TRPV4受体在脑缺血再灌注损伤中的作用及其分子机制研究

发布时间：2018-05-11 22:14

本文选题：瞬时感受器电位香草素受体亚家族Ⅳ型(TRPV4) + 脑缺血再灌注损伤　；参考：《南京医科大学》2014年博士论文

【摘要】：随着世界人口进入老龄化的速度加快,脑卒中(缺血性脑血管病)的发病率逐年升高,现已成为威胁人类生命的最主要疾病之一。因脑卒中具有发病率高、致残率高和死亡率高的特点,给个人、家庭和社会带来巨大的精神压力和经济负担。虽然尽早溶栓治疗通过恢复脑血氧供应能保护神经元,但是缺血后再灌注往往也会加重脑组织的损伤(再灌注损伤),导致神经元坏死。因此,研究缺血诱导脑损伤的病理生理机制,寻找有效的治疗靶点,对提高脑卒中患者的治愈率具有重要意义。缺血再灌注诱导脑损伤主要与谷氨酸兴奋性毒作用、胞内钙(intracellular Ca2+concentration,[Ca2+]i)超载、自由基与一氧化氮的损伤、脑水肿等病理机制有关。其中,谷氨酸兴奋性毒作用被认为是脑缺血诱导神经元死亡的重要机制。然而,临床资料显示阻断谷氨酸受体对脑卒中并未能取得理想的疗效。因此,探究非谷氨酸依赖性钙离子超载诱导的神经元损伤机制将非常有意义。瞬时感受器电位(transient receptor potential, TRP)受体家族是一类重要的非选择性阳离子通道。瞬时感受器电位香草素受体亚家族Ⅳ型(TRP vanilloid4, TRPV4)在中枢神经系统广泛分布,主要表达在海马、大脑皮层、丘脑、小脑等脑区。TRPV4受体作为一种以钙离子为主的阳离子通道,被激活后能引起以Ca2+为主的内向电流。近年来,越来越多的研究报道了TRP受体家族成员参与脑缺血再灌注损伤的病理过程。TRP家族中的nelastati家族Ⅱ型、Ⅶ型和canonical家族Ⅵ型等成员已被报道介导了脑缺血引起的神经元死亡。TRPV4受体自发现以来,因其可被低渗、温热、机械、花生四烯酸及其代谢物等多种刺激激活而越来越受到人们关注。脑缺血时,由于能量代谢障碍所引起的细胞水肿可以通过改变细胞膜机械张力激活TRPV4受体；能量代谢障碍所产生的大量花生四烯酸也可以通过其代谢产物5,6-环氧二十碳三烯甘油酸等激活TRPV4受体。提示缺血再灌注引起脑损伤的病理过程有可能与TRPV4受体的激活有关。阻断TRPV4受体可以减轻氧化应激对海马星形胶质细胞的损伤,对氧糖剥夺导致的海马CA1神经元损伤也具有保护作用,提示阻断TRPV4受体有可能减轻缺血导致的脑损伤。我们前期在初级感觉神经元上的研究发现,TRPV4受体激活后通过影响其下游的信号通路(PKA、PKC、PKG等)调节细胞膜上的电压依赖性离子通道和TRPV1受体的功能,提高神经元的兴奋性。在视网膜神经节细胞上的研究发现TRPV4受体激动剂能够剂量依赖性增加[Ca2+]i,提高细胞的兴奋性,诱导细胞凋亡。此外,激活TRPV4受体能够增加培养的海马神经元之间的微小兴奋性突触后电流的频率,提示脑缺血再灌注通过激活TRPV4受体有可能促进谷氨酸的释放。因此,本课题将通过在体实验首先明确TRPV4受体是否参与脑缺血再灌注损伤的病理过程,然后重点研究阻断TRPV4受体对脑缺血再灌注损伤是否有保护作用及其分子机制,为今后预防和治疗脑缺血再灌注损伤提供新的靶点和思路。研究目的 1.明确TRPV4受体是否参与脑缺血再灌注诱导的损伤； 2.阐明阻断TRPV4受体保护脑缺血再灌注损伤的分子机制。第一部分TRPV4受体在脑缺血再灌注损伤中的作用材料与方法 1.脑缺血再灌注小鼠模型制备：用鱼线阻塞右侧大脑中动脉60min,制备大脑中动脉栓塞(middle cerebral artery occlusion, MCAO)再灌注小鼠模型。 2.实时定量RT-PCR和Western blot:MCAO再灌注后不同时间点取出海马组织提取mRNA和蛋白,检测TRPV4受体的nRNA和蛋白水平。 3.TTC染色：MCAO再灌注24h的脑组织,冠状切片后置TTC溶液中反应,正常脑组织呈红色而梗死组织呈白色,白色脑组织体积即为MCAO再灌注后梗死体积。 4.甲苯胺蓝染色：脑组织用多聚甲醛固定后石蜡包埋切片,用甲苯胺蓝染色,进行海马CA1锥体神经细胞的计数。结果 1. MCAO再灌注后2-72hTRPV4受体的mRNA的表达增加；MCAO再灌注4-48hTRPV4受体蛋白水平增加,MCAO再灌注后18hTRPV4受体蛋白水平达到峰值,之后逐渐下降,MCAO再灌注后72h恢复到正常水平。 2.侧脑室给予TRPV4受体特异性阻断剂HC-067047,在0.1-30μM/2μL/只的剂量范围内,能够浓度依赖性地减小MCAO再灌注后24h脑梗死的体积；HC-067047(10μM/2μL/只)对脑缺血再灌注损伤神经保护作用的有效时间窗长达12h。侧脑室给予TRPV4受体非特异性阻断剂钌红也能够有效减小MCAO再灌注后24h脑梗死的体积。 3.与对照组小鼠相比,侧脑室注射TRPV4受体激动剂4a-PDD能够引起海马CA1神经元的死亡。结论脑缺血再灌注后4-48h海马TRPV4受体表达明显增加。阻断TRPV4受体对脑缺血再灌注损伤有保护作用。