Protective Effects Of ATP Sensitive Potassium Channel Openers On Rats Following Focal Cerebral Ischemia/reperfusion And Its Signaling Transduction Mechanism

PrefaceNowadays ischemic cerebral vascular disease has seriously threatened the health of human being. To find effectively protective drugs for brain is always crucial for clinical studys. ATP sensitive potassium channel (KATP) opener is a new protective drug, but its protective mechanism is still not very clear. The key to treat cerebral ischemia is to alleviate apoptosis in ischemic penumbra. Death receptor signaling pathway and mitochondria signaling pathway are the main signaling transduction pathways. Death receptor signaling pathway and mitochondria signaling pathway are seperatedly characterized by the activation of caspase - 8 and caspase - 9 which then activate down - streamed caspase - 3 and induce neuronal apoptosis. Our experiment aimed at studying the protective effects of K_ATP opener on infarct volumes, scores of neurological function deficits, neuronal apoptosis as well as the expression of caspase - 3, caspase - 8, caspase -9 mRNA and proteins, and investigating its signaling transduction mechanism following focal cerebral ischemia /reperfusion.Materials and Methods220 male Wistar rats were randomly divided into four groups: A ( sham -operated group);B (ischemia/reperfusion group);C (K_A1P opener treatment group) and D ( K_ATP opener and blocker treatment group). The middle cerebral artery occlusion (MCAO) models were established by using the Nagasawa in-traluminal suture occlusion method, the infarct volumes were studied by TTGstaining, scores of neurological function deficits were evaluated, neuronal apop-tosis were detected by TUNEL staining, the expression of caspase - 3, caspase - 8, caspase - 9 mRNA and proteins were detected by immunohistochemical, in situ hybridization and RT - PCR methods, to investigate the protective effects of KATP openers and its signaling transduction mechanism. All data were expressed as mean ± SD. Statistical analysis was carried out by One - way ANOVA.Results1. Infarct volumesThere were not infarcts in A group. The infarcts in B, C and D groups were mainly located in right frontal - parietal cortex, caudate nucleus and putamen nucleus. The infarct volumes of C group were significantly less than B and D groups ( P < 0.01). There were no differences between B and D groups ( P > 0. 05).2. Evaluation of neurological function deficitsCompared with A group, the scores of B, C, D groups were significantly different (P<0.01), the scores of C group were significantly less than B and D groups (P <0.05) , there were no differences between B and D groups (P >0. 05).3. Neuronal apoptosisApoptotic neurons were mainly located around ischemic tissues, that was, ischemic penumbra. Apoptotic cells were rare in the ischemic core region. There were a few apoptotic cells in A group. In B, C and D groups, the numbers of apoptotic cells increased after 6h following cerebral ischemia/ reperfu-sion, then gradually increased as the time went on, at 24h reached the peak, then decreased gradually. The number of apoptotic cells of C group at 6h was not significantly different from B and D groups ( P > 0.05 ) , The number of apoptotic cells of C group at other times were significantly less than B and D groups ( P < 0. 01, P < 0. 05 ) , there were no differences between B and D groups at each time (P>0.05).4. ImmunohistochemistryThere were a few positive cells in A group. The positive cells of B, C and D groups were mainly located in ischemic penumbra. The number of positive cells increased after 6h, then increased as the time went on, at 24h reached the peak, then decreased gradually. The expression of caspase - 8 protein of C group at 6h was not significantly different from B and D groups ( P > 0.05 ) , The expressions of caspase - 3, caspase - 8 and caspase - 9 proteins of C group at other times were significantly lower than B and D groups (P<0.01,P<0.05), there were no differences between B and D groups at each time (P>0.05).5. In situ hybridizationThere were a few positive cells in A group. The positive cells of B, C and D groups were mainly located in ischemic penumbra. The numbers of positive cells increased after 6h, then increased as the time went on, at 24h reached the peak, then decreased gradually. The expression of caspase - 9 mRNA of C group at 6h was not significantly different from B and D groups (P >0.05) , The expressions of caspase - 3, caspase - 8 and caspase - 9 mRNA of C group at other times were significantly lower than B and D groups (P<0.01,P<0.05) , there were no differences between B and D groups at each time (P>0.05).6. Reverse transcription - polymerase chain reaction ( RT - PCR)The expressions of caspase - 3, caspase - 8 and caspase - 9 mRNA in A group were weak. The expressions of caspase - 3, caspase - 8 and caspase - 9 mRNA in B,C,D groups increased at 6h, then increased as the time went on, at 24h reached the peak, then decreased gradually. The expressions of caspase -3, caspase - 8 and caspase - 9 mRNA in C group at all times were significantly less than B and D groups (P <0.01) , there were no differences between B and D groups at each time ( P > 0.05 ).Conclusion1. KATP openers can significantly decrease the infarct volumes, improve neurological function deficits, this result indicates that KATP openers have a protective effect on cerebral ischemia /reperfusion.2. KATP openers have a protective effect on cerebral ischemia /reperfusionby decreasing neuronal apoptosis in ischemia penumbra.3. KATP openers can significantly decrease the expressions of caspase -3, caspase - 8, caspase - 9 mRNA and proteins following cerebral ischemia /reper-fusion, this result indicates that KATP openers can decrease neuronal apoptosis by both death receptor signaling and mitochondria signaling pathways.