腺苷A2受体活化诱导心肌再灌注损伤保护作用的线粒体机制

发布时间：2018-04-29 15:56

本文选题：腺苷A2受体 + Src酪氨酸激酶　；参考：《天津医科大学》2016年博士论文

【摘要】：目的1.观察腺苷A2受体活化对心肌再灌注时线粒体氧化应激损伤的保护作用。2.探讨腺苷A2受体活化诱导心肌再灌注损伤保护作用的线粒体机制。方法建立离体大鼠心脏缺血/再灌注(Ischemia/reperfusion,I/R)损伤模型和心肌H9c2细胞系模拟I/R损伤模型。采用透射电镜和高分辨率呼吸测定仪评定分离线粒体的结构和功能改变。利用线粒体荧光探针JC-1、总活性氧(Reactive oxygen species,ROS)荧光探针DCFH-DA、线粒体超氧化物荧光探针Mitosox Red分别测定分离心肌线粒体和细胞的线粒体膜电位(Mitochondrial membrane potential,△Ψm)、总ROS和线粒体超氧化物水平。使用Amplex red过氧化氢/过氧化物酶检测试剂盒(Amplex red hydrogen peroxide/peroxidase assay kit)测定H2O2含量及过氧化氢酶(Catalase)活性。用线粒体肿胀实验评定线粒体通透性转换孔(Mitochondrial permeablity transition pore,mPTP)的开放程度。用蛋白羰基化检测确定线粒体蛋白的氧化应激损伤程度。Western blotting法检测蛋白表达水平。利用复合体I酶活性检测试剂盒(Complex I enzyme activity dipstick assay kit)检测线粒体呼吸链复合体I的活性。免疫沉淀法富集线粒体复合体I并检测其蛋白酪氨酸磷酸化水平,并用质谱确定酪氨酸磷酸化水平发生变化的目标复合体亚单位,用NetPhosK 1.0确定目标复合体亚单位的潜在酪氨酸磷酸化位点。结果1.与sham组相比,I/R组的呼吸控制率(Respiratory control ratio,RCR)下降,肿胀破裂的线粒体增多,△Ψm降低,线粒体对钙离子的抵抗能力减弱,但用非选择性腺苷受体激动剂5’-N-乙基酰胺基腺苷(5’-(N-ethylcarboxamido)adenosine,NECA)处理后这些变化均减轻(均P0.05)。选择性腺苷A2A受体拮抗剂SCH58261(SCH)和A2B受体拮抗剂MRS1706(MRS)能够不同程度地减弱NECA对线粒体的保护作用(均P0.05)。这些结果表明,腺苷A2A和A2B受体均参与NECA对I/R诱发的心肌线粒体损伤的保护作用。2.I/R后线粒体蛋白羰基化,线粒体总ROS、H2O2增多,提示再灌注后线粒体ROS增多引起氧化应激损伤(均P0.05)。给予NECA能够明显抑制线粒体ROS的增多,减轻线粒体的氧化应激损伤,但此作用被SCH或MRS抑制。以上结果表明,腺苷A2A或A2B受体活化能够抑制再灌注早期ROS的增多而减轻线粒体氧化应激损伤。3.细胞实验证实,NECA能够抑制I/R引起的△Ψm减低和ROS增多;线粒体超氧化物歧化酶Peg-SOD具有与NECA类似的作用;二者联合应用对△Ψm的保护作用增强(均P0.05)。与sham组相比,I/R组超氧化物歧化酶(Superoxide dismutase,SOD)的活性降低,但NECA对其活性没有影响;而NECA能够明显增强过氧化氢酶的活性但此作用被SCH或MRS抑制(均P0.05)。这些结果表明腺苷A2A或A2B受体活化通过增加过氧化氢酶的活性而促进H2O2的分解并减轻线粒体氧化应激损伤。4.与sham组相比,I/R时线粒体呼吸链复合体I活性增强,但被NECA、CGS或BAY所抑制(均P0.05),表明腺苷A2A或A2B受体活化可能通过抑制再灌注期复合体I的活性而减少ROS的产生。5.与对照组相比,NECA能够增加线粒体Src酪氨酸激酶活性,但此作用被MRS抑制;选择性Src酪氨酸激酶拮抗剂PP2能够减弱NECA对I/R引起的心肌线粒体△Ψm的降低、线粒体对钙离子的抵抗能力减弱以及ROS产生增多等作用(均P0.05)。另外与NECA组相比,PP2+NECA组复合体I的活性降低。以上结果表明,腺苷A2受体活化通过激活线粒体Src酪氨酸激酶而抑制复合体I的活性并抑制再灌注期ROS增多,从而减轻I/R诱导的心肌线粒体损伤。6.质谱和NetPhosK 1.0分析推断出复合体I NDUFV2亚单位的Tyr-118,191和192位点,NDUFS3的Tyr-146,245位点以及NDUFS2的Tyr-151位点可能起负性调节复合体I活性的作用。结论1、腺苷A2A或A2B受体激活后,一方面通过抑制复合体Ⅰ的活性减少再灌注时ROS的产生,另一方面通过增加过氧化氢酶的活性而促进ROS的分解,抑制mPTP的开放,进而减轻再灌注时线粒体的氧化应激损伤。2、NECA通过腺苷A2受体激活线粒体Src酪氨酸激酶,从而抑制复合体I活性并抑制ROS的产生,减轻线粒体氧化应激损伤。3、复合体I NDUFV2亚单位的Tyr-118,191和192位点,NDUFS3的Tyr-146,245位点以及NDUFS2的Tyr-151位点可能具有负性调节复合体Ⅰ活性的作用。
[Abstract]:Objective 1. to observe the protective effect of adenosine A2 receptor activation on oxidative stress damage of mitochondria during myocardial reperfusion.2. to explore the mitochondrial mechanism of the protective effect of adenosine A2 receptor activation induced myocardial reperfusion injury. Methods the isolated rat model of myocardial ischemia / reperfusion (Ischemia/reperfusion, I/R) injury and myocardial H9c2 cell line simulation I/R were established. The structure and function of isolated mitochondria were assessed by transmission electron microscopy and high-resolution breathing apparatus. The mitochondrial fluorescence probe JC-1, Reactive oxygen species (ROS) fluorescent probe DCFH-DA, and mitochondrial superoxide fluorescent probe Mitosox Red were used to separate mitochondria and mitochondria from mitochondria and mitochondria respectively. Membrane potential (Mitochondrial membrane potential, delta m), total ROS and mitochondrial superoxide levels. Determine the content and catalase activity using the Amplex red peroxide / peroxidase assay kit (Amplex red hydrogen peroxide/peroxidase assay). The mitochondrial permeability test was used to evaluate the mitochondrial permeability. The opening degree of Mitochondrial permeablity transition pore (mPTP) was detected by protein carbonylation to determine the oxidative stress damage degree of mitochondrial protein,.Western blotting method was used to detect protein expression level. The detection of mitochondrial respiration by the complex I enzyme activity detection kit (Complex I enzyme activity) The activity of the chain complex I. The immunoprecipitation method enriched the mitochondrial complex I and detected its protein tyrosine phosphorylation level, and determined the target complex subunit of the tyrosine phosphorylation level by mass spectrometry. The potential tyrosine phosphorylation site