pH通过AMPK调控心肌细胞自噬的分子机制研究
发布时间：2018-08-27 09:52
【摘要】：研究背景酸碱平衡是维持细胞内环境稳定,保证细胞正常代谢与功能的前提,而受诸多因素的影响,机体pH会发生一定的波动。pH改变会引发细胞内一系列信号变化,参与多种病理生理过程的调控。例如严重烧创伤、严重感染、慢性肾衰及代谢性疾病等发生后,细胞产酸大于排酸,导致酸中毒,造成细胞损伤、组织脏器功能障碍。而胞浆碱化与细胞增殖密切相关,是恶性肿瘤重要特征之一。这提示我们pH对细胞功能状态具有重要调控作用。心脏是循环系统的动力器官,严重烧伤后早期即出现心脏损伤。心脏受损不仅可引起心功能不全,还可进一步加重全身其它组织器官缺血缺氧性损害。因此,烧伤后心肌损害的研究具有极其重要的理论与临床意义。既往研究表明pH下降在介导心脏损伤中发挥重要作用,酸中毒可导致心肌兴奋-收缩耦联障碍,造成心肌收缩力减弱,还可直接损害心肌细胞超微结构造成器质性损害。但pH如何引起心肌损伤,其发生规律及具体分子机制仍不完全清楚。自噬是指溶酶体介导的胞浆物质被降解循环再利用的过程,可更新细胞内衰老、变性或错误折叠的蛋白,清除多余或受损的细胞器,以维持细胞内环境稳态。既往研究证明,自噬在心肌组织中广泛存在,涉及到许多心脏疾病的心肌病理学过程,对于稳定心脏结构和功能有着重要的作用。例如在缺血性心肌病、心力衰竭、心肌炎及心肌肥厚等疾病过程中,心肌细胞的自噬活动会增强。但自噬是否参与调控了pH异常引起心肌损伤及发挥何种作用目前尚不清楚。有研究表明,细胞外pH的改变会影响MCF-10A、MCF-7、Hela等细胞的自噬活性,Marino等的研究发现酸性环境能激活人黑色素瘤细胞的自噬,从而促进细胞存活,这提示我们pH是影响细胞自噬重要因素,且具有细胞特异性。因此我们推测:pH是否通过调控自噬引起心肌细胞功能改变。细胞自噬的诱导和调控是一个非常复杂而精密的过程,许多因素均可以诱导细胞产生自噬,如缺氧、营养压力、氧化应激压力均可激活细胞自噬,同时又有许多信号分子参与自噬的调控,如能量信号主要通过AMPK调控自噬,丝裂原信号主要通过mTORC1影响自噬活性,其他的还有p53、beclin1等信号通路均可参与自噬的调控。一磷酸腺苷激活的蛋白激酶(AMP-activated protein kinase,AMPK)是一种进化保守的、功能强大的丝/苏氨酸蛋白激酶,在心脏中,多种损伤如饥饿、缺血缺氧以及氧化应激等均可导致AMPK激活。活化的AMPK在维持细胞能量代谢和细胞存活中发挥重要作用。其中,自噬就是AMPK依赖的一种重要的适应和存活机制。然而关于AMPK如何调控自噬,目前存在不同的说法。较早的研究认为AMPK活化后能够通过抑制哺乳动物雷帕霉素靶点(Mammalian target of rapamycin,m TOR)活性激活自噬。随着对AMPK功能及作用机制的研究深入,越来越多的研究发现AMPK可以直接作用于ULK1调控自噬,而且大量研究证明AMPK-ULK1是诱导自噬的关键。然而在酸、碱处理条件下AMPK调控心肌细胞自噬的下游信号仍不清楚。本课题旨在研究pH对心肌细胞自噬活性的影响及其相关分子机制,为pH改变所致的心肌损伤的临床防治提供新的思路。研究方法1.通过调控培养基pH模拟细胞外酸、碱化条件,采用CCK8及LDH释放检测酸、碱处理条件下心肌细胞活力的改变。采用蛋白免疫印记(Western blot,WB)和免疫荧光(Immunofluorescence,IF)检测酸、碱处理条件下心肌细胞自噬活性的变化。然后采用荧光分子探针检测酸、碱处理条件下溶酶体酸度的改变,进一步用巴佛洛霉素A1(bafilomycin A1,Baf A1)抑制溶酶体酸化后,检测酸、碱处理条件下自噬活性的变化。2.首先采用WB检测酸、碱处理条件下心肌细胞AMPK活性的变化。使用重组腺病毒转染技术构建了低表达AMPKα2的心肌细胞模型。然后干预AMPK,采用WB和IF检测酸、碱处理条件下心肌细胞自噬水平的变化。此外,采用CCK8、LDH释放及SYTOX green检测酸、碱处理条件下AMPK及其调控的自噬对心肌细胞活力的影响。3.首先采用WB检测酸、碱处理条件下心肌细胞mTORC1活性的变化。干预AMPK,采用WB检测酸、碱处理条件下心肌细胞mTORC1活性的改变。进一步干预mTORC1,检测其对酸、碱处理条件下心肌细胞AMPK活性及自噬水平的影响。采用WB检测了酸、碱处理条件下ULK1活性的改变。干预AMPK,检测酸、碱处理条件下心肌细胞ULK1活性的变化。建立了低表达ULK1的心肌细胞模型。干预ULK1,采用WB和IF检测酸、碱处理条件下心肌细胞自噬水平的变化。最后采用CCK8、LDH释放及SYTOX green染色检测酸、碱处理条件下ULK1及其调控的自噬对心肌细胞活力的影响。结果1.酸化处理3h后,即发现心肌细胞活力明显下降,且随着处理时间的延长,心肌细胞损伤加重;碱化处理后早期,细胞活力增加,持续碱化作用可导致细胞活力下降。酸化处理后,LC3-I向LC3II转换减少;碱化处理诱导LC3-I向LC3II转换增多。酸、碱化处理后,与自噬活性负相关的p62蛋白表达均下降。免疫荧光显示LC3和LAMP1共定位良好;溶酶体检测结果提示细胞外pH改变后6 h,溶酶体的酸度未见明显下降;采用BafA1处理后,酸、碱处理条件下心肌细胞LC3-II蛋白及p62蛋白均显著积累。2.酸化处理抑制心肌细胞AMPK活性,碱化处理诱导心肌细胞AMPK激活。酸化处理6 h后,细胞自噬水平下降,而使用Met激活AMPK后细胞自噬水平升高,使用AMPKα2 sh RNA抑制AMPK活性可逆转Met诱导的自噬活性上调;碱化处理后6h自噬水平升高,使用AMPKα2 sh RNA抑制AMPK活性可显著阻断碱化处理诱导的自噬水平上调。酸化处理6 h后,心肌细胞活力下降,死细胞数目增加,而使用Met激活AMPK和自噬活性后,酸化处理诱导的细胞损伤减轻,死细胞数目减少,使用AMPKα2 sh RNA抑制AMPK活性可阻断这一效应;碱化处理6 h后,细胞活力升高,死亡细胞减少,使用AMPKα2 sh RNA抑制AMPK活性后细胞活力下降,细胞死亡数目增多。3.酸化处理下调心肌细胞mTORC1活性,碱化处理诱导mTORC1激活。酸、碱处理条件下,使用Met和AMPKα2 sh RNA激活/抑制AMPK6 h后,mTORC1活性无明显变化。酸、碱处理条件下,使用m EGF/RAPA激活/抑制mTORC16 h后,AMPK活性和自噬水平均无显著变化。酸化处理抑制心肌细胞ULK1活性,碱化处理诱导ULK1激活。酸化处理6 h后,ULK1活性下降,而使用Met激活AMPK后细胞ULK1活性增强,使用AMPKα2 sh RNA抑制AMPK活性可逆转Met诱导的ULK1激活;碱化处理上调ULK1活性,使用AMPKα2 sh RNA干扰后可抑制这一效应。酸化处理6 h后,自噬水平下降,而使用Met激活AMPK和ULK1后细胞自噬水平上调,进一步使用ULK1 sh RNA抑制ULK1活性可逆转Met诱导的自噬活性增高;碱化处理诱导自噬水平升高,使用ULK1 sh RNA敲降ULK1表达可显著阻断这一过程。酸化处理6h后,细胞活力下降,死亡细胞增加,而使用Met激活AMPK和自噬活性可减轻酸化诱导的细胞损伤,进一步使用ULK1 sh RNA敲降ULK1表达可阻断这一效应;碱化处理6 h后,细胞活力升高,死亡细胞减少,使用ULK1 sh RNA敲降ULK1表达后细胞活力下降,细胞死亡数目增多。结论1.酸化处理导致细胞损活力下降,碱化处理后早期细胞活力升高,持续作用细胞活力下降。酸化处理后,心肌细胞自噬活性明显下降;碱化处理诱导自噬激活,酸、碱化处理后均不伴有自噬通量的损伤。2.pH可调控心肌细胞AMPK的活性,AMPK介导了pH对原代心肌细胞自噬活性及细胞活力的调控。3.pH可调控心肌细胞中mTORC1的活性,酸、碱处理条件下,AMPK和mTORC1的相互作用不明显,mTORC1信号通路在pH介导的心肌细胞自噬调控中不发挥主要作用。酸、碱处理条件下,ULK1可能是AMPK的重要下游靶标之一,AMPK可能通过调控ULK1信号通路介导心肌细胞自噬活性和细胞活力的改变。4.酸、碱处理条件下,早期发生的细胞自噬均有保护心肌的作用,抑制自噬活化会加重心肌细胞损伤。5.在严重烧伤抗休克治疗中,如何使血液酸碱度控制在既不影响血红蛋白释放氧,又不影响细胞生命活动的范围内,对减轻组织细胞缺血缺氧损害,提高抗休克治疗效果具有非常重要的临床指导意义。6.本课题在一定程度上阐明了pH对心肌细胞自噬的影响及其相关机制,为pH改变所致的心肌损伤的临床防治提供新的思路。
