【作者】 李杨；

【导师】 康焰；

【作者基本信息】 四川大学 ， 重症医学， 2021， 博士

【摘要】 目的:重症患者经常出现胰岛素抵抗及应急性高血糖,而这会导致很多不良后果。而ICU中强化胰岛素治疗又会引起患者的血糖出现剧烈波动。研究中发现血糖波动可以导致肝细胞、内皮细胞及血管损伤,已经成为评估重症患者预后的独立危险因素。由于葡萄糖是大脑主要代谢底物,越来越多的研究开始关注中枢神经系统,发现相对于持续低糖和高糖,血糖波动对大脑的损伤更大。但是血糖波动引起神经元细胞损伤的具体机制不明。一般认为血糖波动诱导的细胞损伤与线粒体活性氧（ROS）产生增加和抗氧化系统失衡有关。线粒体不仅是细胞内的能量库,还是细胞内重要的钙池。Ca²⁺作为第二信使对线粒体的功能完整具有重要意义,线粒体活性氧的产生/清除均与其密切相关。而线粒体钙离子单向转运体（MCU）是Ca²⁺进入线粒体通道的主要组成成分,其对Ca²⁺内流具有门控作用。但是其具体的调控机制目前尚不明确。miRNAs是机体内不能编码蛋白的一组小分子RNA,但是可通过结合靶基因m RNA的3’UTR从而负向调节m RNA的翻译最终影响靶基因的蛋白表达。在几乎所有的细胞生理过程中都起到了关键作用,如细胞死亡、生存。本研究拟通过体内模型验证血糖波动对大鼠认知功能和海马组织损伤入手,进一步在细胞水平对miR-129-3p在血糖波动所致神经元损伤中通过调控MCU信号通路发挥的作用及相关机制做进一步探讨。材料与方法:1.通过对高糖大鼠每日分时段多次注射胰岛素建立大鼠血糖波动模型模拟ICU中强化胰岛素治疗后的高血糖患者的血糖变化,结合MCU抑制剂钉红处理大鼠,Morriss水迷宫实验观察大鼠认知功能改变,氧化状态指示剂含量及抗氧化物酶活性观察海马组织氧化应激状态。流式细胞学,Tunel染色观察海马组织凋亡,Western Blot检测凋亡相关蛋白。2.体外分离大鼠海马原代神经元细胞,用不同含量葡萄糖培养液建立血糖波动模型。定量PCR和Western Blot分别检测miR-129-3p和MCU的表达水平,分析其表达在各组之间的差异。双荧光素酶报告基因实验,研究mir R-129-3p对MCU的直接靶向作用。分别用miR-NC,miR-129-3p mimics、miR-129-3p inhibitor、miR-129-3p mimics+MCU、miR-129-3p inhibitor+NAC、miR-129-3p mimics+peptide和miR-NC+peptide处理血糖波动海马神经元细胞,流式细胞检测细胞凋亡,通过免疫荧光技术检测线粒体内钙离子水平、活性氧的产生和YAP1核定位,氧化状态指示剂含量及抗氧化物酶活性评估氧化平衡状态,定量PCR和Western Blot检测呼吸链相关分子表达变化,线粒体膜电位检测技术、ELISA,RT-q PCR和Western Blot研究线粒体相关细胞凋亡和Hippo/YAP1通路活化的情况。结果:通过本实验研究,我们发现1)血糖波动可引起大鼠学习记忆能力及空间探索能力的下降,而MCU抑制剂钉红则可减轻其认知功能损伤。2)血糖波动可引起大鼠海马组织氧化能力增加（MDA含量升高）,抗氧化能力下降（抗氧化酶SOD、GSH-Px、CAT活性下降）,而MCU抑制剂钌红则可减轻血糖所致氧化应激。3)血糖波动可引起大鼠海马组织凋亡增加,促凋亡蛋白升高（Bax、caspase-3）,抗凋亡蛋白凋亡下降（Bcl-2）,而MCU抑制剂钌红则可减轻血糖所致凋亡。4)神经元在血糖波动损伤后,MCU的蛋白表达水平升高,而miR-129-3p的m RNA表达水平下降。5)miR-129-3p可直接靶向MCU并降低其m RNA表达。6)血糖波动可致海马神经元细胞凋亡增加,而过表达miR-129-3p可靶向MCU抑制其所致的神经元细胞损伤。7)血糖波动可致海马神经元细胞线粒体内钙离子增加和活性氧增加,过表达miR-129-3p可靶向MCU抑制血糖波动所致钙离子超载和氧化应激。8)血糖波动可致起线粒体抗氧系统失衡（Mn SOD活性下降,GSH/GSSG升高）,而过表达miR-129-3p可靶向MCU引起线粒体抗氧系统恢复稳态。9)血糖波动可致线粒体膜电位稳定下降,促进细胞色素c从线粒体向胞浆,释放过表达miR-129-3p可靶向MCU抑制血糖波动所致线粒体细胞色素c释放,并抑制caspase-9/cleaved-caspase-9与caspase-3/cleaved-caspase-3信号轴的激活。而过表达miR-129-3p可靶向MCU恢复线粒体膜电位稳定,并抑制细胞色素c外放,并最终抑制caspase通路。8)抑制miR-129-3p可加重血糖波动所致神经元细胞凋亡。而抗氧化剂NAC可减轻神经元损伤。9)抑制miR-129-3p可加重线粒体钙超载和氧化应激。而抗氧化剂可减少钙离子内流。10)miR-129-3p可抑制活性氧产生相关呼吸链复合体活性（ND1与UQCRC2）并提高抗氧化转录因子（NRF2）的表达水平。而抗氧化剂也能产生同等效应。11)miR-129-3p可刺激YAP1核转位和抑制Hippo/YAP1通路的激活（促进YAP1和LATS1表达,抑制p-YAP1、p-LATS1、MST1和MST2表达）。12)miR-129-3p通过抑制Hippo/YAP1通路从而减轻神经元细胞的氧化应激状态（MDA含量升高,SOD、GSH-Px、CAT活性升高）。结论:综合以上结果可以发现,血糖波动可致大鼠认知功能下降,发生氧化应激和细胞凋亡。而miR-129-3p可直接靶向MCU调节线粒体内钙离子稳态,降低呼吸链活性氧产生相关复合体活性,恢复抗氧化系统稳态,通过减少ROS堆积抑制线粒体相关的细胞凋亡,从而在血糖波动诱导的神经元损伤中起到保护作用。并且miR-129-3p可通过对Hippo/YAP1通路减少细胞的氧化应激。miR-129-3p/MCU轴有望成为治疗血糖波动引起神经元损伤的潜在靶点。更多还原

【Abstract】 Objective:ICU patients often suffer insulin resistance and acute hyperglycemia,which can lead to many adverse consequences.Intensive insulin treatment（ITT）will cause drastic blood glucose fluctuations.Researchers have found that blood glucose fluctuations can lead to injury for liver cells,endothelial cells and blood vessels,which has become an independent risk factor for evaluating the prognosis of ICU patients.Since glucose is the main metabolite of the brain,researchers have focused on the central nervous system and found that glucose fluctuations are more damaging to the brain than persistent low and high glucose.However,the specific mechanism of blood glucose fluctuations causing neuronal cells damage is unknown.It is generally believed that the cell damage induced by glucose fluctuation is related to the increased production of mitochondrial reactive oxygen