内质网应激在内源性心肌细胞保护中的作用及其信号转导机制研究

发布时间：2018-08-26 16:59

【摘要】： 心肌缺血是心肌重构和心力衰竭的主要原因，减轻心肌缺血损伤是防治心力衰竭的关键，尽早恢复缺血心肌血流是减轻心肌缺血损伤的根本措施。但是，经过一定时间缺血的组织器官在恢复血液灌注后，会出现不可逆性再灌注损伤。探索激发机体内源性保护机制，减轻缺血再灌注(ischemia/reperfusion, I/R)损伤是防治心肌缺血的关键。缺血预处理(ischemic preconditioning, IPC)及缺血后处理(ischemic postconditioning, I-postC)是近年发现的重要内源性保护机制，可明显缩小缺血心肌的梗死面积，减轻I／R损伤。探讨IPC和I-postC心脏保护的共同及不同机制具有重要的科学意义，并将为其临床应用提供新的思路。内质网(endoplasmic reticulum, ER)是真核细胞中重要细胞器，I／R过程中的多种刺激因素，包括缺氧、酸中毒、ATP耗竭、氧化应激、钙超载等均可导致内质网功能失调，即内质网应激(ER stress, ERS)。一定程度的ERS可诱导葡萄糖调节蛋白类(glucose-regulated proteins, GRPs)、钙网蛋白(calreticulin, CRT)、折叠酶等ER伴侣分子表达上调，增强ER处理未折叠蛋白的能力，促进ER功能恢复；ERS持续存在或过强时将诱导caspase-12、CHOP／GADD153等促凋亡因子的表达／活化，触发ER相关性凋亡信号途径，诱导细胞凋亡。持续而严重的ERS是由I／R诱导并导致组织细胞损伤的重要机制。IPC、I-postC是否可通过调节ERS反应，增强细胞对应激因素的耐受能力，缓解ER应激程度，从而减轻I／R损伤?本工作采用乳鼠心肌细胞缺氧／复氧(hypoxia／reoxygenation, H／R)模型模拟在体I／R损伤，观察缺氧预处理(hypoxic preconditioning, HPC)和缺氧后处理(hypoxic postconditioning, H-postC)对于ER应激分子GRP78、CRT表达及caspase-12活化的影响及其与心肌细胞保护的关系，并探讨HPC和H-postC调节ERS、介导心肌细胞保护的细胞信号转导机制。主要方法与结果如下：原代培养的Sprague-Dawley乳鼠心肌细胞缺氧2 h复氧14 h复制H／R模型，HPC组细胞缺氧20 min复氧24 h后进行H／R操作；H-postC组细胞缺氧2h后先进行3轮5 min复氧／5 min缺氧的缺氧后处理，再进行持续复氧14 h结束实验。 1．HPC和H-postC对于心肌细胞H／R损伤的影响采用台盼蓝排斥实验检测细胞存活率；采用LDH活性测试盒检测细胞培养液中LDH漏出情况；采用Annexin V-FITC细胞凋亡检测试剂盒检测细胞凋亡率。结果显示：HPC、H-postC可明显提高H／R心肌细胞的存活率，减轻细胞凋亡和LDH漏出。 2．HPC、H-postC对ERS分子表达／活化的影响采用逆转录聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)方法检测细胞GRP 78 mRNA水平；Western blot分析检测CRT表达和caspase-12活化水平。结果表明：H／R诱导细胞GRP 78 mRNA、CRT表达和caspase-12活化上调，HPC及H-postC使H／R细胞GRP 78 mRNA上调程度增加，减轻H／R后CRT过表达程度及caspase-12活化水平，但H-postC对H／R心肌细胞caspase-12活化的抑制效果较HPC差。 3．HPC、H-postC调节ERS的信号途径研究于HPC、H-postC前应用p38 MAPK特异性抑制剂SB203580及JNK特异性抑制剂SP600125，分别在HPC及H-postC处理前加入细胞培养液(终浓度分别为5μmol／L、25μmol／L，37℃预孵育5-10 min)。结果显示：(1)HPC前应用SB203580可明显抑制CRT表达上调并明显减弱HPC抑制caspase-12活化、减轻心肌细胞H／R损伤的作用；而应用SP600125对CRT表达和caspase-12活化水平和HPC的心肌细胞保护作用均无明显影响。(2)H-postC前应用SB203580可明显抑制CRT表达上调，并降低H-postC对细胞H／R损伤的保护，但仅轻度减弱H-postC对caspase-12活化的抑制作用；H-postC前应用SP600125对CRT表达及H-postC细胞保护作用未产生明显影响，但可进一步降低caspase-12的活化水平。结论：HPC及H-postC均可减轻H／R心肌细胞的损伤及凋亡，，二者对细胞的保护水平相当；其细胞保护机制均涉及p38 MAPK介导的ERS调节及ERS相关细胞凋亡途径的抑制。但HPC对H／R诱导caspase-12活化的抑制效果较H-postC为强。JNK活化可促进caspase-12激活，可能参与H／R诱导的过度ERS反应及ERS介导的细胞凋亡。
[Abstract]:Myocardial ischemia is the main cause of myocardial remodeling and heart failure. Reducing myocardial ischemic injury is the key to prevent and treat heart failure. Early recovery of ischemic myocardial blood flow is the fundamental measure to reduce myocardial ischemic injury. The key to preventing and treating myocardial ischemia is to stimulate the endogenous protective mechanism of organism and to reduce ischemia/reperfusion (I/R) injury. Ischemic preconditioning (IPC) and ischemic postconditioning (I-postC) are important endogenous protective mechanisms discovered in recent years, which can significantly reduce the ischemic myocardium. It is of great scientific significance to explore the common and different mechanisms of IPC and I-postC cardioprotection and to provide new ideas for its clinical application.
