Protective Effects Of Iptakalim On Cerebral Ischemia In Rats And Its Pharmacological Properties

Cerebrovascular disease, a kind of severe health-threatening disorder, may be divided into haematological and ischemic cerebrovascular abnormalty. The latter is a leading cause of death and permanent disability of humankind. The mechanisms of ischemia-evoked irreversible neuronal damage are complex. Although many neuroprotective agents have been proven efficacious in animal models, so far none of them show significant benefit in patients with acute ischaemic stroke on primary endpoint measures. It is very pressing and indispensable to develop novel potent neuroprotective drugs.The ATP-sensitive potassium channel (K_Atp) was originally discovered in cardiac muscle and thought to be an important therapeutic targets for myocardial ischemia. Katp subunits are widely expressed throughout different brain regions such as cerebral cortex, hippocampus, striatum, substantial nigra, and nucleus accumbens. This suggests that K_atp may be an ideal target of cerebral ischemia.Iptakalim (Ipt) a novel Katp opener (KCO) which designed and synthesized by native researchers. Previous studies have indicated that Ipt possessed protective effect on global cerebral ischemia-evoked in gerbil. In the present study, we investigated the protective effects of Ipt on cerebral ischemia injury and its pharmacological properties on the bases of the series work. The experimental models we chosed include: the permanent focal cerebral ischemia model in rats, the focal cerebral ischemia-reperfusion model in rats and the whole cerebral ischemia model in mice. Functional behavioural assessment, tissue pathology detection, blood rheology determination and molecular biology experiment were employed to investigate the experimental therapeutic value of Ipt on ischemic damage and its pharmacological properties, so as to obtain the basic evidence todevelop Ipt into a safe and effective therapeutic drug in ischemia therapy.l.Protective effects of Ipt on experimental cerebral ischemia injury.1.l.Effects of Ipt on brain damage after permanent focal cerebral ischemia in ratsTo investigate the therapeutic effects of Ipt on brain damage induced by permanent focal cerebral ischemia.The rat model of focal cerebral ischemia was induced by middle cerebral artery occlusion using a suture method. Infarct size was evaluated by TTC staining, and neurological deficit score was evaluated by Longa's method. Cerebral water content of the ischemic hemisphere was measured using wet-and-dry weight method.The infarct size of the brain was (24.75 + 6.66)% of contralateral hemisphere 6h after focal cerebral ischemia. Pretreatment of Ipt 1.0, 2.0, 4.0 mg-kg"1 (given inperitoneal, 30min before operation) decreased the infarct size by 0.95%, 6.6%(P<0.05), 14.34%(P<0.01), respectively. The neurological deficit scores and the cerebral water content of the ischemic hemisphere were decreased by Ipt 2.0 and 4.0 mg-kg"1. Nim 0.3 mg-kg"1 was also found to reduce infarct size, neurological deficit scores and the cerebral water content.The treatment window of opportunity of Ipt 4.0 mg-kg"'was 3h, neither the infarction volumes nor the deficits of sensorimotor behaviors were significantly improved when Ipt was given at 4h after the onset of permanent MCAO. Nim 0.3 mg-kg"'has no apparent treatment effect.These results show that Ipt has protective effects on brain damage induced by permanent focal cerebral ischemia. 1.2.Protective effects of Ipt on focal cerebral ischemia-reperfusion injury in ratsTo study the cerebral protective effects of Ipt on focal cerebral ischemia -reperfusion injury(CIRI) in rats.The model of acute reperfusion injury after cerebral ischemia in rats was made by MCAO. After 60min ischemia occurred with Id, 3d and 7d reperfusion(ischemia model divided into 3 groups), 60min after MCAO and before reperfusion ip Ipt 4.0 mg-kg"1, thereafter once every day (Ipt divided into 3 groups: Id, 3d and 7d) .Neurological deficit score and infarct size was evaluated as before.Rats after CIRI revealed obvious neurological syndrome. Ipt could statistically improved the neurological funcation of rats,and decreased the Infarct size .This suggestthat Ipt had protective effects on CIRI.1.3.Protective effects of Ipt on cerebral ischemia in miceTo study the protective effects of Ipt against cerebral ischemia of mice.Mice were given different dosage (0.5 ^ 2.(h 8.0mg-kg^' ig) of drugs for three weeks, three weeks later, we made model of brain anoxia of mice by Scissors and record the breathing time, the GSH-PX activity , SOD activity and the MDA content in the brain homogenates were investigated. The erythrocyte membrane fluidity was studied by the fluorescence polarization technique.The results showed that compared with the control group, Ipt 0.5, 2.0, 8.0mg-kg"' ig markedly prolonged the breathing time after mice being headed, increased the fluidity of erythrocyte membrane and revealed dosage related. Ipt had no significant effect on the activity of GSH-PX and SOD. The protective effects of Ipt against cerebral ischemia is related to its effects of increasing the fluidity of erythrocyte membrane and decreasing blood viscosity.2.The pharmacological properties of Ipt on anticerebral ischemia injury 2.1.The