Protection Of Iptakalim On Neurovascular Unit Against Ischemic Injury And Its Mehcanisms

Brain is an important organ with active metabolism. Interruption and decrease incerebral blood fluid leads to ischemia and or hypoxia to brain then the consequence isneuronal dysfunction and cerebral diseases. Cerebral ischemia/hypoxia injury is astrengthening problem to health with high mobility and mortality. Once brainischemia and hypoxia happened, it caused increase of oxygen radicals, induction ofreactive oxygen species,overproduction of peroxides, loss of mitochondria membranepotential and so on. Cerebral ischemia and hypoxia injury leads to the self-damage inneurons, astrocytes and microvascular endothelials, then necrosis and apoptosis, astogether became the cellular basic for brain impairment.Apoptosis is one regarded way for cell death. Apoptosis is a kind of energy-consumption, programmed process. As energy factory, mitochondria play crucial rolein apoptosis. It is known that ischemia and hypoxia can cause cell mitochondriadysfunction then to activate pre-apoptotic proteinases to induce apoptosis.The study on neuroprotection once focused on the protection of only neuron but itfailed to work. Recently, the neuroprotection means something new, which containsnot only protection for neurons but protections for astrocytes and endothelial cellsalso. Neurovascular unit as a functional module, become the core of neuroprotection.The definition of neurovascular unit is the neurons, astrocytes, endothelial cells andcell matrix even the interation and signal transduciton among them. It is a conceptionemphasize the dynamic homostasis among all elements.The neuroprotecion ofiptakalim, a K_ATPopener, on ischemia and hypoxia were confirmed in differentmodels in vivo and in vitro.How about effects of iptakalim on neurovascular unitsunder hypoxia and its property? The meanings of study the effects of iptakalim onneurovascular units under hypoxia are to confirm its pharmacological characteristics,to witness the development of neuroprotectant and to find effective intervention forbrain ischemia and hypoxia injury.Our study will apply with in vivo brain injury model contains ratMCAO/reperfusion model and unilaterally occluding the carotid artery feeding in aatmospheric chamber with oxygen concentration at10%for12h simulated high altitude equivalent to5000m leading to ischemic brain injury. In vitro, cell ischemiccondition induced by sodium dithionite to verify the neuroprotection of iptakalim onNVU cells including neurons, astrocytes and microvascular endothelial cells, andfurther explore the different elements and multiple ways involved with theneuroprotection of iptakalim and its molecular mechanism.Results were as follows,Part1Protection of iptakalim on neurovascular unit against ischemia injury1. Protection of iptakalim on main component cells of NVU against ischmic injury1.1Difference in expression of K_ATPsubtypes among NVU cellsTo mRNA expression of Kir6.1: ECs <neurons<astrocytes; To mRNAexpression of SUR2B: neurons<astrocytes <ECs;1.2Ipt restrains cell death under ishemic conditionsTreatment with0.01,0.1, or1μmol/l Ipt inhibited neurons cell death underischemic conditions. Similar results were obtained for astrocytes and endothelialcells under the same conditions, and treatment with Ipt from0.01μmol/lsignificantly inhibited cell death under ischemic conditions in these cells.1.3Ipt suppresses LDH release from cells under ischemic conditionThe level of LDH released into the culture medium for the three cell types underischemic conditions was strikingly higher compared with control groups.Pretreatment with0.01,0.1, or1μmol/l Ipt significantly decreased the level ofLDH released for all three cell types compared with cells under ischemicconditions. Glibenclamide, a K_ATPchannel blocker, at a concentration of1mol/lreversed the protective effects of Ipt on the three types of cells. This suggeststhat Ipt protects cells from death via activating K_ATPchannels.2. Protection of iptakalim on rat ischemic brain injury1.1Iptakalim Attenuates pathological process of brain tissue2,4,8mg/kg iptakalim attenuated the brain edema, improved the neuron survivaland maintained the function of brain against ischemia/hypoxia brain injurycompared with ischemia/hypoxia injury groups.1.2Iptakalim maintains BBB integrity under ischemic brain injuryTreatment with Ipt at doses of2,4, or8mg/kg/day can suppress the perivascularspace widening and endothelial cell swelling compared