Astrocytic Kir6.1 Knockout Aggravates Ischemia/Reperfusion Injury By Enhancing Endoplasmic Reticulum Stress In Mice

Stroke is an acute brain blood circulation disorder with highest morbidity and highest mortality.Nowadays it is a serious threat to human health.There are two main types of stroke:ischemic,due to lack of blood flow,and hemorrhagic,due to bleeding.About 80%of strokes are ischemic.Recombinant tissue plasminogen activator is still the main treatment in clinic,but its application is seriously limited because of the short therapeutic time window and the risk of bleeding.Unfortunately,almost all clinical studies to find new drugs with neuroprotective effects for stroke failed in recent years.Studies have shown that the protection of neurological function can't be limited to the protection of single neurons and reseachers should pay more attention to the neurovascular units consisting of neurons,astrocytes,endothelial cells.Therefore,the development of new stroke therapy strategies is especially urgent for stroke patients.Astrocytes are the most abundant cells in the CNS and provide guarantee for the funtion of neurons.Astrocytes have multiple important biological functions such as participating in formation of synapse,transporting neurotransmitters,maintaining the ion homeostasis and signal transduction.The importance of astrocytes in cerebral ischemia has been gradually understood in recent years.They proliferated,activated and the specific markers of collagen fibrillary acidic protein(GFAP)were upregulated during the ischemia.Astrocytes are more tolerant than neuron and have a dual role in ischemic state:releasing glutathione,neurotrophic factors,producing erythropoietin to protect the neurons and also damage the neurons by releasing excitatory amino acids,inflammatory factors and reducing the gap junction.Thus,astrocytes play a crucial role in the progress of stroke and it is important to determine the role and mechanism of astrocytes which may provide a new target for the treatment of ishcemic stroke.ATP-sensitive potassium(K-ATP)channels couple energy metabolism with electric activity,providing an unique link between cellular energetics and electric excitability.These channels,which are usually closed under normal conditions,are activated rapidly in response to and decrease in intracellular ATP/ADP ratio,which plays important roles in brain diseases including stroke.K-ATP channels are eight heteromultimers composed of four inwardly rectifying potassium channel(Kir6.x)and four sulfonylurea receptors(SURs).Kir6.x are pore-forming subunits,including Kir6.1 and Kir6.2,determining the selectivity of ions transportion.Astrocytes primarily express Kir6.1 submit rather than Kir6.2 submit.SURs,including SUR1 and SUR2,are the submits regulating channel function.Our previous studies demonstrated that Kir6.1/K-ATP channel was associated with ischemic stroke.Kir6.1 knockdown exacerbated cerebral I/R-induced brain damages and K-ATP opener nicorandil protected primary astrocytes from OGD/R induced cell injury,which suggested that astrocytic Kir6.1/K-ATP channel participated in the development and progression of ischemic stroke.Therefore,in this part of our work,astrocytic Kir6.1 knockout mice were used to investigate the effects of astocytic Kir6.1 knockout on the cerebral ischemia/reperfusion induced brain injury in the model of tMCAO.We used the Kir6.1 siRNA to silenced the expression of Kir6.1 on the primary astrocytes to explore the cell activity and apoptosis induced by OGD/R injury.The results showed that astocytic Kir6.1 knockout accelebrated tMCAO-induced cerebral ischemia/eperfusion injury and aggravated penumbra endoplasmic reticulum stress,autophage and apoptosis.Astrocytic Kir6.1 knockdown exacerbated cellular damage and apoptosis caused by OGD/R,increased endoplasmic reticulum stress,autophage and apoptosis and endoplasmic reticulum stress inhibitor 4-PBA reversed the deteriorative effects due to astrocytic Kir6.1 knockdown.These results suggested that astrocytic Kir6.1 knockout accelebrated cerebral ischemic reperfusion injury by enhancing endoplasmic reticulum stress-dependent autophage and apoptosis.AIM:To explore the effects and mechanisms of astrocytic Kir6.1/K-ATP channel on ischemia/reperfusion-induced brain injury in mice.METHODS:1).We used CRE-LOXP systerm to establish astrocytic Kir6.1 knockout mice.Two-month-old Con(KCNJ8f/f)and CKO(KCNJ8f/fGFAPCre)male mice were subjected to cerebral ischemia by tMCAO with 1 h of ischemia followed by 24 h of reperfusion.The neruological deficits,infarct volumes and brain water contents were measured in each group after 24 h of reperfusion to investigate the effects of astrocytic Kit6.1 on ischemia/reperfusion injury.2).The mice were subjected to tMCAO and the brains were taken out after 24 h or 72 h of reperfusion respectively.We used immunohistochemistry staining to observe the changes of neurons,microglia,astrocytes and neutovascular substrates.3).We examined the endoplasmic reticulum stress,autophage and apoptosis-related protein levels after 24 h of reperfusion by Western Blot.4).We cultured primary astrocytes and infected with Kir6.1 siRNA to determine the interference efficient by Western Blot.After being infected with Kir6.1 siRNA,the astrocytes were exposed to OGD/R.We examined the endoplasmic reticulum stress and apoptosis-related pretein levels by Western Blot.5).After being infected with Kir6.1 siRNA,the astrocytes were treated with endoplasmic reticulum stress inhibitor 4-PBA and exposed to OGD/R.we detected cell vitality with MTT and cell apoptosis with Fluorescent cell sorting.RESULTS:1)Astrocytic Kir6.1 knockout aggravated tMCAO-induced neruological deficits,increased infarct volumes and brain water contents.2)Astrocytic Kir6.1 knockout enhanced neuron loss,strengthened astrocyte and microglia proliferation and activation and destructed neutovascular substrates integrity in the cortex penumbra of mice.3)Astrocytic Kir6.1 knockout enhanced endoplasmic reticulum stress and increased the expression of autophagy and apoptosis related proteins in the cortex penumbra of mice.5)Astrocytic Kir6.1 knockdown exacerbated the endoplasmic reticulum stress and apoptosis induced by OGD/R in primary astrocytes.4)Astrocytic Kir6.1 knockdown aggravated cellular damage and apoptosis induced by OGD/R and 4-PBA reversed these effects of Kir6.1 down-regulation.CONCLUSION:Astrocytic Kir6.1 knockout aggravated cerebral ischemia/reperfusion injury through enhancing endoplasmic reticulum stress-depended apoptosis.The major contribution of the present study lies in:Astrocytic Kir6.1/K-ATP channel protects cerebral ischemia injury via modulating endoplasmic reticulum stress-dependent apoptosis.