调控线粒体形态与功能恢复高糖状态下七氟醚后处理心肌保护作用的机制研究

发布时间：2018-01-15 04:17

本文关键词：调控线粒体形态与功能恢复高糖状态下七氟醚后处理心肌保护作用的机制研究　出处：《新疆医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:心血管疾病严重威胁围术期麻醉与手术安全。缺血再灌注损伤(IRI)是围术期心脏不良事件发生的主要原因。通过在再灌注早期应用七氟醚实现心肌保护效应的方法被称为七氟醚后处理(Sevoflurane Postconditionning,SPostC)。SPostC可产生类似缺血/缺氧预处理的心肌保护作用,且已经证实对于年轻健康的心肌所发生的IRI具有良好的心肌保护效果,是极具希望的抗心肌损伤的治疗手段。然而,糖尿病状态下,SPostC心肌保护效果缺失或弱化,严重制约了SPostC的转化应用。并且使用胰岛素使糖尿病大鼠血糖水平正常后仍不能恢复SPostC的心肌保护作用。如何才能恢复现有心肌保护手段对糖尿病等病理性心肌的保护作用,是目前围术期医学亟待解决的重要临床问题。本研究期望通过探讨七氟醚后处理的线粒体保护机制,寻找保护效应弱化的关键,并观察高糖对七氟醚后处理调控线粒体融合分裂作用的干扰与影响;最终通过调控线粒体融合分裂平衡恢复高糖状态下SPostC的心肌保护作用。方法:建立在体大鼠IRI模型与离体Langendorff心脏缺血再灌模型。在体大鼠IRI模型,缺血30min再灌注2h,测定心肌梗死面积、ATP含量与JAK2、STAT3的磷酸化水平。离体Langendorff心脏缺血再灌模型,复灌末测定线粒体3态呼吸、线粒体呼吸控制率(RCR),与线粒体呼吸酶活性[细胞色素C氧化酶(Cc O)、NADH氧化酶(NADHO)及琥珀酸氧化酶(SUCO)]。透射电镜观察心肌细胞线粒体超微结构。原代培养乳鼠心肌细胞,分别在低糖与高糖浓度下培养48h后建立缺氧/复氧损伤模型,实施SPost C(复氧开始时吸入2.4%七氟醚)。缺氧或缺血前给予YC-1(终浓度为100μmol/L)、2ME2(2μmol/L)、环孢素A(终浓度0.2μmol/L)、Mdivi-1(终浓度50μmol/L或100μmol/L)进行干预,观察心肌细胞复氧后的死亡率、LDH水平、细胞存活率,线粒体形态改变、mPTP开放程度及线粒体膜电位变化。应用western blotting或免疫荧光法测定线粒体融合分裂关键蛋白(Drp1、Fis1、Mfn1、Mfn2与Opa1)的表达水平,HIF-1α表达水平。结果:在体IRI模型中,SPost C通过增加JAK2/STAT3的磷酸化水平降低再灌注后心梗面积,保持线粒体完整结构(P0.05,SPostC组vs I/R组)。离体灌流模型中,SPostC稳定HIF-1α恢复线粒体呼吸功能与呼吸酶活性(P0.05,SPost C组vs I/R组)。细胞实验缺氧复氧损伤后,SPostC通过增加线粒体内部连接度与拉长度,抑制mPTP开放、维持线粒体膜电位从而降低LDH水平与细胞死亡率,增加细胞存活率发挥心肌保护作用(P0.05,SPostC组vs H/R组)。但是HIF-1α抑制剂消除了这一保护作用(P0.05,YC-1组vs SPostC组)。高糖浓度下,CsA未能降低缺氧/复氧损伤后的LDH水平与细胞死亡率(P0.05),线粒体平均面积/周长比与线粒体膜电位水平变化也无统计学差异(P0.05);但应用Drp1抑制剂Mdivi-1恢复了高糖浓度下SPostC对线粒体形态的影响(P0.05),挽救了高糖状态下SPost C对细胞死亡率、LDH水平与细胞存活率的作用(P0.05)。结论:SPostC通过激活大鼠JAK2-STAT3信号通路抵抗缺血/再灌注损伤发挥心肌保护作用。SPost C通过调节线粒体融合分裂关键蛋白Mfn2、Opa1、Drp1的表达,增加线粒体连接度(平均面积/周长)与拉长度(反圆度),维持线粒体融合分裂平衡,抑制mPTP通道开放同时稳定线粒体膜电位。HIF-1α是SPostC维持线粒体融合分裂平衡,改善心肌线粒体呼吸功能及呼吸酶活性发挥心肌保护作用的关键分子。体外细胞实验,高糖降低线粒体连接性与拉长度、促进线粒体分裂致mPTP通道开放和线粒体膜电位的降低,消除了SPostC的心肌保护效果,并且此作用与Drp1增高及Opa1表达不足有关。缺氧前应用环孢素A抑制m PTP开放无法恢复该保护效应,但应用Drp1抑制剂Mdivi-1干预心肌细胞线粒体融合分裂过程,抑制高糖状态下的线粒体过度分裂恢复了SPostC的心肌保护作用。
[Abstract]:Objective: cardiovascular disease is a serious threat to perioperative anesthesia and operation safety. Ischemia reperfusion injury (IRI) is a major cause of perioperative adverse cardiac events. Through the method of myocardial protection effect in the early stage of reperfusion of sevoflurane is known as sevoflurane (Sevoflurane Postconditionning, SPostC.SPostC) can produce similar ischemia hypoxia preconditioning for myocardial protection, which has been confirmed to occur for young healthy myocardial IRI has myocardial protective effect is very good, hope that the treatment of anti myocardial injury. However, the condition of diabetes, the effect of SPostC deletion or weakening of myocardial protection, seriously restricts the transformation and application of SPostC and protective effect. The use of insulin to the blood glucose levels in diabetic rats after myocardial recovery still cannot SPostC. How can the existing means of myocardial protection on the recovery of diabetes The protective effect of pathological myocardial, is an important clinical problem in the perioperative medicine needs to be solved. This research expects mitochondrial protection mechanism of sevoflurane by postprocessing, find the key protective effect of weakening, and observe the effect and effects of interference on sevoflurane postprocessing regulation of mitochondrial fusion division; finally through the regulation of mitochondrial fission and fusion balance recovery myocardial protective effect of SPostC under high glucose condition. Methods: establish the rat model of IRI and Langendorff from ischemia reperfusion heart model in rats. IRI model, 2h ischemia and 30min reperfusion, myocardial infarct size was determined, the contents of ATP and JAK2, the phosphorylation level of STAT3 from the Langendorff heart ischemia. At the end of reperfusion and reperfusion model, determination of mitochondrial state 3 respiration, mitochondrial respiratory control rate (RCR), oxidase and mitochondrial respiratory enzyme activity [cytochrome C (Cc O), NADH oxidase (NADHO) Succinate oxidase (SUCO) and observe the mitochondrial ultrastructure of myocardial cells. TEM. Primary cultured neonatal rat myocardial cells were cultured in 48h, low sugar and high glucose concentration after the hypoxia / reoxygenation injury model, the implementation of SPost C (inhalation of 2.4% sevoflurane reoxygenation started). Lack of oxygen or given before ischemia (YC-1 the final concentration of 100 mol/L), 2ME2 (2 mol/L), cyclosporin A (final concentration 0.2 mol/L), Mdivi-1 (final concentration of 50 mol/L or 100 mol/L) intervention, myocardial cells were observed after reoxygenation mortality, the level of LDH, the survival rate of cells, mitochondrial morphology change, change the opening degree and the mitochondrial membrane potential of mPTP. Determination of mitochondrial fission and fusion protein by Western or blotting key immunofluorescence (Drp1, Fis1, Mfn1, Mfn2 and Opa1) the expression level of HIF-1 alpha expression. Results: in the IRI model, SPost C by increasing the phosphorylation level of JAK2/STAT3 decreased again After reperfusion the myocardial infarction area, maintain complete mitochondrial structure (P0.05, SPostC group, vs group I/R). In vitro perfusion model, mitochondrial respiratory function and enzyme activity of SPostC alpha stable HIF-1 (P0.05, SPost group C recovery vs I/R group). Cells of experimental hypoxia reoxygenation injury after SPostC by increasing mitochondrial internal connectivity with the length of pull, inhibiting the opening of mPTP, maintaining mitochondrial membrane potential so as to reduce the level of LDH and the rate of cell death and increased cell survival rate play a role in myocardial protection (P0.05, SPostC group vs group H/R). But HIF-1 alpha inhibitors eliminates this protective effect (P0.05, YC-1 group vs group SPostC). Under high concentration of glucose, CsA to reduce the level of LDH cells to hypoxia / reoxygenation injury and mortality of oxygen (P0.05), average area / perimeter ratio changes of mitochondria and mitochondrial membrane potential level had no significant difference (P0.05); but the application of Drp1 inhibitor Mdivi-1 recovered under high concentration of glucose SPo Effect of stC on mitochondrial morphology (P0.05), to save the death rate of SPost cells C under high glucose condition, the level of LDH and the survival rate of cell function (P0.05). Conclusion: SPostC can activate the JAK2-STAT3 signaling pathway in rats against ischemia / reperfusion injury play a role in myocardial protection of.SPost C through the regulation of mitochondrial fission and fusion protein Mfn2. Opa1, Drp1 expression, increased mitochondrial connectivity (average area / perimeter) and length of pull (anti roundness), maintain the balance of mitochondrial fusion fission, inhibition of mPTP channel opening and the stability of mitochondrial membrane potential.HIF-1 SPostC alpha is to maintain mitochondrial fusion and fission balance of key molecules to improve mitochondrial respiratory function and respiratory enzyme activity in myocardium of myocardial preservation role. In vitro experiments, high glucose decreased mitochondrial connectivity and length of pull, reduce mitochondrial fission induced by mPTP channel opening and the mitochondrial membrane potential, elimination In addition to the myocardial protective effect of SPostC, and this effect with the increase of Drp1 and Opa1 expression insufficiency. Using cyclosporine A inhibits m PTP open not be able to restore the protective effect of hypoxia, but the application of mitochondrial Drp1 inhibitor Mdivi-1 on myocardial cell fusion fission process, inhibition of glucose under the situation of excess mitochondrial fission restored the myocardial protective effect of SPostC.

【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R614

【相似文献】