species（ROS）and the imbalance of antioxidant system.Mitochondria are not only the energy reservoir,but also the important calcium pool.Ca²⁺as the second messenger,plays an important role in the functional integrity of mitochondria,and is closely related to the production/clearance of ROS.Mitochondrial calcium uniport（MCU）is a one-way Ca²⁺transporter located on the mitochondrial inner membrane,which is important for controlling Ca²⁺influx.But the exact regulation mechanism is not clear yet.miRNAs are a group of small non-coding RNAs that negatively regulate gene expression by binding to the 3’UTR of m RNA targets.which plays an important role in different cellular processes,such as cell death.This study intends to verify the effect of blood glucose fluctuation on cognitive function and hippocampal tissue injury in vivo,and further explore the role and related mechanism of miR-129-3p regulating MCU signal pathway in neuronal injury caused by blood glucose fluctuation in vitro.Materials and Methods:1.High glucose rats were injected with insulin several times a day to establish a rat blood glucose fluctuation model,simulating the blood glucose changes of hyperglycemic patients after intensive insulin treatment in ICU.Rat with applying with MCU inhibitor-ruthenium red,With Morris water maze experiment,we observe the changes of cognitive function,the content of oxidation state indicator and antioxidant enzyme activity,and observe the oxidative stress state of hippocampus.Apoptosis in hippocampus was observed by flow cytometry and TUNEL staining,and apoptosis related proteins were detected by Western blot.2.Primary rat hippocampal neurons were isolated in vitro,and the blood glucose fluctuation model was established with different content of glucose culture medium.The expression levels of miR-129-3p and MCU were detected by quantitative PCR and Western blot,respectively.The direct targeting effect of miR-129-3p on MCU was studied by double luciferase reporter gene experiment.Hippocampal neurons with blood glucose fluctuation were treated with miR-NC,miR-129-3p mimics,miR-129-3p inhibitor,miR-129-3p mimics+MCU and miR-129-3p inhibitor+NAC,miR-129-3p mimics+peptide and miR-NC+peptiderespectively.Cell apoptosis was detected by flow cytometry,calcium ion level and production of reactive oxygen species in mitochondria and nuclear localization of YAP1 were detected by immunofluorescence technique,and oxidation balance was evaluated by the content of oxidation state indicator and antioxidant enzyme activity,Quantitative PCR and Western blot were used to detect the expression of respiratory chain related molecules.Mitochondrial membrane potential detection technology,ELISA,RT q PCR and Western blot were used to study the activation of mitochondrial related apoptosis and Hippo/YAP1 pathway.Results:Through this experimental study,we found that 1)blood glucose fluctuation can caused the decline of learning and memory ability and spatial exploration ability of rats,while MCU inhibitor-ruthenium-red reduced the damage of cognitive function.2)Blood glucose fluctuation can increased the oxidative capacity（increased the content of MDA）and decreased the antioxidant capacity（decreased the activities of antioxidant enzymes SOD,GSH-Px and CAT）,while ruthenium red,an MCU inhibitor,can reduce the oxidative stress caused by blood glucose.3)Blood glucose fluctuation can increased the