[Abstract]:BACKGROUND Acid-base balance is the premise of maintaining the stability of intracellular environment and ensuring the normal metabolism and function of cells. Influenced by many factors, the body pH will fluctuate. pH changes can cause a series of intracellular signal changes and participate in the regulation of various pathophysiological processes, such as severe burns, severe infection, chronic renal failure and so on. After the occurrence of metabolic diseases, cells produce more acid than they expel it, which leads to acidosis, cell damage and organ dysfunction. Cytoplasmic alkalization is closely related to cell proliferation, which is one of the important characteristics of malignant tumors. This suggests that pH plays an important role in regulating cell function. Cardiac injury occurs early after injury. Cardiac injury can not only cause cardiac insufficiency, but also aggravate ischemia-hypoxia damage in other tissues and organs of the whole body. Therefore, the study of myocardial damage after burns has extremely important theoretical and clinical significance. Toxicity can lead to disturbance of excitation-contraction coupling, weaken the contractility of myocardium, and directly damage the ultrastructure of myocardial cells, resulting in organic damage. Autophagy has been proven to be widespread in myocardial tissues and involved in myocardial pathological processes in many heart diseases. It plays an important role in stabilizing cardiac structure and function, such as ischemia. It is not clear whether autophagy is involved in the regulation of myocardial injury caused by abnormal pH and what role it plays. Studies have shown that changes in extracellular pH can affect the autophagy of MCF-10A, MCF-7, Hela and Marino. It was found that acidic environment can activate autophagy of human melanoma cells and promote cell survival. This suggests that pH is an important factor affecting cell autophagy and has cell specificity. Many factors can induce autophagy, such as hypoxia, nutritional stress and oxidative stress. At the same time, many signal molecules participate in the regulation of autophagy. For example, energy signal mainly regulates autophagy through AMPK, mitogen signal mainly affects autophagy activity through mTORC1, and other factors include p53, becl. AMP-activated protein kinase (AMPK) is an evolutionarily conserved and powerful serine/threonine protein kinase. In the heart, a variety of damage such as starvation, ischemia and hypoxia, and oxidative stress can lead to the activation of AMPK. Autophagy is an important adaptation and survival mechanism of AMPK dependence. However, there are different opinions about how AMPK regulates autophagy. Earlier studies suggested that AMPK activation can inhibit mammalian target of rapamycin (m). TOR) Activated autophagy. With the further study of the function and mechanism of AMPK, more and more studies have found that AMPK can directly affect ULK1 to regulate autophagy, and a large number of studies have proved that AMPK-ULK1 is the key to induce autophagy. However, the downstream signal of AMPK regulating cardiomyocyte autophagy under acid and alkali treatment