| Objective: Cerebral ischemia diseases are common and frequently-occurring illnesses. People pay close attention to these diseases because of higher morbility, higher mutilation rate, higher death rate, even pharmacal treatment limitation. Therefore, it is important to find new pharmacal targets and develop ideal medicines to prevent and cure cerebral ischemia diseases. Many researches find that ATP sensitive potassium channels (KATP) play a significant protective effect not only in myocardial ischemia reperfusion, but also in cerebral ischemia. KATP will become a new target to prevent and cure the cerebral ischemia injury. ATP sensitive potassium channel includes surface ATP sensitive potassium channel (sKATP) and mitochondrial ATP sensitive potassium channel (mitoKATP), KATP contains two parts, which are inwardly rectifying potassium channel (Kir) and sulfonylurea receptor (SUR), the former shapes ion channel, the latter determines the function. It is reported that Kir6.1 mRNA and SUR mRNA express in rodents brain. However, whether the KATP regulators affect the subunit of KATP, whether KATP opener protect the neuron, and what difference between the KATP openers need further study and research. Our research adopts mitoKATP opener (diazoxide),mitoKATP blocker (5-HD),nonselective KATP opener (pinacidil) and nonselective KATP blocker (Glibenclamide) to pretreat gerbil in order to study the expression changes of kir6.1,SUR 1,SUR 2 three subunits after ischemia reperfusion. And we also adopt regulators to pretreat hippocampal neurons to observe changes of their morphology,survival rate,apoptosis rate and LDH contents in vitro.Methods:1. The I-R models of gerbil were performed by occlusion common carotid artery for 10 min and reperfusion for 60 min. 48 gerbils were randomly divided into eight groups: sham-operated group, I-R group, I-R+diazoxide pretreatment group, I-R+5-HD pretreatment group, I-R+diazoxide+5-HD pretreatment group, I-R+pinacidil pretreatment group, I-R+glibenclamide pretreatment group, I-R+pinacidil+glibenclamide pretreatment group. The gerbil was injected with KATP openers and blockers respectively, the morphology changes of brain tissue was observed after HE dyeing, the expressions of Kir6.1, SUR1, SUR2 mRNA in brain tissue were detected by RT-PCR, and the expression of Kir6.1 protein was detected by Western Blot.2. Primary cultured newborn rat hippocampal neurons were treated with 95% CO2 and 5%N2. They were randomly divided into ten groups: Normal group, hypoxia-reperfusion group, H-R+diazoxide pretreatment group , H-R+5-HD pretreatment group, H-R+diazoxide+5-HD pretreatment group, H-R+pinacidil pretreatment group, H-R+glibenclamide pretreatment group, H-R+pinacidil+glibenclamide pretreatment group, H-R+diazoxide+ glibenclamide pretreatment group, H-R+pinacidil+5-HD pretreatment group. Trypan blue stain,Hoechst33342 stain,flow cytometry were used to detect cell survival rate,apoptosis rate and LDH contents.Results:1. The effect of KATP channel regulators on the histomorphology and the expression of subunits on the reperfusion injury in gerbil brain:①Histomorphology: the brain tissue of sham-operated group is normal, endochylema and nucleus are clear; compared with sham-operated group, I-R group and regulators pretreated group display neuron swelled and neurocytes arranged disorder; compared with I-R group, I-R+D group and I-R+P group neuron injury ligher; compared with I-R+D group, I-R+D+5HD group displays unevenness colouration and neuron arranged more disorder, I-R+P group neuron injury a little severe; compare with I-R+P group, I-R+P+G group disorders obviously.②RT-PCR: Compared with sham-operated group, the expression of Kir6.1 mRNA in I-R group was increased obviously. Compared with I-R group, the expression of Kir6.1 mRNA in diazoxide pretreatment group was increased significantly, whereas that in glibenclamide treatment group was decreased obviously (P<0.01). Compared with sham-operated group, the expression of SUR2 mRNA was increased both in I-R groups and pretreatment groups; however, there was no difference among Katp opener and blocker groups. And the expression of SUR1 mRNA was no difference in sham-operated group, I-R group and pretreatment groups.③Western Blot results: The expression of Kir 6.1 protein was no difference in sham-operated group, I-R group and pretreatment groups.2. The effect of Katp channel regulators on the survival rate, apoptosis rate and LDH contents of hippocampal neuron on the hypoxia reperfusion injury:①Neuron survival rate: Compared with normal group ( 94.7±2.28 )%, H-R group and pretreatment group significantly decreased (P<0.01); compared with H-R group (73.2±3.51 ) %, H-R+D group ( 85.2±4.71 ) % and H-R+P group ( 84.6±1.94) % significantly increased (P<0.01); compared with H-R+D group, H-R+D+5HD group (75.6±3.14) % and H-R+D+G group (68.2±5.11) % significantly decreased (P<0.01), H-R+P group decreased (P>0.05); compared with H-R+D+5HD group, H-R+D+G group significantly decreased, while H-R+P+5HD group ( 74.3±6.08 ) % decrease but not obviously (P<0.05).②Neuron apoptosis rate: Compared with normal group ( 12.40±0.07) % , H-R group and pretreatment group significantly increased (P<0.01); compared with H-R group (49.51±2.58 ) %, H-R+D group ( 23.82±0.14) % and H-R+P group ( 37.05±0.67)% significantly decreased (P＜0.01); compared with H-R+D group, H-R+D+5HD group( 34.81±0.83 )% , H-R+P group and H-R+D+G group ( 50.48±0.56 ) % significantly increased (P＜0.01); compared with H-R+D+5HD group, H-R+D+G group significantly increased (P<0.01), while H-R+P+5HD group (35.65±0.49) % increased but not obviously (P>0.05).③LDH contents: Compared with normal group ( 76.84±10.53 ) U/L, H-R group and pretreatment group significantly increased (P<0.01); compared with H-R group (255.45±26.23)U/L, H-R+D group (133.29±15.00)U/L and H-R+P group (193.47±3.39)U/L significantly decreased (P＜0.01); compared with H-R+D group, H-R+D+5HD group (302.90±16.61)U/L, H-R+P group and H-R+D+G group (378.26±7.63)U/L significantly increased (P<0.01); compared with H-R+D+5HD group, H-R+D+G group significantly increased (P<0.01), while H-R+P+5HD group (329.59±10.31)U/L increased but not obviously (P＜0.05).Conclusion:1. The mitoKATP opener Diazoxide and the nonselective KATP opener Pinacidil both lessened the ischemia reperfusion injury in gerbil brain, and the protective role of the mitoKATP opener diazoxide was better than Pinacidil.2. During hypoxia reperfusion injury of cultured hippocampal neurons, Diazoxide and Pinacidil both increased neuron survival rate, decreased neuron apoptosis rate and LDH contents in medium.As well, the protective role of Diazoxide was better than Pinacidil in decreasing neuron apoptosis rate and LDH contents.3. During ischemia reperfusion injury of gerbil brain, the expression of Kir6.mRNA was increased by pretreated diazoxide obviously, however, the expression of SUR1 and SUR2 mRNA were not influenced by the KATP opener and blocker. It suggested that mitoKATP, which subunit is Kir6.1, plays an important protective role in cerebral ischemia;4. Our research found in whole level,cellular level and molecular level, that KATP, especially mitoKATP, played an important protective role in cerebral ischemia reperfusion, and mitoKATP will become a new target to prevent and cure the cerebral ischemia injury.
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