| During cerebral ischemia-reperfusion,harmful products,especially oxygen free radicals,attacked DNA and DNA repairing system,which influenced cerebral ischemia-reperfusion injury.Protecting DNA usually could relieve cerebral ischemia-reperfusion injury.Because of disturbation of oxygen supply and energy metabolism after cerebral ischemia-reperfusion,a amount of harmful products damaged DNA,such as DNA breaks.DNA breaks included DNA single strand breaks(DNA SSBs) and DNA double strand breaks(DNA DSBs),DNA DSBs caused by endonuclease were irreversible,the character of apoptosis.DNA SSBs by oxidative stress were reversible,DNA SSBs were threshold determined fate of damaged cells.So,to study the mechanisms of repairing DNA SSBs had impotant theoritical and practical significance.The harmful factors of DNA was damaging factors of DNA repairing proteins,which destroyed "second defence"that prevented DNA from damage,for example rapid reduction of APE/Ref-1,Ku70 and Ku80 after cerebral ischemia-reperfusion.To enhance DNA repairing-related proteins could decrease DNA damage.Studies above shown that decrease of DNA repairing-related proteins were causes of damaged DNA unrepaired and cell death.XRCC1(X-ray repair cross-complementing group 1)which played a important role in the DNA single strand breaks repairing path.interacted with enzymatic components of each stage of DNA single strand break repairing .The relationship between XRCC1 and DNA SSBs and apoptosis remained unknown and the problems should be solved.Oxygen free radicals put impacts on DNA damage after cerebral ischemia- reperfusion.Studies shown anti-oxidative treatments could relieve cerebral ischemia-reperfusion injury.Melatonin (N-acetyl-5-methoxytryp tamine) was a potent free radical scavenger as well as indirect antioxidant. It was produced mainly in pineal gland.Studies indicated administration of melatonin before ischemia could prevent DNA from oxidative injury after cerebral ischemia- reperfusion.The one of possible reasons that melatonin prevented DNA from damage was to enhance DNA repairing capacities.But correct mechanism that melatonin protected DNA was unknown.Effect of melatonin on XRCC1 after cerebral ischemia-reperfusion was unclear.To study these problems was necessary to understand the protective mechanism of melatonin after cerebral ischemia- reperfusion and to supply experimental base for clinical medicine.Method:Wister rats (male, weight 250～300g),were randomly divided into 3 groups:(1) false operation group (FO group),5 rats,(2) ischemia-reperfusion group (IR group,in which the rats were subjected to middle cerebral artery occlusion(MCAO) for 2h,then reperfusion for 10min,1,4,12,24 and 48h,5 rats every time point,(3) melatonin therapy group (MT group),5 rats every time point.The MCAO modle of rats according to the method of Liu Kangding, were employed in this study.XRCC1 protein expression was analyzed by immunohistochemistry and Western Blot;DNA SSBs were evaluated by single-cell gel electrophoresis assay,apoptosis by flow cytometry(FCM);Effect of melatonin on ultrastructures of neural cell were observed through transmission electron microscope(TEM).Quantitative data were expressed by Mean±SD;Comparsion between groups were evaluated by t test.Software of SPSS 10.0 was utilized to statistical analysis.Result:1.DNA SSBs slightly increased at 10min after cerebral ischemia- reperfusion,and it elevated to peak at 12 h after cerebral ischemia-reperfusion and remained in relatively high level at 24-48h.There were more DNA SSBs in the basal ganglia than in the cortex.2.Apoptosis cell appeared at 10min after cerebral ischemia- reperfusion, reached the peak at 24h,remained high level at 48h after ischemia-reperfusion. In ischemia brain tissue,The number of apoptosis cell in the cortex was lower than in the basal ganglia. 3.The XRCC1 protein was constitutively expressed in the entity region of the normal rat brain,mainly expressed in the hippocampus,XRCC1 in the cortex was more than that in basal ganglia. immunohistoc...
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