is still unclear. To study the effects of pH on autophagy of cardiomyocytes and its related molecular mechanisms, and to provide new ideas for the clinical prevention and treatment of myocardial injury induced by pH changes. Methods 1. The extracellular acidification and alkalization conditions were simulated by adjusting the pH of the medium, and the changes of cardiomyocyte viability were detected by CCK8 and LDH release. Western blot (WB) and immunofluorescence (IF) were used to detect the changes of autophagy activity of cardiac myocytes under acid and alkali treatment. Then the changes of lysosomal acidity were detected by fluorescent molecular probe under acid and alkali treatment. After inhibition of lysosomal acidification by bafilomycin A1 (Baf A1), the acid was detected. The changes of autophagy activity under alkali treatment were studied. 2. The changes of AMPK activity in cardiomyocytes under acid and alkali treatment were detected by WB. A myocardial cell model with low expression of AMPK alpha2 was constructed by recombinant adenovirus transfection technique. CCK8, LDH release and SYTOX green were used to detect the effect of AMPK and its regulated autophagy on myocardial cell viability under alkali and acid treatments. WB was used to detect the changes of ULK1 activity. Intervention of AMPK was performed to detect the changes of ULK1 activity in cardiomyocytes under acid and alkali treatment. A myocardial cell model with low expression of ULK1 was established. Intervention of ULK1 with WB and IF was used to detect the changes of ULK1 activity in cardiomyocytes under acid and alkali treatment. The changes of autophagy level of cardiomyocytes were detected by CCK8, LDH release and SYTOX green staining. The effect of ULK1 and its autophagy on the viability of cardiomyocytes was detected under alkaline treatment. Results 1. After 3 hours of acidification treatment, the viability of cardiomyocytes decreased significantly, and the damage of cardiomyocytes was aggravated with the prolongation of treatment time. After acidification, the conversion of LC3-I to LC3II was decreased; after alkalization, the conversion of LC3-I to LC3II was increased; after acidification, the expression of p62 protein negatively correlated with autophagic activity was decreased. Immunofluorescence showed that LC3 and LAMP1 were well co-located; lysosome assay showed that the expression of Lysozyme was positive. The results showed that the acidity of lysosome did not decrease significantly 6 hours after the change of extracellular pH, and the accumulation of LC3-II protein and p62 protein in cardiac myocytes under acid and alkaline treatment was significant after BafA1 treatment. 2. Acidification inhibited the activity of AMPK in cardiac myocytes, alkaline treatment induced the activation of AMPK in cardiac myocytes. The autophagy level of AMPK activated by Met increased, and the inhibition of AMPK activity by AMPK alpha 2sh RNA reversed the up-regulation of autophagy induced by Met; the increase of autophagy level 6 h after alkalinization treatment, and the inhibition of AMPK activity by AMPK alpha 2sh RNA significantly blocked the up-regulation of autophagy induced by alkalinization treatment. The number of AMPK cells increased, but the number of dead cells decreased and the damage of AMPK cells induced by acidification was alleviated after activation of AMPK and autophagy by