of Bcl-2,PKAc and p-CREB was up-regulated,and the percentage of PKAc and p-CREB positive cells was increased in HP and PS groups,and the expression of Bax was down-regulated (P＜0.05),the hippocampal cells were arranged neatly,the cytoplasm was abundant,and the nuclei were visible in HP group.Compared with HP group,the expression of cleaved caspase-3 was significantly up-regulated,the expression of Bcl-2,PKAc and p-CREB protein was down-regulated and the percentage of PKAc and p-CREB positive cells was decreased (P＜0.05),the cells had irregular arrangement and shrinked,and nuclear condensation was found in cells in HPH group.Conclusion The mechanism by which hypoxic preconditioning reduces propofol-induced central neurotoxicity may be related to activating cAMP-PKA-CREB signaling pathway in the developing rats.
ABSTRACT
Objective To investigate the role of hypoxia-inducible factor-1α/ hexokinase Ⅱ (HIF-1α/HKⅡ) signaling pathway in the inhibition of hypoxia/reoxygenation (H/R)-induced apoptosis in the rat cardiomyocytes by hypoxic preconditioning (HPC).Methods Primarily cultured cardiomyocytes obtained from the neonatal rats were seeded in culture dishes at the density of 5× 105cells/ml.The cardiomyocytes were attached to the wall for 72 h and then randomly divided into 6 groups (n=15 each) using a random number table:control group (group C);group H/R;group HPC;HIF-1o inhibitor YC-1 group (group YC-1);group HPC + YC-1;dimethyl sulfoxide (DMSO) group.The ceils were exposed to D-Hank solution saturated with 95％ N2 and 5％ CO2 for 4 h,and then cultured in the normal culture atmosphere for 2 h.The cells in YC-1 and DMSO groups were incubated in the culture medium containing 10 μmol/l YC-1 and 0.1％ DMSO (100 μl) for 24 b,respectively.HPC was induced by 3 cycles of 10 min hypoxia followed by 30 min reoxygenation,and H/R injury model was then established in group HPC.In group HPC+YC-1,the cells were incubated for 5 min in the M199 culture medium supplemented with 10 μmol/L YC-1 and 10％ fetal bovine serum,and the other treatments were similar to those previously described in group HPC.After the end of treatments,the apoptosis in cardiomyocytes was detected by TUNEL,the expression of HIF-1α,HKⅡ and cytochrome c was detected by Western blot,and the mitochondrial membrane potential was quantitatively measured using fluorescence.Apoptotic rate was calculated.Results Compared with group C,the apoptotic rate was significantly increased,the mitochondrial membrane potential was significantly decreased,and the expression of HIF-1α,HKⅡ and cytochrome c was significantly up-regulated in group H/R (P＜0.05).Compared with group H/R,the apoptotic rate was significantly decreased,and the mitochondrial membrane potential was significantly increased,and the expression of HIF-1αt and HKⅡ was significantly up-regulated,and the expression of cytochrome c was significantly down-regulated in group HPC (P＜0.05).Compared with group HPC,the apoptotic rate was significantly increased,the mitochondrial membrane potential was significantly decreased,the expression of HIF-1α and HKⅡ was significantly down-regulated,and the expression of cytochrome c was significantly up-regulated in group HPC+YC-1 (P＜0.05).Conclusion The mechanism by which HPC inhibits H/R-induced apoptosis in rat cardiomyocytes is associated with activation of HIF-1α/HKⅡ signaling pathway.