apoptosis of rat hippocampus,increased the expression of pro-apoptotic protein（Bax,caspase-3）and decrease the expression of anti-apoptotic protein（Bcl-2）,while ruthenium red,an MCU inhibitor,can reduce the apoptosis caused by blood glucose.4)After blood glucose fluctuation injury,the protein expression level of MCU increased,while the m RNA expression level of miR-129-3p decreased.5)miR-129-3p can directly targeted MCU and reduced its m RNA expression.6)Glucose fluctuation can increase the apoptosis of hippocampal neurons,and overexpression of mir-129-3p can targeted MCU to inhibit the neuronal damage caused by it.7)Blood glucose fluctuation can increased calcium and reactive oxygen species in mitochondria of hippocampal neurons.Overexpression of miR-129-3p can targeted MCU to inhibit calcium overload and oxidative stress caused by blood glucose fluctuation.8)Blood glucose fluctuation can caused mitochondrial antioxidant system imbalance（Mn SOD activity decreased and GSH/GSSG increased）,while overexpression of miR-129-3p can targeted MCU to restore the homeostasis of mitochondrial antioxidant system.9)Blood glucose fluctuation can leaded to the stable decrease of mitochondrial membrane potential and promote the release of cytochrome c from mitochondria to cytoplasm.Overexpression of miR-129-3p can targeted MCU to inhibit the release of mitochondrial cytochrome C caused by blood glucose fluctuation and inhibited the activation of caspase-9/cleaved-caspase-9 and caspase-3/cleaved-caspase-3signal axis.Overexpression of miR-129-3p can targeted MCU to restore mitochondrial membrane potential stability,inhibit cytochrome c efflux,and finally inhibit caspase pathway.8)Inhibition of miR-129-3p can aggravated neuronal apoptosis caused by blood glucose fluctuation.The antioxidant（NAC）can reduce neuronal damage.9)Inhibition of miR-129-3p can aggravated mitochondrial calcium overload and oxidative stress.Antioxidants can reduced calcium influx and oxide.10)miR-129-3p can inhibited the activity of respiratory chain complex proteins related to reactive oxygen species production（ND1 and UQCRC2）and increased the expression level of antioxidant transcription factor（Nrf2）,of which antioxidant-NAC-showed the same effect.11)miR-129-3p can stimulated YAP1 nuclear accumulation and inhibited the activation of Hippo/YAP1pathway（increased the expression of p-YAP1 and LATS1,decreased the expression of p-YAP1,p-LATS1,MST1 and MST2）.12)miR-129-3p reduced the oxidative stress state of cells by inhibiting Hippo/YAP1 pathway（increased the content of MDA,decreased the activities of antioxidant enzymes SOD,GSH-Px and CAT）.Conclusion:Based on the above results,it can be found that blood glucose fluctuation can induced cognitive decline,oxidative stress and cell apoptosis in rats.miR-129-3p can directly targeted MCU to regulate calcium homeostasis in mitochondria,reduced the activity of complex related to the production of reactive oxygen species in respiratory chain,restored the homeostasis of antioxidant system,and inhibitd mitochondria related apoptosis by reducing ROS accumulation,so as to play a protective role in neuron injury induced by blood glucose fluctuation.Moreover,miR-129-3p can reduced the oxidative stress of cells through the Hippo/YAP1 pathway.miR-129-3p/MCU axis is expected to be a potential target for the treatment of neuronal damage caused by blood glucose fluctuation.更多还原