relationship of the protective effects of Ipt on cerebral ischemia and the ATP sensitive potassium channelTo study the influence of Gli(ATP-sensitive potassium channel antagonist) on the protective effects of Ipt on cerebral ischemia in mice,investigate the mechanism of Ipt and compare the characteristic effect of IpU Nim and Pin.The result suggested: compared with the control group, Ipt(2.0mg-kg"1-d^I, ig 3 weeks) could markedly prolong gasp duration of decapitative mice, increased the fluidity of erythrocyte membrane, decreased blood viscosity. On the same time, Gli SOmg-kg^-d"1, ig had no effect on the breathing time, LFU and blood viscosity of decapitative mice, but it could antagonized the effect of Ipt. Nim 2.0mg-kg¹-d¹, ig and Pin 2.0mg-kg"'-d"1, ig also markedly prolonged gasp duration of decapitative mice, increased the fluidity of erythrocyte membrane, decreased blood viscosity. Nim could apparent decreased the content of MDA, improve the activity of GSH-PX and SOD, Iptand Pin had no apparent effect on MDA, GSH-PX and SOD. The result above elucidated that the protective effect of Ipt against cerebral ischemia of mice is related to the ATP sensitive potassium channel opening. 2.2.Regulation on brain Kir6.X expression in rats after focal cerebral ischemiaTo investigate the effect of Ipt on Kir6.1 and Kir6.2mRNA expression in rat brain of focal cerebral ischemia.The result revealed: compared with sham-operated group, the rats in ischemic group exhibited obvious abnormal neurological deficit, the expression of Kir6.2mRNA significantly upregulated. Iptl.O, 2.0, 4.0 mg-kg"1 ip exhibited significant improving effects on neurological function and statistically augmented the expression of Kir6.2mRNA compared with ischemic group. In the same time,Nim 0.3mg-kg"' ip could improving effects on neurological function,but had no obvious effect on the expression of Kir6.2 mRNA.2.3.The effects of Ipt on the expression of apoptosis related protein in rat brain with focal cerebral ischemiaIn this study,haematoxylin and eosin(HE) staining were employed to detect the histological protective effects of Ipt on rats with focal cerebral ischemia,and use immunohistochemical technique to detect the expression of apoptosis related protein Bax, Bcl-2 and caspase-3 in neuron and investigating the anti-apoptosis mechanism of Ipt.The result shown that: there were no obviously pathological changes in sham -operated group. In ischemic group, many neurons disappeared in the center of the infarcts,the nuclei of the remaining neurons were pyknotic and theneural fibers became vacuolar.neuron defects in the infracted center of Ipt group(Ipt 4.0mgkg"1 ip 30min before MCAO and 3h after MCAO) and Nim group(Nim OJmg-kg"1 ip) were significantly slighter than that of ischemic group.The Bax and Bcl-2 positive cells in ischemic group were more expressed compared with sham-operated group. In Ipt group and Nim group the Bcl-2 positive cells increased and the Bax positive cells decreased,and the effect of Ipt group has statistically profile compared with ischemic group. MCAO 60min and reperfusion 3d,the expression of caspase-3 in rat brain were obviously increased ,Ipt could statistically decreased the over expression of caspase3 compared with the control.The result suggested that the depressed effect of Ipt on neuron apoptosis is closelycorrelated to the expression of Bax,Bd-2 and caspase-3.2.4.Effects of Ipt on blood rheology after focal cerebral ischemia in ratsTo investigate the effects of Ipt on blood rheology after cerebral ischemia, in model of MCAO.The erythrocyte membrane fluidity was studied by the fluorescence polarization technique,the erythrocyte deformability and aggregation changes detected by instruments.Focal cerebral ischemia for 6h induced a significant decrease in erythrocyte deformability and the fluidity of erythrocyte membrane,an increase in erythrocyte aggregation and blood viscosity. Pretreatment of Ipt 2.0,4.0mg-kg'' and Nim 0.3mg-kg"' can improve those index of blood rheology. 3.Effects of Iptakalim derivatives on the glutamate induced toxity in PC12 cells.To investigate the effects of newly synthesized Iptakalim (2,3-dimethyl-2-butylamine ) derivatives on the injury of neuron cell induced by glutamate.The PC 12 cells were cultured in vitro. When glutamate injury, MTT test showed a reduced production of formazan obviously, indicading that the PC 12 cells were seriously damaged. When the cells were pretreated with those compounds with three to five carbon branched alkane or with possess conjugation system phenol or heterocyclic compounds, it was shown that the number of surviving PC 12 cells increased significantly. Pretreated with those compounds with one to two carbon branched alkane or benzene, have no effects on the injury of neuron cell induced by glutamate or aggravate the injury in PC 12 cells.This results suggest that only those compounds with specific structures had the potential of protect PC 12 cells against glutamate injury. 4.Conclusion4.1.Iptakalim exerted effective functional protection in permanent focal cerebral ischemia, focal cerebral ischemia-reperfusion and the whole cerebral ischemia model, Alleviate the brain edema, reduced the infarct size and improve the neurological funcation of rat under experimental ischemia model.