第二部分阻断TRPV4受体保护脑缺血再灌注损伤的分子机制研究材料与方法 1.脑缺血再灌注损伤的细胞模型建立：脑缺血时,由于能量代谢障碍会引起脑水肿。我们的前期研究已证明,细胞水肿可以激活TRPV4受体。为了研究脑缺血后细胞水肿激活TRPV4受体导致细胞死亡的机制,本研究采用将细胞外液渗透压降低到240mOsm/kg(低渗刺激)模拟脑水肿细胞模型。 2.场电位记录：制备海马冠状切片,检测海马Schaffer支-CA1突触的兴奋性突触后电位(excitatory post-synaptic potentiation, EPSP)和双脉冲易化(paired-pulse facilitation, PPF)。 3.全细胞膜片钳记录：制备海马冠状切片,检测脑片海马CA1锥体神经元微小兴奋性突触后电流(miniature excitatory postsynaptic current, mEPSC)、NMDA受体介导的电流(NMDA-indcued current,INMDA)和海马突触前锥体神经元高电压激活钙电流(high voltage-gated calcium current,Ica)。 4.甲苯胺蓝染色：同第一部分。结果 1.场电位记录结果显示,TRPV4受体激动齐4α-PDD进行海马脑片灌流能引起海马Schaffer侧支-CA1突触EPSP的斜率增加伴有PPF的比值降低,提示TRPV4受体活化能增加突触前神经递质的释放。当降低海马脑片细胞外灌流液的渗透压到240mOsm/kg(低渗刺激),海马Schaffer侧支-CA1突触EPSP斜率显著增加,而PPF的比值降低。TRPV4受体阻断剂(HC-067047)能够阻断低渗突触前神经递质的释放。 2.全细胞膜片钳记录结果显示,低渗增加海马脑片CA1锥体细胞mEPSC的频率和幅度,提示低渗刺激不仅能增加突触前神经递质的释放,还能增加突触后膜谷氨酸受体的活性。 3.低渗刺激对海马CA1突触前锥体神经元的电压门控性钙电流(Ica)的幅度、电流—电压曲线、激活曲线和失活曲线无明显作用。给予N型或P/Q型电压门控性钙通道阻断剂不能影响低渗刺激增加EPSP斜率。 4. TRPV4受体激动剂4a-PDD能增加海马CA1锥体神经元INMDA的幅度。同样低渗刺激也能增加海马CA1锥体神经元INMDA的幅度。低渗刺激增加TMDA受体活性的作用能完全被TRPV4受体阻断剂HC-067047所阻断,提示低渗刺激通过激活TRPV4受体能增强谷氨酸NMDA受体功能活性。 5.与]TRPV4受体激动剂4α-PDD的作用相同,低渗刺激能够增加INMDA量效曲线中的最大反应能力,但对ECso值无明显影响,并对INMDA电流的I-V曲线无明显作用,提示低渗刺激激活TRPV4受体能增强谷氨酸INMDA受体离子通道的开放。 6.NR2B亚基特异性阻断剂ifenprodil能有效阻断低渗刺激对INMDA的增强作用,而NR2A亚基阻断剂NVP-AAM007对低渗刺激的作用无明显影响。CaMKII的阻断剂能够阻断低渗刺激对INMDA的增强作用。PKC和CKII的阻断剂对低渗刺激的作用无影响。 7. NMDA受体阻断剂MK801能减轻TRPV4受体过激活诱导海马CA1神经元的损伤。结论缺血再灌注后TRPV4受体过表达和脑水肿增强TRPV4受体活性能增加TRPV4受体的钙离子内流,以促进谷氨酸的释放,同时激活CaMKII通过提高NR2B亚基磷酸化水平增加NMDA受体的钙内流。结果提示缺血再灌注后TRPV4受体功能增加可以引起钙超载,激活细胞凋亡信号通路,进而造成神经元死亡。因此,本研究提出阻断TRPV4受体对脑缺血再灌注损伤有保护作用。
[Abstract]:With the rapid growth of the population of the world, the incidence of cerebral apoplexy (ischemic cerebrovascular disease) is increasing year by year. It has become one of the most important diseases that threaten human life. Because of the high incidence of stroke, high disability rate and high mortality, stroke has brought great mental pressure and economic burden to individuals, families and society. Although thrombolytic therapy can protect neurons by restoring cerebral oxygen supply as early as possible, reperfusion after ischemia may also aggravate the injury of brain tissue (reperfusion injury) and lead to necrosis of neurons. Therefore, it is important to study the pathophysiological mechanism of cerebral ischemia induced brain injury and find effective therapeutic targets for the improvement of the cure rate of stroke patients. It's meaning.
