线粒体自噬在对比剂所致急性肾损伤中的作用及机制研究
发布时间：2018-09-08 09:11
【摘要】：目的:1.建立新型CI-AKI大鼠动物模型。2.观察CI-AKI大鼠肾组织中自噬、线粒体自噬水平的变化,以及氧化应激水平、线粒体损伤和肾小管上皮细胞凋亡情况的改变。3.探讨雷帕霉素预处理增强自噬、线粒体自噬活性对对比剂所致线粒体损伤、氧化应激损伤及肾小管上皮细胞凋亡情况的影响。方法:1.健康雄性SD大鼠54只,体重210-230 g,采用计算机随机法将其随机分成9组(n=6):正常组(A组):予以尾静脉注射同等剂量的生理盐水;不同剂量尾静脉注射组(B1-3组):尾静脉分别注射碘海醇3.5,5.25,8.75 g I/kg;不同剂量腹腔注射组(C1-3组):腹腔分别注射碘海醇5.25,8.75,12.25 g I/kg;尾静脉多次注射组(D1-2组):连续两天分别予以尾静脉注射碘海醇5.25,7.0g I/kg。观察对比剂或生理盐水注射24 h后对比剂对大鼠肾功能及肾组织病理的影响,探索新型CI-AKI大鼠动物模型。2.健康雄性SD大鼠24只,体重210-230 g,采用计算机随机法将其随机分成4组(n=6):正常组(Con组):予以腹腔注射同等剂量的生理盐水;CI-AKI模型组(CI-AKI组):予以腹腔注射超大剂量的碘海醇(12.25 g I/kg);低剂量雷帕霉素+对比剂组(Rap1组):注射同等剂量对比剂之前予以连续腹腔注射雷帕霉素(2 mg/kg/d)一周;高剂量雷帕霉素+对比剂组(Rap2组):注射同等剂量对比剂之前予以连续腹腔注射雷帕霉素(5 mg/kg/d)一周;对比剂或生理盐水注射24 h后处死大鼠,观察雷帕霉素对CI-AKI大鼠肾功能及肾组织病理的影响。采用Western-blot检测肾组织中LC3、Beclin-1、Pink-1、胞浆及线粒体中Cyt c的表达水平;免疫荧光双染检测LC3标记的自噬小体和LAMP-2标记的溶酶体或TOMM20标记的线粒体的共表达情况,检测细胞自噬、线粒体自噬活性;JC-1法检测线粒体膜电位(Mitochondrial membrane potential,MMP);TUNEL法检测肾组织中肾小管上皮细胞凋亡程度;ELISA法检测大鼠肾组织中MDA含量,探讨不同剂量雷帕霉素预处理对CI-AKI大鼠的作用及其机制。结果:1.与A组比较,B1-3组,C1-2组,D1-2组血肌酐轻度升高,而C3组血肌酐明显升高(A组vs C3组,P0.05)。与A组比较,B1-3组,C1-2组,D1-2组仅见轻度肾小管变性,C3组大鼠肾组织重度损伤,可见严重的肾小管上皮细胞变性、坏死脱落,以及大量空泡变性。2.肾功能及组织病理:与Con组比较,CI-AKI组血肌酐明显升高,肾组织重度损伤(Con组vs CI-AKI组,P0.05),而雷帕霉素预处理显著且剂量依赖性地减轻对比剂所致肾功能及组织病理损伤(Rap1/2组vs CI-AKI组,P0.05;Rap1组vs Rap2组,P0.05)。3.自噬水平:与Con组比较,CI-AKI组大鼠肾组织中自噬相关蛋白LC3和Beclin-1的表达水平增加(Con组vs CI-AKI组,P0.05),LC3标记的自噬小体与LAMP-2标记的溶酶体共定位信号表达增加,对比剂诱导自噬激活。而雷帕霉素预处理显著且剂量依赖性地增强了自噬相关蛋白的表达水平(Rap1/2组vs CI-AKI组,P0.05;Rap1组vs Rap2组,P0.05),及自噬小体与溶酶体的共定位表达信号,增强自噬活性。4.线粒体自噬水平:与Con组比较,CI-AKI组大鼠肾组织中线粒体自噬相关蛋白pink-1的表达水平增加(Con组vs CI-AKI组,P0.05),LC3标记的自噬小体与TOMM-20标记的线粒体共定位信号表达增加,对比剂诱导线粒体自噬激活。而雷帕霉素预处理显著且剂量依赖性地增强了自噬小体与线粒体的共定位表达信号,增强线粒体自噬活性。5.氧化应激水平:与Con组比较,CI-AKI组大鼠肾组织中MDA含量增多(Con组vs CI-AKI组,P0.05),而雷帕霉素预处理显著且剂量依赖性地降低了肾组织中MDA含量(Rap1/2组vs CI-AKI组,P0.05;Rap1组vs Rap2组,P0.05)。6.线粒体损伤情况:与Con组比较,CI-AKI组大鼠肾组织中胞浆/线粒体Cyt c水平增加(Con组vs CI-AKI组,P0.05),MMP降低(Con组vs CI-AKI组,P0.05),对比剂导致肾组织中线粒体损伤。而雷帕霉素预处理显著且剂量依赖性地抑制了线粒体中Cyt c的释放及MMP的降低,延缓了对比剂所致线粒体损伤(Rap1/2组vs CI-AKI组,P0.05;Rap1组vs Rap2组,P0.05)。7.肾小管上皮细胞凋亡情况:与Con组比较,CI-AKI组肾组织中肾小管上皮细胞凋亡率增加(Con组vs CI-AKI组,P0.05),而雷帕霉素预处理明显且剂量依赖性地降低了对比剂所致的肾小管上皮细胞凋亡率(Rap1/2组vs CI-AKI组,P0.05;Rap1组vs Rap2组,P0.05)。结论:1.超大剂量腹腔注射对比剂可成功建立新型CI-AKI大鼠动物模型。2.对比剂可导致肾组织线粒体损伤并激活自噬及线粒体自噬活性。但激活的自噬及线粒体自噬不足以清除受损的线粒体,过度累积的受损线粒体将导致大量ROS和Cyt c释放,导致肾组织氧化应激损伤及肾小管上皮细胞凋亡。3.雷帕霉素预处理可增强CI-AKI大鼠肾组织中自噬及线粒体自噬水平。增强了的自噬及线粒体自噬水平可以减轻对比剂所致线粒体损伤及其所致的氧化应激损伤和肾小管上皮细胞凋亡,从而延缓CI-AKI大鼠肾脏损伤程度。
[Abstract]:AIM: 1. To establish a new animal model of CI-AKI. 2. To observe the changes of autophagy, mitochondrial autophagy, oxidative stress, mitochondrial damage and apoptosis of renal tubular epithelial cells in rats with CI-AKI. 3. To investigate the effects of rapamycin pretreatment on autophagy and mitochondrial autophagy induced by contrast agents. Methods: Fifty-four healthy male SD rats weighing 210-230 g were randomly divided into 9 groups (n=6): normal group (group A): the same dose of saline was injected into caudal vein; different doses of saline were injected into caudal vein (group B1-3): iodine sea was injected into caudal vein respectively. 3.5,5.25,8.75 g I/kg of iohexol; different doses of intraperitoneal injection group (C1-3 group): intraperitoneal injection of iohexol 5.25,8.75,12.25 g I/kg; tail vein multiple injection group (D1-2 group): two consecutive days were given tail vein injection of iohexol 5.25,7.0 g I/kg. To explore a new animal model of CI-AKI. 2. 