Experimental Therapeutic Effects Of ATP-Sensitive Potassium Channel Openers On Local Cerebral Ischemia/Reperfusion-induced Stroke In Rats

Stroke is a suddenly occurring cerebral blood circulation disorders with high incidence of morbidity and mortality, which seriously endanger human health. It can be divided into ischemic stroke, which accounts for the vast majority of stroke, and hemorrhagic stroke. So far, already stated mechanisms of ischemia-reperfusion brain injury include:energy depletion, calcium overload, excitotoxicity, oxidative stress, inflammation and apoptosis. In recent decades, although the study of the pathogenesis of ischemia-reperfusion injury of brain has been made a great progress, almost all of the transformations from basic research to clinical application were failed. Currently, there is a lack of ideal drugs for the therapeutic of cerebral ischemia.The only one, which has been approved by FDA, is still recombinant tissue plasminogen activator (recombinant tissue plasminogen activator, r-tPA).However, the strict treatment time window of r-tPA and its severe side effects greatly limit the clinical use of it. Therefore, the study of the pathogenesis of stroke induced nervous damage; the finding of effective therapeutic targets and the development of an ideal therapy are urgently needed. Researches show that the focus of recovery of brain function should not only been on protecting neurons, but also the protection of the entire neurovascular unit, including neurons, glias and endothelial cells, and the neurogenesis after cerebral ischemia.ATP-sensitive potassium (K-ATP) channels are unique biological receptors, which couple cellular energy metabolism to the electrical activity in the body. The intracellular ATP/ADP level is the gating factor of this special non-voltage-dependent potassium ion channel. K-ATP channels are widely distributed in the brain. The molecular diversity of this channel on different cells in the brain is the structural basis of being a target for protecting neurovascular unit. Under physiological conditions, the K-ATP channel is closed in brain. K-ATP channels will be opened when the brain tissue is under ischemia, hypoxia or oxidative stress, makes K+efflux, cell hyperpolarization, and inhibits excitatory neurotoxicity, which plays a protective effect for neurons. Moreover, in vitro study showed that opening of K-ATP channel can regulate the function of astrocytes, inhibit microglia and activate the proliferation of stem cells. Thus, the opening of K-ATP channel is an important endogenous protective mechanism of the brain.Iptakalim is a novel small molecule compounds with an aliphatic amine structure, which makes it pass through blood-brain barrier easily. It is a selective agonist of SUR2type K-ATP channels. Both in vivo and in vitro studies show that iptakalim has neuroprotective effects against ischemic brain injury. In gerbils global cerebral ischemia models, iptakalim ameliorated motor dysfunction caused by ischemia, reduced the release of excitatory neurotransmitters in the brain, thereby reducing neuronal loss. In stroke-prone rats with salt loading, iptakalim reduced the incidence of stroke and mortality, delayed the incidence of stroke and prolonged the survival of animal. In addition, in a rat MCAO model, iptakalim reduced infarct volume and cerebral edema and improved neurological dysfunction. In spite of the stated neuroprotective mechanism of iptakalim, are there any other mechanisms of iptakalim’s neuroprotective effect, which play an important role in ischemia-reperfusion injury? Latest research showed that activation of the inflammasome plays an important role in cerebral ischemia-reperfusion injury. In NLRP3-/-mice focal cerebral ischemia model, knockout of NLRP3can reduce the infarct volume and blood brain barrier damage. Thus, whether the neuroprotective effect of iptakalim against cerebral ischemia-reperfusion injury is associated with NLRP3inflammasome? It has never been reported. Additionally, in vitro studies showed that K-ATP channel can adjust the expression of nutritional factors in astrocytes; K-ATP channel opener had a pro-proliferation effect on stem cells, which would promote the repairing of the brain. The role of K-ATP channel opener in long-term functional recovery after cerebral