Cerebral injury induced by ischemia-reperfusion is mainly related to excitatory toxicity of glutamic acid, intracellular calcium (intracellular Ca2+concentration, [Ca2+]i) overload, damage of free radicals and nitric oxide, brain edema and other pathological mechanisms. It is important to explore the mechanism of neuronal damage induced by non glutamic acid dependent calcium overload.
The transient receptor potential (TRP) receptor family is an important class of non selective cation channels. The transient receptor potential vanillin receptor subfamily (TRP vanilloid4, TRPV4) is widely distributed in the central nervous system, mainly expressed as.TRPV4 receptors in the hippos, cerebral cortex, thalamus, cerebellum and other brain regions. A cation channel based on calcium ions, which is activated, can cause Ca2+ - based introversion current. In recent years, more and more studies have reported that the members of the TRP receptor family involved in the pathological process of cerebral ischemia reperfusion injury, the nelastati family type II in the.TRP family, type VII and canonical family VI have been reported to have been mediated The neuron death.TRPV4 receptor caused by cerebral ischemia has been discovered since it has been activated by a variety of stimuli, such as hypotonic, warm, mechanical, peanut four enoic acid and its metabolites. A large number of arachidic acid produced by metabolic disorders can also activate the TRPV4 receptor through its metabolite 5,6- epoxy twenty carbon three glycic acid. It is suggested that the pathological process of cerebral injury induced by ischemia-reperfusion may be related to the activation of TRPV4 receptor. Blocking the TRPV4 receptor can reduce the impairment of oxidative stress on astrocytes in the hippocampus. Injury also has protective effects on hippocampal CA1 neurons damage induced by oxygen glucose deprivation, suggesting that blocking TRPV4 receptor may alleviate ischemic brain damage.
