曲美他嗪促进AMPK依赖的自噬流减轻心肌细胞缺氧复氧损伤
发布时间：2018-08-19 08:50
【摘要】：研究目的观察曲美他嗪(Trimetazidine,TMZ)预处理能否有效减轻心肌细胞缺氧/复氧(Hypoxia/reoxygenation,H/R)损伤,并探索心肌细胞缺氧/复氧期间,曲美他嗪预处理是否经过单磷酸腺苷活化蛋白激酶(AMP-activated protein kinase,AMPK)通路调控自噬。研究方法第一部分实验:观察H/R过程中曲美他嗪的保护作用,并确定最有效浓度。构建新生大鼠心肌细胞H/R模型,研究对象随机分为对照组、H/R组、H/R+TMZ(10μM)组、H/R+TMZ(50μM)组、H/R+TMZ(100μM)组。采用MTS检测细胞活性,收集培养液并通过试剂盒检测LDH水平以观察细胞损伤程度,比较各浓度疗效,确定最有效浓度。第二部分实验:探明曲美他嗪对H/R过程中自噬的影响及干预自噬表达对曲美他嗪疗效影响本部分实验主要采用氯喹(chloroquine,Cq)抑制自噬流,实验分组为对照组、对照+TMZ 组、对照+Cq 组、H/R 组、H/R+TMZ 组、H/R+Cq 组、H/R+TMZ+Cq组。MTS检测细胞活性,试剂盒检测培养液LDH水平,并通过Western blot半定量分析自噬相关蛋白Beclin-1、LC3和p62的表达,转染病毒mRFP-GFP-LC3并通过共聚焦显微镜以测定细胞自噬相关蛋白分布情况,透射电镜直接观察各组自噬体数量,TUNEL染色检测心肌细胞凋亡水平。第三部分实验:探明H/R过程中干预AMPK对曲美他嗪所调控的自噬的影响本部分实验主要使用compound C(com C)抑制AMPK信号,实验分组为对照组、对照 + com C 组、H/R 组、H/R+TMZ 组、H/R+ com C 组、H/R+TMZ+ com C组。MTS检测细胞活性,试剂盒检测培养液LDH水平,最后通过Western blot半定量分析自噬相关蛋白LC3和p62及AMPK信号通路蛋白p-AMPK/AMPK和p-mTOR/mTOR的表达。结果1.曲美他嗪预处理减轻心肌细胞H/R损伤与对照组相比,H/R组的细胞活性显著性下降(P0.05);各曲美他嗪预处理组细胞活性均比H/R组高(P0.05),H/R+TMZ(50μM)组细胞活性比H/R+TMZ(10μM)组高(P0.05),但与 H/R+TMZ(100μM)组无统计学差异(P0.05)。因此50μM为曲美他嗪的最有效浓度。2.曲美他嗪预处理在心肌细胞H/R期间促进自噬流H/R处理后各自噬相关蛋白均表达上调(P0.05,C vs.H/R组),然而曲美他嗪预处理后Beclin-1、LC3-Ⅱ/LC3-Ⅰ比值均进一步增强,p62减少(P0.05,H/R vs.H/R + TMZ组);透射电镜结果显示,相比对照组,H/R组细胞浆内出现更多自噬体,(P0.05);而相比H/R组,H/R+TMZ组自噬体数量进一步增加(P0.05)。共聚焦显微镜下,对照组无明显LC3聚集现象;H/R处理后,自噬体数量明显增加(P0.05,C vs.H/R组);与H/R组相比,H/R+TMZ组的自噬溶酶体更多(P0.05)。3.Cq预处理抵消曲美他嗪对心肌细胞的保护作用Cq 预处理后,LC3-Ⅱ/LC3-Ⅰ、p62 的表达均增加(P0.05,H/R + TMZ vs.H/R + TMZ + Cq组);共聚焦显微镜下,Cq预处理显著减少自噬溶酶体(P0.05,H/R + TMZ vs.H/R + TMZ + Cq 组);与 H/R+TMZ 组相比,H/R+TMZ+Cq组细胞活性明显下降(P0.05);4.曲美他嗪预处理减少H/R中细胞凋亡TUNEL染色结果显示,与对照组相比,H/R组凋亡细胞显著性增加(P0.05);曲美他嗪预处理后,凋亡细胞数量明显减少(P0.05,H/Rvs.H/R + TMZ组);然而H/R + TMZ+ Cq与H/R + TMZ组相比,凋亡细胞显著性增加(P0.05)。5.曲美他嗪预处理通过AMPK-mTOR调控H/R中的自噬与H/R组相比,曲美他嗪预处理显著增加p-AMPK/AMPK的比值以及减少p-mTOR/mTOR 的比值(P0.05,H/Rvs.H/R + TMZ 组);与 H/R + TMZ 组相比,compound C 减少 p-AMPK/AMPK 比值,增加 p-mTOR/mTOR 比值(P0.05);此外,compound C减少LC3-Ⅱ/LC3-Ⅰ比值并增加了 p62的表达;H/R +TMZ+comC与H/R + TMZ组相比,其细胞活性减少,LDH水平增加(P0.05),结论曲美他嗪预处理通过AMPK-mTOR信号通路促进自噬流减轻心肌细胞H/R损伤
[Abstract]:Objective To investigate whether trimetazidine (TMZ) preconditioning can effectively alleviate hypoxia/reoxygenation (H/R) injury in cardiomyocytes and whether trimetazidine preconditioning can be regulated by AMP-activated protein kinase (AMPK) pathway during hypoxia/reoxygenation in cardiomyocytes. METHODS The first part of the experiment was to observe the protective effect of trimetazidine during H/R and determine the most effective concentration.The H/R model of neonatal rat cardiomyocytes was established. The level of LDH was measured to observe the degree of cell injury and compare the therapeutic effects of different concentrations to determine the most effective concentration. Part II: Experiments: To explore the effect of trimetazidine on autophagy in H/R and the effect of autophagy on trimetazidine. This part of the experiment mainly used chloroquine (Cq) to inhibit autophagy. The experiment was divided into control group and control group + TMZ. MTS assay was used to detect cell activity, LDH level in culture medium, and the expression of autophagy-related proteins Beclin-1, LC3 and p62 was semi-quantitatively analyzed by Western blot. The distribution of autophagy-related proteins was detected by confocal microscopy. The number of autophages was observed by transmission electron microscopy and the level of myocardial apoptosis was detected by TUNEL staining. Part III: To explore the effect of AMPK intervention on autophagy regulated by trimetazidine during H/R. In this part, compound C (com C) was used to inhibit AMPK signal. The experiment was divided into control group, control + com C group, H/R group, H/R + TMZ group. Group H/R+com C, H/R+TMZ+com C, H/R+TMZ+com C. MTS was used to detect cell activity and LDH level in culture medium. The expressions of