with the untreatedischemic group by transmission electron microscope.That means ipt maintainedBBB intergrity.Part2The molecular mechanism of protection of iptakalim on NVU against ischemicinjury 1.1Iptakalim Attenuates Neuronal Apoptosis in vitro1.1.1Ipt regulated the mRNA expression of bcl-2/bax and down-regulate thecaspase-3protein expression under ischemic condition.The mRNA expressionratio of bcl-2/bax was reduced in ischemic group compared to the normal one. Iptcan significantly increase its ratio. About caspase-3, Ipt can down-regulate itsprotein expression after hypoxia in NVU cells compared to model ones.1.1.2Ipt inhibits cellular apoptosis in morphology under ischemic conditionsHoechst33258was applied to neurons, astrocytes, and endothelial cells. Controlgroups for each cell type showed integral nuclei. Thus, cells treated with ischemicsolution exhibited characteristics of apoptotic cells such as nuclei condensationand fragmentation. In contrast, pretreatment with1μmol/l Ipt reduced theapoptosis induced by the ischemic solution.1.1.3Ipt inhibits cellular apoptosis under ischemic conditionsCertain concentrations of Ipt can reduce the rate of apoptosis in NVU cell.1.2Iptakalim Attenuates Neuronal Apoptosis in vivoCaspase-3activity in the cortex was increased in ischemic/hypoxia(H/I) groupcompared with the sham group. In the ipt-treatment group, there was a significantdecrease in caspase-3activity in the2,4,8mg/kg groups compared with that in H/Igroup. Bcl-2and Bax activity were also measured, because activation of Bcl-2incells is a marker for an endogenous protective pathway in anti-apoptosis, whereasthe appearance of Bax indicates that more cells have initiated programmed celldeath. Thus, the ratio of the expression of these two proteins demonstrates thebalance between cellular protection and cell death. The ratio of Bcl-2/Bax wasreduced in ischemic group compared to the sham operation group. Treatment with2,4,8mg/kg iptakalim significantly increased this ratio to different extent. Iptakalimcould arrest apoptosis in rats after cerebral H/I injury.2.Iptakalim affects brain mitochondria function2.1Iptakalim maintained the morphology of brain mitochondria under hypoxiainjury2.2Iptakalim improves the function of brain mitochondriaIpt can improve the function of brain mitochondria under hypoxia injury byincreasing the ATP synthesis rate, raising the P/O ratio and elevating the RCR.2.3Ipt prevents loss of the MMP under ischemic conditionsApplication of0.01,0.1, and1μmol/l Ipt to the three different cell typesprevented the loss of MMP under ischemic conditions.2.4Ipt reduced the production of ROS in NVU cells3Iptakalim affects the interation among NVU cells 3.1Ipt enhances BDNF secretion in neurons and astrocytes under ischemiccondition and improves the interaction among NVU cellsIpt can enhances BDNF secretion in neurons, astrocytes and ECsunder ischemiccondition.BDNF, as a brain derivative neurotrophyic factor,can improves theinteraction among NVU cells.3.2Ipt can enhance the interaction among NVU cells through the effects on ECMand ACMECM and ACM can increase the neuron survival rate under ischemic condition.Iptcan enhance their effects on neurons survival by strengthening the secretion ofneurotrophic factors.3.3Ipt influences the BDNF,TrkB expression in brain tissue underischemia/hypoxia brain injury.Given with Ipt at doses of2,4, or8mg/kg/day can suppress down-regulation ofBDNF, TrkB protein expression in cerebral cortex.To summarize as conclusions,1. Activation of K_ATP,iptakalim plays the neuroprotection role not only in cerebralischemia/reperfusion but also in ischemia/hypoxia brain injury.2. Iptakalim can protect NVU cells such as neurons,astrocytes and endothelial cells,components of neurovascular unit, from ischemic condition induced by sodiumdithionite. Besides, iptakalim can also enhance the secretion of BDNF to strengthenthe interaction of components of NVU.This protection originates its enhancement ofsecrection of BDNF, which initiates the inner protection pathway.3. The protection of iptakalim on NVU is the bottom of its neuroprotecion againstischemia/hypoxia brain injury. The possible mechanisms may include as follows,a. its antiapoptosis pathwayb. the effect on remaining the function of brian mitochondria, maybe involved withactivation of mito K_ATP.c. Ipt has selective effect on NVU cells particular on ECs, this is possible related todifferent intensity of m RNA expression of the subtypes of K_ATPon of NVU cells.