Our previous study on primary sensory neurons found that the activation of the TRPV4 receptor was enhanced by regulating the voltage dependent ion channels and the function of the TRPV1 receptor on the cell membrane by affecting the downstream signal pathways (PKA, PKC, PKG, etc.). A study on the retinal deity ganglion cells found that TRPV4 receptor agonists could be used. Dose dependence increases [Ca2+]i, increases cell excitability and induces apoptosis. In addition, activation of TRPV4 receptors can increase the frequency of small excitatory postsynaptic currents between cultured hippocampal neurons, suggesting that cerebral ischemia-reperfusion may promote the release of glutamic acid by activating the TRPV4 receptor.
Therefore, the subject will first clarify whether the TRPV4 receptor is involved in the pathological process of cerebral ischemia reperfusion injury in vivo, and then focus on the protection of TRPV4 receptor on cerebral ischemia reperfusion injury and its molecular mechanism, and provide new targets and ideas for the prevention and treatment of cerebral ischemia reperfusion injury in the future.
research objective
1. whether TRPV4 receptor is involved in the injury induced by cerebral ischemia-reperfusion.
2. elucidate the molecular mechanism of blocking TRPV4 receptor in protecting cerebral ischemia-reperfusion injury.
Part 1 the role of TRPV4 receptor in cerebral ischemia-reperfusion injury
Materials and methods
1. mouse model of cerebral ischemia and reperfusion was made: the right middle cerebral artery 60min was blocked with fish line, and the middle cerebral artery occlusion (MCAO) was prepared and the mouse model was reperfused.
2. real-time quantitative RT-PCR and Western blot:MCAO were used to remove hippocampal formation at different time points after reperfusion.
MRNA and protein were extracted to detect the nRNA and protein levels of TRPV4 receptor.
3.TTC staining: MCAO reperfusion of the brain tissue of 24h and reacting in TTC solution after coronary slice, the normal brain tissue is red and the infarct tissue is white, and the volume of white brain tissue is the infarct volume after MCAO reperfusion.
4. toluidine blue staining: paraffin embedded sections of brain tissue were fixed with paraformaldehyde and stained with toluidine blue to carry out counting of hippocampal CA1 pyramidal neurons.
Result
The expression of mRNA in 2-72hTRPV4 receptor increased after 1. MCAO reperfusion, and the level of 4-48hTRPV4 receptor protein in MCAO reperfusion increased. After MCAO reperfusion, the protein level of 18hTRPV4 receptor reached the peak, then gradually decreased, and 72h recovered to normal level after MCAO reperfusion.
The 2. lateral ventricles were given TRPV4 receptor specific blocker HC-067047. Within the dose range of 0.1-30 mu M/2 L/, the volume of 24h cerebral infarction after MCAO reperfusion was reduced in a concentration dependent manner; the effective time window of HC-067047 (10 u M/2 L/ only) on the neuroprotective effect of cerebral ischemia-reperfusion injury was not specific to the TRPV4 receptor. Ruthenium red, a sex blocker, can also effectively reduce the volume of 24h infarction after MCAO reperfusion.
3. compared with control mice, intracerebroventricular injection of TRPV4 receptor agonist 4a-PDD can cause the death of hippocampal CA1 neurons.
conclusion
The expression of TRPV4 receptor in hippocampus of 4-48h increased significantly after cerebral ischemia-reperfusion. Blocking TRPV4 receptor had protective effect on cerebral ischemia-reperfusion injury.
The second part is to block the molecular mechanism of TRPV4 receptor protecting cerebral ischemia-reperfusion injury.
Materials and methods
1. the cell model of cerebral ischemia reperfusion injury is established: cerebral ischemia can cause brain edema due to energy metabolism disorder. Our previous study has proved that cell edema can activate TRPV4 receptor. In order to study the mechanism of cell edema activated by TRPV4 receptor after cerebral ischemia, the osmotic pressure of extracellular fluid is reduced in this study. To 240mOsm/kg (hypotonic stimulation) simulated brain edema cell model.