ischemia-reperfusion injury is still not clear.In view of above, we used male SD rats to develop middle cerebral artery occlusion (MCAO) model to study the effects of iptakalim on local cerebral ischemia-reperfusion injury in the acute stage, reveal new mechanisms involved in its neuroprotective effect. Then, we studied the effects of iptakalim on long-term functional recovery after cerebral ischemia/reperfusion, and explored whether K-ATP channel openers could help repairing the injured brain in the late stage.AIM:To studies impacts of iptakalim, a K-ATP channel opener, on focal cerebral ischemia/reperfusion induced neural injury in acute phase and in repairment stage.METHODS:Transient middle cerebral artery occlusion model was established in male rats by intraluminal suture method for90min, then restore perfusion. Different doses of iptakalim (1、2、4mg/kg, i.p.)was administrated immediately after the onset of ischemia. In acute phase (24h after reperfusion), we compare the neurological deficit between the control group and iptakalim treated group; The2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) staining was used to contrast infarct volume.72after reperfusion, immunohistochemical staining was used to detect the number and morphologic of neurons in boundary between ischemic core and penumbra, and astrocytes surrounding the infarct area. Immunoblot was applied to detect CD11b, the marker of microglia, apoptosis-related proteins caspase-3and inflammation-related protein nuclear factor κB(NF-κB),caspase-1, interleukin-1beta(Il-1β). Real-time quantitative PCR (real-time per) was used to detect the mRNA level of NOD-like receptor pyrin domain containing three (NLRP3). After establishment of rat MCAO model, iptakalim (2mg/kg) was administrated once per day. Respectively, in the first7,14,21,28,35day following ischemia/reperfusion, we used cylinder test and corner test to evaluate the motor-function recovery of rats. Immunohistochemical staining method was applied to detect neurons, reactive astrocytes and cell proliferation in subventricular zone (SVZ).Western blot was used to detect the levels of inflammatory cells in ischemic penumbra.RESULTS:1. Iptakalim can reduce focal cerebral ischemia/reperfusion induced acute injury.MCAO induced neuromotor dysfunction, infract volume in acute phase could be reduced by iptakalim in a dose-dependent manner. Iptakalim (2mg/kg) mitigated the neuronal injury in infract area, and also inhibited the activation and proliferation of glia in peri-infract area.2. Iptakalim ameliorate cerebral ischemia-reperfusion injury induced apoptosis and inflammation.Iptakalim significantly down-regulated the activation of caspase-3, inhibited the nuclear translocation of NF-κB, and prevented the activation of inflammasome.3. Iptakalim (2mg/kg/day×35day) fails to improve sensorimotor dysfunction recovery of rats, and does not affect cell proliferation in SVZ in the late stage after cerebral ischemic/reperfusion injury4. Iptakalim promotes the survival of neuron.Iptakalim (2mg/kg, i.p. once per day) increased the number of mature neuron in SVZ in the late phase after cerebral ischemia-reperfusion injury.5. Iptakalim inhibits the activation and proliferation of glia in the late phase after cerebral ischemia-reperfusion injury.Iptakalim (2mg/kg, i.p. once per day) continuously suppressed the activation and proliferation of astrocytes in SVZ and inhibited the expression of CD11b,the mark of microglia, in ischemia penumbra.CONCLUSIONS:1. Iptakalim significantly ameliorates focal cerebral ischemia/reperfusion induced acute injury in rats, due to inhibiting glial activation and inflammatory response.2. Iptakalim enhances neuronal survival and inhibits the activation and proliferation of glia in the late phase after cerebral ischemia-reperfusion injury in rats, but fails to improve sensorimotor dysfunction recovery and cell proliferation in SVZ.INNOVATIONS:1. The present work clarifies that iptakalim can inhibit the activations of glial cells and NLRP3inflammasome, and thereby protect against cerebral ischemia/reperfusion-induced brain injury.2. Our results reveals that long-term openning of K-ATP channel can continuously suppress the activation of glia after cerebral ischemia/reperfusion injury, and subsequently enhance neuronal survival.