Met. Inhibition of AMPK activity by AMPK alpha 2sh RNA blocked this effect. After 6 hours of alkalinization treatment, the cell viability increased and the dead cells decreased. After inhibition of AMPK activity by AMPK alpha 2sh RNA, the cell viability decreased. Acidification decreased the activity of mTORC1 and alkalinization induced the activation of mTORC1. Under acid and alkaline treatment, the activity of mTORC1 did not change significantly after activation/inhibition of AMPK by Met and AMPK alpha2 sh RNA for 6 h. Under acid and alkaline treatment, the activity of AMPK and the level of autophagy did not change significantly after activation/inhibition of mTORC16 h by m EGF/RAPA. Acidification treatment inhibited ULK1 activity in cardiomyocytes and alkalinization treatment induced ULK1 activation. After 6 hours of acidification treatment, ULK1 activity decreased, while after activation of AMPK by Met, ULK1 activity increased. Inhibition of AMPK activity by AMPK alpha 2 sh RNA reversed Met-induced ULK1 activation; alkalinization treatment increased ULK1 activity, and after AMPK alpha 2 sh RNA interference, ULK1 activity was inhibited. After 6 h of acidification, the autophagy level decreased, while the autophagy level increased after the activation of AMPK and ULK1 by Met. Further inhibition of ULK1 activity by ULK1 sh RNA reversed the increase of autophagy induced by Met, and the increase of autophagy level induced by alkalinization was significantly blocked by knockdown of ULK1 expression by ULK1 sh RNA. After 6 hours of treatment, the cell viability decreased and the dead cells increased, while the activation of AMPK and autophagy by Met alleviated acidification-induced cell injury, which was blocked by ULK1 sh RNA knockdown. After 6 hours of alkalinization treatment, the cell viability increased and the dead cells decreased. After knockdown of ULK1 expression by ULK1 sh RNA, the cell viability decreased. The number of cell death increased. Conclusion 1. Acidification decreased the activity of cell damage, increased the activity of cell in the early stage of alkalization, and decreased the activity of sustained acting cells. AMPK and AMPK mediated the regulation of pH on autophagy and viability of primary cardiomyocytes. 3. pH regulated the activity of mTORC1 in cardiomyocytes. Under acid and alkali treatment, the interaction between AMPK and mTORC1 was not obvious. The mTORC1 signaling pathway did not play a major role in pH-mediated autophagy of cardiomyocytes. ULK1 may be one of the important downstream targets of AMPK. AMPK may mediate the changes of autophagy and cell viability by regulating ULK1 signaling pathway. 4. Under acid and alkali treatment, early autophagy can protect myocardium, and inhibition of autophagy may aggravate myocardial injury. 5. In severe burns, AMPK can resist shock. In the treatment, how to control the blood acidity and alkalinity in the range of not affecting hemoglobin to release oxygen, but also not affecting cell life activity, has very important clinical significance to reduce the damage of tissue and cell ischemia and hypoxia, improve the therapeutic effect of anti-shock. 6. To some extent, this topic clarifies the effect of pH on myocardial autophagy. And its related mechanisms will provide new ideas for clinical prevention and treatment of myocardial injury induced by pH changes.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54