2. field potentials recording: the hippocampal coronary slices were prepared to detect the excitatory postsynaptic potential (excitatory post-synaptic potentiation, EPSP) and double pulse facilitation (paired-pulse facilitation, PPF) of the hippocampal Schaffer branch -CA1 synapses.
3. whole cell patch clamp recording: preparation of hippocampal coronary slices to detect the micro excitatory postsynaptic current (miniature excitatory postsynaptic current, mEPSC) of hippocampal CA1 pyramidal neurons, NMDA receptor mediated current (NMDA-indcued current, INMDA) and high voltage activated calcium current (high voltage-gate) in the hippocampal synaptic pyramidal neurons (high voltage-gate). D calcium current, Ica).
4. toluidine blue staining: same as the first part.
Result
The results of 1. field potential recording showed that the perfusion of TRPV4 receptor excited Qi 4 alpha -PDD in hippocampal slices could cause the increase of the slope of -CA1 synapse EPSP in the lateral branch of the hippocampal Schaffer and the ratio of PPF, suggesting that the activation of TRPV4 receptor can increase the release of presynaptic neurotransmitters. When the osmotic pressure of the fine extracellular perfusion fluid of the hippocampus slices to 240mOsm/kg (low permeability) Stimulated), the slope of -CA1 synapse EPSP in the lateral branch of the hippocampal Schaffer increased significantly, while the ratio of PPF to the.TRPV4 receptor blocker (HC-067047) could block the release of the hypotonic neurotransmitter.
2. whole cell patch clamp recording results showed that low permeability increased the frequency and amplitude of mEPSC in hippocampal slice CA1 pyramidal cells, suggesting that hypotonic stimulation not only increased the release of presynaptic neurotransmitters, but also increased the activity of glutamate receptors in the postsynaptic membrane.
3. hypotonic stimulation has no obvious effect on the amplitude of voltage gated calcium current (Ica), current voltage curve, activation curve, and inactivation curve of hippocampal CA1 presynaptic pyramidal neurons. Giving N or P/Q type voltage gated calcium channel blockers can not affect low permeability stimulation to increase EPSP skew.
4. TRPV4 receptor agonist 4a-PDD can increase the amplitude of INMDA in hippocampal CA1 pyramidal neurons. The same hypotonic stimulation also increases the amplitude of INMDA in hippocampal CA1 pyramidal neurons. The effect of hypotonic stimulation on the activity of TMDA receptor can be completely blocked by the TRPV4 receptor blocker HC-067047, suggesting that hypotonic stimulation can enhance glutamate by activating TRPV4 receptor. The functional activity of NMDA receptor.
5. the action of]TRPV4 receptor agonist 4 alpha -PDD is the same. Hypotonic stimulation can increase the maximum reaction ability in the INMDA dose effect curve, but it has no obvious effect on the ECso value, and has no obvious effect on the I-V curve of INMDA current, suggesting that the activation of the TRPV4 receptor by the hypotonic stimulation can enhance the opening of the glutamate INMDA receptor ion channel.
6.NR2B subdivision antagonist ifenprodil can effectively block the enhancement of INMDA by low permeability stimulation, while NR2A subunit blocker NVP-AAM007 has no significant effect on the effect of low permeability stimulation..CaMKII blockers can inhibit the enhancement of low permeability stimulation to INMDA, and.PKC and CKII have no effect on the effect of low permeability stimulation.
7. NMDA receptor blocker MK801 can alleviate the damage of CA1 neurons induced by TRPV4 receptor over activation.
conclusion
The overexpression of TRPV4 receptor and the enhancement of TRPV4 receptor activity after ischemia reperfusion can increase the calcium influx of TRPV4 receptor, promote the release of glutamate, and activate CaMKII by increasing the level of NR2B subunit phosphorylation to increase the calcium influx of the NMDA receptor. The results suggest that the increase of TRPV4 receptor function after ischemia-reperfusion can cause calcium overload. Activation of apoptotic signaling pathway, which leads to neuronal death, therefore suggests blocking TRPV4 receptors to protect against cerebral ischemia-reperfusion injury.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743

【参考文献】