Endoplasmic reticulum (ER) is an important organelle in eukaryotic cells. Many stimulating factors in I/R process, including hypoxia, acidosis, ATP depletion, oxidative stress, calcium overload and so on, can lead to endoplasmic reticulum dysfunction, that is, endoplasmic reticulum stress (ERS). The up-regulation of ER chaperone molecules such as Ted proteins, GRPs, calreticulin (CRT) and folding enzyme enhances the ability of ER to treat unfolded proteins and promotes the recovery of ER function; the persistence or excessive presence of ERS induces the expression and activation of pro-apoptotic factors such as caspase-12, CHOP/GAD153, triggers ER-related apoptotic signaling pathways and induces cell apoptosis. Apoptosis. Persistent and severe ERS is an important mechanism by which I/R induces tissue and cell damage. Can IPC and I-postC enhance the tolerance of cells to stress factors and alleviate ER stress so as to reduce I/R injury by regulating the response of ERS? The effects of hypoxic preconditioning (HPC) and hypoxic postconditioning (H-postC) on the expression of ER stress molecules GRP78, CRT and caspase-12 activation and their relationship with myocardial cell protection were observed. The relationship between HPC and H-postC regulating ERS and mediating myocardial cell protection was also discussed. The main methods and results are as follows:
The primary cultured Sprague-Dawley neonatal rat cardiomyocytes were hypoxic for 2 h and reoxygenated for 14 h to replicate the H/R model. The HPC group was hypoxic for 20 min and reoxygenated for 24 h, and the H-postC group was hypoxic for 2 h, followed by 3 rounds of 5 min reoxygenation/5 min hypoxic postconditioning followed by 14 h continuous reoxygenation.
1. The effects of HPC and H-postC on H/R injury of cardiomyocytes were detected by Trypan blue rejection assay; LDH leakage was detected by LDH activity test kit; and apoptosis was detected by Annexin V-FITC cell apoptosis test kit. The results showed that HPC and H-postC could significantly improve H/R myocardial cells. The survival rate alleviated cell apoptosis and LDH leakage.
2. The effects of HPC and H-postC on the expression and activation of ERS were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. The up-regulation of se-12 activation, the up-regulation of GRP 78 mRNA in H/R cells by HPC and H-postC, and the down-regulation of CRT overexpression and caspase-12 activation after H/R were alleviated. However, the inhibition effect of H-postC on the activation of caspase-12 in H/R myocardial cells was worse than that of HPC.
3. The signal pathway of regulating ERS by HPC and H-postC was studied before HPC and H-postC were treated with SB203580, a p38 MAPK specific inhibitor, and SP600125, a JNK specific inhibitor. Cell culture medium was added before HPC and H-postC treatment (the final concentration was 5 micromol/L, 25 micromol/L, pre-incubated at 37 C for 5-10 minutes). The results showed that: (1) SB203580 could be used before HPC treatment. SP600125 had no significant effect on CRT expression, caspase-12 activation level and myocardial protective effect of HPC. (2) SB203580 before H-postC significantly inhibited the up-regulation of CRT expression and decreased H/postC on myocardial cells. The protective effect of H-postC on the activation of caspase-12 was slightly weakened, but the protective effect of SP600125 on the expression of CRT and the cytoprotection of H-postC before H-postC was not obvious, but the activation level of caspase-12 was further decreased.
CONCLUSION: Both HPC and H-postC can reduce the injury and apoptosis of H/R cardiomyocytes, and both of them have the same level of protection to H/R cardiomyocytes. The mechanism of cell protection involves the regulation of ERS mediated by p38 MAPK and the inhibition of ERS-related apoptosis pathway. 12 activation may be involved in H / R induced excessive ERS response and ERS mediated apoptosis.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363.2

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