autophagy-associated proteins LC3 and p62, and AMPK signaling pathway proteins p-AMPK/AMPK and p-mTOR/mTOR were analyzed semi-quantitatively by Western blot. Results 1. Trimetazidine pretreatment reduced H/R injury of cardiomyocytes compared with control group. The cell viability of H / R + TMZ group was higher than that of H / R group (P 0.05). The cell viability of H / R + TMZ group was higher than that of H / R + TMZ group (P 0.05), but there was no significant difference between H / R + TMZ group (P 0.05). During R period, the expression of autophagy-related proteins was up-regulated after autophagy H/R treatment (P 0.05, C vs. H/R group). However, after trimetazidine pretreatment, the ratio of Beclin-1, LC3-II/LC3-I was further enhanced and p62 was decreased (P 0.05, H/R vs. H/R + TMZ group). Compared with H/R group, the number of autophages in H/R+TMZ group was further increased (P 0.05). There was no obvious LC3 aggregation in control group under confocal microscope; the number of autophages in H/R group was significantly increased (P 0.05, C vs. H/R group); the number of autophagic lysosomes in H/R+TMZ group was more than that in H/R group (P 0.05). 3. Cq pretreatment counteracted the protective effect of trimetazidine on cardiomyocytes. After pretreatment, the expression of LC3-II/LC3-I and p62 increased (P 0.05, H/R+TMZ vs.H/R+TMZ+Cq group); under confocal microscope, Cq pretreatment significantly decreased autophagy lysosome (P 0.05, H/R+TMZ vs.H/R+TMZ+Cq group); compared with H/R+TMZ+Cq group, the activity of H/R+TMZ+Cq group decreased significantly (P 0.05); Apoptotic TUNEL staining showed that the number of apoptotic cells in H/R group was significantly increased (P 0.05); the number of apoptotic cells was significantly decreased after trimetazidine pretreatment (P 0.05, H/Rvs.H/R+TMZ group); however, the number of apoptotic cells in H/R+TMZ+Cq group was significantly increased compared with that in H/R+TMZ group (P 0.05). 5. Trimetazidine pretreatment regulated H/R by AMPK-mTOR. Compared with H/R group, trimetazidine pretreatment significantly increased the p-AMPK/AMPK ratio and decreased the p-mTOR/mTOR ratio (P 0.05, H/Rvs.H/R+TMZ group); compared with H/R+TMZ group, compound C decreased the p-AMPK/AMPK ratio, increased the p-mTOR/mTOR ratio (P 0.05); in addition, compound C decreased the LC3-II/LC3-I ratio and increased the p62 table. Compared with H/R+TMZ+comC group, H/R+TMZ+comC group showed decreased cell activity and increased LDH level (P 0.05). Conclusion Trimetazidine preconditioning can alleviate H/R injury of cardiomyocytes by promoting autophagy through AMPK-mTOR signaling pathway.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
本文编号：2191160
[Abstract]:Objective To investigate whether trimetazidine (TMZ) preconditioning can effectively alleviate hypoxia/reoxygenation (H/R) injury in cardiomyocytes and whether trimetazidine preconditioning can be regulated by AMP-activated protein kinase (AMPK) pathway during hypoxia/reoxygenation in cardiomyocytes. METHODS The first part of the experiment was to observe the protective effect of trimetazidine during H/R and determine the most effective concentration.The H/R model of neonatal rat cardiomyocytes was established. The level of LDH was measured to observe the degree of cell injury and compare the therapeutic effects of different concentrations to determine the most effective concentration. Part II: Experiments: To explore the effect of trimetazidine on autophagy in H/R and the effect of autophagy on trimetazidine. This part of the experiment mainly used chloroquine (Cq) to inhibit autophagy. The experiment was divided into control group and control group + TMZ. MTS assay was used to detect cell activity, LDH level in culture medium, and the expression of autophagy-related proteins Beclin-1, LC3 