Inosine inhibits apoptosis and cytochrome C mRNA expression in rat neurons after cerebral ischemia/reperfusion

Jinrong Wang; Mingjun Bi; Qin Li

摘要：BACKGROUND: It has been demonstrated that adenosine can induce glial cell to release cytochrome C,enhance expression of apoptotic gene bax,inhibit anti-apoptotic gene bcl-2,and activate caspase-3 to apoptosis;Whereas inosine can inhibit neuronal apoptosis which is similar to bil-2.OBJECTIVE: To observe the affects of inosine on neuronal apoptosis and expression of cytochrome C mRNA in rats after focal cerebral ischemia/reperfusion,and analyze the pathway of its neuroprotective effect.DESIGN: A randomised controlled animal trial.SETTINGS: Department of Neurology,Rongcheng Second People's Hospital;Department of Neruology,Affiliated Union Hospital,Tongji Medical College,Huazhong University of Science and Technology.MATERIALS: Sixty-eight rats,weighing 230-280 g and clean grade,were used.TdT-mediated dUTP-biotin nick end labeling(TUNEL)and cytochrome C mRNA in situ hybridization kits and DAB staining kit were purchased from Wuhan Boster Biological Co.,Ltd;Inosine injection[200mg(2ml)each] from Qingdao First Pharmaceutical Factory.METHODS:The experiment was accomplished in the animal experimental center in Tongji Medical College of Huazhong University of Science and Technology from December 2003 to June 2005.①Sixty-four rats were made into focal ischemia by middle cerebral artery occlusion(MCAO)with a nylon monofilament suture.The successfully induced rats were assigned to inosine group(n=32)and model group(n=32)at random.Rats in the inosine group were intraperitoneally administrated with inosine in dose of 100mg/kg preoperatively.twice a day,7 days in all.The rats in the control group were injected with the same dose of saline solution by the similar way preoperatively.Each group was randomized into ischemia/reperfusion 2,6,12,24 hours,2,3,7 and 14 days subgroups consisted of 4 rats.The other 4 rats were taken as the sham-operated group,the rats were given the same treatment except for not introduced the filament into the external carotid artery stump.and brain tissue was removed at 2 hours of reperfusion. ②In situ hybridization was performed to examine the expression of cytochrome C mRNA while TUNEL staining was made to characterize apoptosis.③The t test was used to compare the difference of measurement data.MAIN OUTCOME MEASURES:①Neuronal apoptosis in the different regions of the ischemic brain tissue;②Expression of cytochrome C mRNA in the different regions at different time points after MCAO.RESULTS:All the 68 rats were involved in the analysis of results.①Neuronal apoptosis:A small number of TUNEL-positive cells were detected in the sham-operated brain and non-ischemic brain.The number of apoptotic cells in the ischemic cortex peaked at 24 hours of reperfusion[(72.00±1.98)cells]and that in the striatum peaked at 2 days[(94.75±3.57)cells],then decreased to the level of sham-operated group at 14 days.Inosine could reduce apoptotic cells from 12 hours to 7 days of reperfusion as compared with the model group (t:6.19-26.67,P＜0.01).②Cytochrome C mRNA expression:There was weak expression of cytochrome C mRNA in both sham-operated brain and contralateral brain. Cytochrome C was detected at 2 hours of reperfusion in ischemic brain[(25.75±3.50),(39.75±2.49)cells],and strongly increased to a peak at 12 hours and 24 hours of reperfusion in cortex and striatum[(122.50±6.69), (119.25±5.12)cells], respectively.Furthermore,inosine could significantly decrease cytochrome C expression in cortex at 12 hours to 14 days of reperfusion after ischemic reperfusion and that in striatum at 12 hours to 3 days(t=8.67-43.26,P＜0.01).CONCLUSION:Inosine can exert a neuroprotective effect by inhibiting apoptosis and cytochrome C mRNA expression.