本文编号：2206918
[Abstract]:BACKGROUND Acid-base balance is the premise of maintaining the stability of intracellular environment and ensuring the normal metabolism and function of cells. Influenced by many factors, the body pH will fluctuate. pH changes can cause a series of intracellular signal changes and participate in the regulation of various pathophysiological processes, such as severe burns, severe infection, chronic renal failure and so on. After the occurrence of metabolic diseases, cells produce more acid than they expel it, which leads to acidosis, cell damage and organ dysfunction. Cytoplasmic alkalization is closely related to cell proliferation, which is one of the important characteristics of malignant tumors. This suggests that pH plays an important role in regulating cell function. Cardiac injury occurs early after injury. Cardiac injury can not only cause cardiac insufficiency, but also aggravate ischemia-hypoxia damage in other tissues and organs of the whole body. Therefore, the study of myocardial damage after burns has extremely important theoretical and clinical significance. Toxicity can lead to disturbance of excitation-contraction coupling, weaken the contractility of myocardium, and directly damage the ultrastructure of myocardial cells, resulting in organic damage. Autophagy has been proven to be widespread in myocardial tissues and involved in myocardial pathological processes in many heart diseases. It plays an important role in stabilizing cardiac structure and function, such as ischemia. It is not clear whether autophagy is involved in the regulation of myocardial injury caused by abnormal pH and what role it plays. Studies have shown that changes in extracellular pH can affect the autophagy of MCF-10A, MCF-7, Hela and Marino. It was found that acidic environment can activate autophagy of human melanoma cells and promote cell survival. This suggests that pH is an important factor affecting cell autophagy and has cell specificity. Many factors can induce autophagy, such as hypoxia, nutritional stress and oxidative stress. At the same time, many signal molecules participate in the regulation of autophagy. For example, energy signal mainly regulates autophagy through AMPK, mitogen signal mainly affects autophagy activity through mTORC1, and other factors include p53, becl. AMP-activated protein kinase (AMPK) is an evolutionarily conserved and powerful serine/threonine protein kinase. In the heart, a variety of damage such as starvation, ischemia and hypoxia, and oxidative stress can lead to the activation of AMPK. Autophagy is an important adaptation and survival mechanism of AMPK dependence. However, there are different opinions about how AMPK regulates autophagy. Earlier studies suggested that AMPK activation can inhibit mammalian target of rapamycin (m). TOR) Activated autophagy. With the further study of the function and mechanism of AMPK, more and more studies have found that AMPK can directly affect ULK1 to regulate autophagy, and a large number of studies have proved that AMPK-ULK1 is the key to induce autophagy. However, the downstream signal of AMPK regulating cardiomyocyte autophagy under acid and alkali treatment is still unclear. To study the effects of pH on autophagy of cardiomyocytes and its related molecular mechanisms, and to provide new ideas for the clinical prevention and treatment of myocardial injury induced