24 healthy male SD rats weighing 210-230 g were randomly divided into four groups (n=6): normal group (Con group): intraperitoneal injection of the same dose of normal saline; CI-AKI model group (CI-AKI group): intraperitoneal injection of excessive doses of iohexol (12.25 g I/kg); low doses of iohexol (12.25 g I/kg); Rapamycin + contrast group (Rap1 group): rapamycin (2 mg / kg / d) was injected intraperitoneally for one week before the same dose of contrast medium was injected; rapamycin + contrast medium group (Rap2 group): rapamycin (5 mg / kg / d) was injected intraperitoneally for one week before the same dose of contrast medium was injected; rapamycin (5 mg / kg / d) was injected intraperitoneally for 24 hours after the same dose of contrast medium or saline. To observe the effect of rapamycin on renal function and histopathology in CI-AKI rats, the expression of LC3, Beclin-1, Pink-1, Cyt C in cytoplasm and mitochondria was detected by Western blot, and the co-expression of autophagosomes labeled with LC3 and lysosomes labeled with LAMP-2 or mitochondria labeled with TOMM20 was detected by immunofluorescence double staining. Cell autophagy, mitochondrial autophagy activity; JC-1 method to detect mitochondrial membrane potential (MMP); TUNEL method to detect renal tubular epithelial cell apoptosis; ELISA method to detect the content of MDA in rat kidney tissue, to explore the effect of different doses of rapamycin preconditioning on CI-AKI rats and its mechanism. Compared with group A, group B1-3, group C1-2 and group D1-2 showed mild elevation of serum creatinine, while group C3 showed marked elevation of serum creatinine (vs C3, P 0.05). Compared with group A, group B1-3, group C1-2, group D1-2 showed only mild tubular degeneration, group C3 showed severe renal tissue damage, severe tubular epithelial cell degeneration, necrosis and loss, and a large number of vacuole degeneration.2. Histopathology: Compared with Con group, serum creatinine was significantly elevated and renal tissue was severely damaged in CI-AKI group (vs CI-AKI group, P 0.05), while rapamycin preconditioning significantly and dose-dependent reduced renal function and histopathological injury induced by contrast medium (vs CI-AKI group, P 0.05, Rap 1 vs Rap 2 group, P 0.05).3. Autophagy level: Compared with Con group, CI-AKI preconditioning significantly and dose-dependent decreased renal function and histopathological injury induced by contrast medium (vs CI-AKI group, P 0.05, Rap 1/ The levels of autophagy-associated protein LC3 and Beclin-1 in renal tissues of rats in group C increased (Con vs CI-AKI, P 0.05), the co-localization signal expression of LC3-labeled autophagosomes and LAMP-2-labeled lysosomes increased, and autophagy was activated by contrast medium. Rapamycin pretreatment significantly and dose-dependent enhanced the expression of autophagy-related protein in water. Mitochondrial autophagy level: Compared with the control group, the expression level of mitochondrial autophagy-related protein pink-1 in renal tissue of rats in CI-AKI group increased (vs CI-AKI group, P Compared with the control group, the pretreatment with rapamycin significantly and dose-dependent enhanced the co-localization signal of autophagy and mitochondria, and enhanced the autophagy activity of mitochondria. 5. Oxidative stress level: Compared with the control group, MD in renal tissue of rats in CI-AKI group increased significantly and dose-dependent. The content of MDA increased (vs CI-AKI group, P 0.05), while rapamycin preconditioning significantly and dose-dependent decreased the content of MDA in renal tissue (Rap1/2 vs CI-AKI group, P 0.05; Rap1 vs Rap2 group, P 0.05). 