of the target complex subunit was determined by NetPhosK 1. Results 1. compared with the sham group, the I/R group was compared with the group I/R. The respiratory control rate (Respiratory control ratio, RCR) decreased, the swelling and ruptured mitochondria increased, delta m decreased, and the mitochondrial resistance to calcium decreased, but these changes were reduced after treatment with non selective adenosine receptor agonist 5 '-N- ethyl amidosyladenosine (5' - (N-ethylcarboxamido) adenosine, NECA). Sexual adenosine A2A receptor antagonist SCH58261 (SCH) and A2B receptor antagonist MRS1706 (MRS) can weaken the protective effect of NECA on mitochondria in varying degrees (P0.05). These results suggest that adenosine A2A and A2B receptors are involved in the protective action of NECA on mitochondrial damage induced by I/R, mitochondria protein carbonylation. 2 increased, suggesting that the increase of mitochondrial ROS caused oxidative stress injury (all P0.05). NECA could inhibit the increase of mitochondrial ROS and reduce oxidative stress damage of mitochondria, but this effect was inhibited by SCH or MRS. The above results showed that the activation of adenosine A2A or A2B receptor can inhibit the increase of early ROS in reperfusion and reduce the grain size. .3. cell experiments of oxidative stress damage confirmed that NECA could inhibit the decrease of delta m and ROS increase caused by I/R; mitochondrial superoxide dismutase Peg-SOD had a similar role to NECA; the two combined application of the protection of delta m was enhanced (P0.05). The activity of I/R group superoxide dismutase (Superoxide) was compared with the sham group. Sex decreased, but NECA had no effect on its activity, while NECA could significantly enhance the activity of catalase, but this effect was inhibited by SCH or MRS (P0.05). These results suggest that the activation of adenosine A2A or A2B receptor activates the decomposition of H2O2 by increasing the activity of catalase and reduces the oxidative stress of linear particles compared to sham groups, I/R. The mitochondrial respiratory chain complex I activity was enhanced, but was inhibited by NECA, CGS or BAY (P0.05), indicating that the activation of adenosine A2A or A2B receptor may reduce ROS production by inhibiting the activity of the reperfusion phase complex I, and that NECA can increase the activity of mitochondrial Src tyrosine kinase, but this effect is inhibited; selective tyrosine The acid kinase antagonist PP2 can weaken the decrease of NECA induced I/R induced mitochondrial delta m, the decrease of mitochondrial resistance to calcium and the increase of ROS production (P0.05). In addition, the activity of I in the PP2+NECA complex is reduced compared with the NECA group. The results show that the activation of adenosine A2 receptor activates the mitochondrial Src tyrosine. The kinase inhibits the activity of the complex I and inhibits the increase of ROS in the reperfusion period, thus reducing the I/R induced myocardial mitochondrial damage by.6. mass spectrometry and NetPhosK 1 analysis to deduce the Tyr-118191 and 192 loci of the complex I NDUFV2 subunit, and the NDUFS3 Tyr-146245 loci and NDUFS2 Tyr-151 positions may play a negative role in regulating the activity of the complex. Conclusion 1, after the activation of adenosine A2A or A2B receptor, one side reduces the production of ROS by inhibiting the activity of complex I, on the other hand, by increasing the activity of the catalase, it promotes the decomposition of ROS, inhibits the opening of mPTP, and then reduces the mitochondrial oxygenation stress damage.2, NECA activates the grain by adenosine A2 receptor. Src tyrosine kinase, which inhibits the activity of complex I and inhibits the production of ROS, reduces the mitochondrial oxidative stress damage.3, the Tyr-118191 and 192 loci of the I NDUFV2 subunit of the complex, and the Tyr-146245 site of NDUFS3 and the Tyr-151 site of NDUFS2 may have a negative regulation of the activity of complex I activity.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54

【相似文献】