by pH changes. Methods 1. The extracellular acidification and alkalization conditions were simulated by adjusting the pH of the medium, and the changes of cardiomyocyte viability were detected by CCK8 and LDH release. Western blot (WB) and immunofluorescence (IF) were used to detect the changes of autophagy activity of cardiac myocytes under acid and alkali treatment. Then the changes of lysosomal acidity were detected by fluorescent molecular probe under acid and alkali treatment. After inhibition of lysosomal acidification by bafilomycin A1 (Baf A1), the acid was detected. The changes of autophagy activity under alkali treatment were studied. 2. The changes of AMPK activity in cardiomyocytes under acid and alkali treatment were detected by WB. A myocardial cell model with low expression of AMPK alpha2 was constructed by recombinant adenovirus transfection technique. CCK8, LDH release and SYTOX green were used to detect the effect of AMPK and its regulated autophagy on myocardial cell viability under alkali and acid treatments. WB was used to detect the changes of ULK1 activity. Intervention of AMPK was performed to detect the changes of ULK1 activity in cardiomyocytes under acid and alkali treatment. A myocardial cell model with low expression of ULK1 was established. Intervention of ULK1 with WB and IF was used to detect the changes of ULK1 activity in cardiomyocytes under acid and alkali treatment. The changes of autophagy level of cardiomyocytes were detected by CCK8, LDH release and SYTOX green staining. The effect of ULK1 and its autophagy on the viability of cardiomyocytes was detected under alkaline treatment. Results 1. After 3 hours of acidification treatment, the viability of cardiomyocytes decreased significantly, and the damage of cardiomyocytes was aggravated with the prolongation of treatment time. After acidification, the conversion of LC3-I to LC3II was decreased; after alkalization, the conversion of LC3-I to LC3II was increased; after acidification, the expression of p62 protein negatively correlated with autophagic activity was decreased. Immunofluorescence showed that LC3 and LAMP1 were well co-located; lysosome assay showed that the expression of Lysozyme was positive. The results showed that the acidity of lysosome did not decrease significantly 6 hours after the change of extracellular pH, and the accumulation of LC3-II protein and p62 protein in cardiac myocytes under acid and alkaline treatment was significant after BafA1 treatment. 2. Acidification inhibited the activity of AMPK in cardiac myocytes, alkaline treatment induced the activation of AMPK in cardiac myocytes. The autophagy level of AMPK activated by Met increased, and the inhibition of AMPK activity by AMPK alpha 2sh RNA reversed the up-regulation of autophagy induced by Met; the increase of autophagy level 6 h after alkalinization treatment, and the inhibition of AMPK activity by AMPK alpha 2sh RNA significantly blocked the up-regulation of autophagy induced by alkalinization treatment. The number of AMPK cells increased, but the number of dead cells decreased and the damage of AMPK cells induced by acidification was alleviated after activation of AMPK and autophagy by Met. Inhibition of AMPK activity by AMPK alpha 2sh RNA blocked this effect. After 6 hours of alkalinization treatment, the cell viability increased and the dead cells decreased. After inhibition of AMPK activity by