6. Mitochondrial injury: Compared with Con group, the level of cytoplasmic/mitochondrial Cyt C in renal tissue of rats in CI-AKI group increased (vs CI-AKI group, P 0.0). 5) decreased MMP (vs CI-AKI group, P 0.05), and mitochondrial damage was induced by contrast medium. Rapamycin preconditioning significantly and dose-dependent inhibited the release of Cyt C and the decrease of MMP in mitochondria, and delayed the mitochondrial damage induced by contrast medium (vs CI-AKI group, P 0.05, Rap1 vs Rap2 group, P 0.05). Cell apoptosis: Compared with Con group, the apoptosis rate of renal tubular epithelial cells in CI-AKI group increased (vs CI-AKI group, P 0.05), while rapamycin pretreatment significantly and dose-dependent decreased the apoptosis rate of renal tubular epithelial cells induced by contrast medium (Rap 1/2 vs CI-AKI group, P 0.05; Rap 1 vs Rap 2 group, P 0.05). Intraperitoneal injection of contrast agent can successfully establish a new animal model of CI-AKI. 2. Contrast agent can induce mitochondrial damage and activate autophagy and mitochondrial autophagy. However, activated autophagy and mitochondrial autophagy are insufficient to remove damaged mitochondria. Overaccumulating damaged mitochondria will result in the release of large amounts of ROS and Cytc, leading to the release of renal tissue. Oxidative stress injury and apoptosis of renal tubular epithelial cells. 3. Rapamycin pretreatment can enhance the levels of autophagy and mitochondrial autophagy in renal tissue of CI-AKI rats. Enhanced levels of autophagy and mitochondrial autophagy can alleviate contrast-induced mitochondrial injury, oxidative stress injury and tubular epithelial cell apoptosis, thereby delaying CI-AKI rats. The degree of renal injury in AKI rats.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692
[Abstract]:AIM: 1. To establish a new animal model of CI-AKI. 2. To observe the changes of autophagy, mitochondrial autophagy, oxidative stress, mitochondrial damage and apoptosis of renal tubular epithelial cells in rats with CI-AKI. 3. To investigate the effects of rapamycin pretreatment on autophagy and mitochondrial autophagy induced by contrast agents. Methods: Fifty-four healthy male SD rats weighing 210-230 g were randomly divided into 9 groups (n=6): normal group (group A): the same dose of saline was injected into caudal vein; different doses of saline were injected into caudal vein (group B1-3): iodine sea was injected into caudal vein respectively. 3.5,5.25,8.75 g I/kg of iohexol; different doses of intraperitoneal injection group (C1-3 group): intraperitoneal injection of iohexol 5.25,8.75,12.25 g I/kg; tail vein multiple injection group (D1-2 group): two consecutive days were given tail vein injection of iohexol 5.25,7.0 g I/kg. To explore a new animal model of CI-AKI. 2. 24 healthy male SD rats weighing 210-230 g were randomly divided into four groups (n=6): normal group (Con group): intraperitoneal injection of the same dose of normal saline; CI-AKI model group (CI-AKI group): intraperitoneal injection of excessive doses of iohexol (12.25 g I/kg); low doses of iohexol (12.25 g I/kg); Rapamycin + contrast group (Rap1 group): rapamycin (2 mg / kg / d) was injected intraperitoneally for one week before the same dose of contrast medium was injected; rapamycin + contrast medium group (Rap2 group): rapamycin (5 mg / kg / d) was injected intraperitoneally for one week before the same dose of contrast medium was injected; rapamycin (5 mg / kg / d) was