and p62 was semi-quantitatively analyzed by Western blot. The distribution of autophagy-related proteins was detected by confocal microscopy. The number of autophages was observed by transmission electron microscopy and the level of myocardial apoptosis was detected by TUNEL staining. Part III: To explore the effect of AMPK intervention on autophagy regulated by trimetazidine during H/R. In this part, compound C (com C) was used to inhibit AMPK signal. The experiment was divided into control group, control + com C group, H/R group, H/R + TMZ group. Group H/R+com C, H/R+TMZ+com C, H/R+TMZ+com C. MTS was used to detect cell activity and LDH level in culture medium. The expressions of autophagy-associated proteins LC3 and p62, and AMPK signaling pathway proteins p-AMPK/AMPK and p-mTOR/mTOR were analyzed semi-quantitatively by Western blot. Results 1. Trimetazidine pretreatment reduced H/R injury of cardiomyocytes compared with control group. The cell viability of H / R + TMZ group was higher than that of H / R group (P 0.05). The cell viability of H / R + TMZ group was higher than that of H / R + TMZ group (P 0.05), but there was no significant difference between H / R + TMZ group (P 0.05). During R period, the expression of autophagy-related proteins was up-regulated after autophagy H/R treatment (P 0.05, C vs. H/R group). However, after trimetazidine pretreatment, the ratio of Beclin-1, LC3-II/LC3-I was further enhanced and p62 was decreased (P 0.05, H/R vs. H/R + TMZ group). Compared with H/R group, the number of autophages in H/R+TMZ group was further increased (P 0.05). There was no obvious LC3 aggregation in control group under confocal microscope; the number of autophages in H/R group was significantly increased (P 0.05, C vs. H/R group); the number of autophagic lysosomes in H/R+TMZ group was more than that in H/R group (P 0.05). 3. Cq pretreatment counteracted the protective effect of trimetazidine on cardiomyocytes. After pretreatment, the expression of LC3-II/LC3-I and p62 increased (P 0.05, H/R+TMZ vs.H/R+TMZ+Cq group); under confocal microscope, Cq pretreatment significantly decreased autophagy lysosome (P 0.05, H/R+TMZ vs.H/R+TMZ+Cq group); compared with H/R+TMZ+Cq group, the activity of H/R+TMZ+Cq group decreased significantly (P 0.05); Apoptotic TUNEL staining showed that the number of apoptotic cells in H/R group was significantly increased (P 0.05); the number of apoptotic cells was significantly decreased after trimetazidine pretreatment (P 0.05, H/Rvs.H/R+TMZ group); however, the number of apoptotic cells in H/R+TMZ+Cq group was significantly increased compared with that in H/R+TMZ group (P 0.05). 5. Trimetazidine pretreatment regulated H/R by AMPK-mTOR. Compared with H/R group, trimetazidine pretreatment significantly increased the p-AMPK/AMPK ratio and decreased the p-mTOR/mTOR ratio (P 0.05, H/Rvs.H/R+TMZ group); compared with H/R+TMZ group, compound C decreased the p-AMPK/AMPK ratio, increased the p-mTOR/mTOR ratio (P 0.05); in addition, compound C decreased the LC3-II/LC3-I ratio and increased the p62 table. Compared with H/R+TMZ+comC group, H/R+TMZ+comC group showed decreased cell activity and increased LDH level (P 0.05). Conclusion Trimetazidine preconditioning can alleviate H/R injury of cardiomyocytes by promoting autophagy through AMPK-mTOR signaling pathway.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
【参考文献】
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1 Eirini Pantazi;Mohamed Amine Zaouali;Mohamed Bejaoui;Emma Folch-Puy;Hassen Ben Abdennebi;Ana Teresa Varela;Anabela Pinto Rolo;Carlos Marques Palmeira;Joan Roselló-Catafau;;Sirtuin 1 in rat orthotopic liver transplantation:An I GL-1 preservation solution approach[J];World Journal of Gastroenterology;2015年06期
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