AMPK alpha 2sh RNA, the cell viability decreased. Acidification decreased the activity of mTORC1 and alkalinization induced the activation of mTORC1. Under acid and alkaline treatment, the activity of mTORC1 did not change significantly after activation/inhibition of AMPK by Met and AMPK alpha2 sh RNA for 6 h. Under acid and alkaline treatment, the activity of AMPK and the level of autophagy did not change significantly after activation/inhibition of mTORC16 h by m EGF/RAPA. Acidification treatment inhibited ULK1 activity in cardiomyocytes and alkalinization treatment induced ULK1 activation. After 6 hours of acidification treatment, ULK1 activity decreased, while after activation of AMPK by Met, ULK1 activity increased. Inhibition of AMPK activity by AMPK alpha 2 sh RNA reversed Met-induced ULK1 activation; alkalinization treatment increased ULK1 activity, and after AMPK alpha 2 sh RNA interference, ULK1 activity was inhibited. After 6 h of acidification, the autophagy level decreased, while the autophagy level increased after the activation of AMPK and ULK1 by Met. Further inhibition of ULK1 activity by ULK1 sh RNA reversed the increase of autophagy induced by Met, and the increase of autophagy level induced by alkalinization was significantly blocked by knockdown of ULK1 expression by ULK1 sh RNA. After 6 hours of treatment, the cell viability decreased and the dead cells increased, while the activation of AMPK and autophagy by Met alleviated acidification-induced cell injury, which was blocked by ULK1 sh RNA knockdown. After 6 hours of alkalinization treatment, the cell viability increased and the dead cells decreased. After knockdown of ULK1 expression by ULK1 sh RNA, the cell viability decreased. The number of cell death increased. Conclusion 1. Acidification decreased the activity of cell damage, increased the activity of cell in the early stage of alkalization, and decreased the activity of sustained acting cells. AMPK and AMPK mediated the regulation of pH on autophagy and viability of primary cardiomyocytes. 3. pH regulated the activity of mTORC1 in cardiomyocytes. Under acid and alkali treatment, the interaction between AMPK and mTORC1 was not obvious. The mTORC1 signaling pathway did not play a major role in pH-mediated autophagy of cardiomyocytes. ULK1 may be one of the important downstream targets of AMPK. AMPK may mediate the changes of autophagy and cell viability by regulating ULK1 signaling pathway. 4. Under acid and alkali treatment, early autophagy can protect myocardium, and inhibition of autophagy may aggravate myocardial injury. 5. In severe burns, AMPK can resist shock. In the treatment, how to control the blood acidity and alkalinity in the range of not affecting hemoglobin to release oxygen, but also not affecting cell life activity, has very important clinical significance to reduce the damage of tissue and cell ischemia and hypoxia, improve the therapeutic effect of anti-shock. 6. To some extent, this topic clarifies the effect of pH on myocardial autophagy. And its related mechanisms will provide new ideas for clinical prevention and treatment of myocardial injury induced by pH changes.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54
【参考文献】
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1 姚伟,钱桂生,杨晓静;NHE-1与大鼠肺动脉平滑肌细胞增殖和凋亡(英文)[J];Chinese Medical Journal;2002年01期
，本文编号：2206918
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