injected intraperitoneally for 24 hours after the same dose of contrast medium or saline. To observe the effect of rapamycin on renal function and histopathology in CI-AKI rats, the expression of LC3, Beclin-1, Pink-1, Cyt C in cytoplasm and mitochondria was detected by Western blot, and the co-expression of autophagosomes labeled with LC3 and lysosomes labeled with LAMP-2 or mitochondria labeled with TOMM20 was detected by immunofluorescence double staining. Cell autophagy, mitochondrial autophagy activity; JC-1 method to detect mitochondrial membrane potential (MMP); TUNEL method to detect renal tubular epithelial cell apoptosis; ELISA method to detect the content of MDA in rat kidney tissue, to explore the effect of different doses of rapamycin preconditioning on CI-AKI rats and its mechanism. Compared with group A, group B1-3, group C1-2 and group D1-2 showed mild elevation of serum creatinine, while group C3 showed marked elevation of serum creatinine (vs C3, P 0.05). Compared with group A, group B1-3, group C1-2, group D1-2 showed only mild tubular degeneration, group C3 showed severe renal tissue damage, severe tubular epithelial cell degeneration, necrosis and loss, and a large number of vacuole degeneration.2. Histopathology: Compared with Con group, serum creatinine was significantly elevated and renal tissue was severely damaged in CI-AKI group (vs CI-AKI group, P 0.05), while rapamycin preconditioning significantly and dose-dependent reduced renal function and histopathological injury induced by contrast medium (vs CI-AKI group, P 0.05, Rap 1 vs Rap 2 group, P 0.05).3. Autophagy level: Compared with Con group, CI-AKI preconditioning significantly and dose-dependent decreased renal function and histopathological injury induced by contrast medium (vs CI-AKI group, P 0.05, Rap 1/ The levels of autophagy-associated protein LC3 and Beclin-1 in renal tissues of rats in group C increased (Con vs CI-AKI, P 0.05), the co-localization signal expression of LC3-labeled autophagosomes and LAMP-2-labeled lysosomes increased, and autophagy was activated by contrast medium. Rapamycin pretreatment significantly and dose-dependent enhanced the expression of autophagy-related protein in water. Mitochondrial autophagy level: Compared with the control group, the expression level of mitochondrial autophagy-related protein pink-1 in renal tissue of rats in CI-AKI group increased (vs CI-AKI group, P Compared with the control group, the pretreatment with rapamycin significantly and dose-dependent enhanced the co-localization signal of autophagy and mitochondria, and enhanced the autophagy activity of mitochondria. 5. Oxidative stress level: Compared with the control group, MD in renal tissue of rats in CI-AKI group increased significantly and dose-dependent. The content of MDA increased (vs CI-AKI group, P 0.05), while rapamycin preconditioning significantly and dose-dependent decreased the content of MDA in renal tissue (Rap1/2 vs CI-AKI group, P 0.05; Rap1 vs Rap2 group, P 0.05). 6. Mitochondrial injury: Compared with Con group, the level of cytoplasmic/mitochondrial Cyt C in renal tissue of rats in CI-AKI group increased (vs CI-AKI group, P 0.0). 5) decreased MMP (vs CI-AKI group, P 0.05), and mitochondrial damage was induced by contrast medium. Rapamycin preconditioning significantly and dose-dependent inhibited the release of Cyt C and the decrease of MMP in mitochondria, and delayed the mitochondrial damage induced by contrast medium (vs CI-AKI group, P 0.05, Rap1 vs Rap2 group, P 0.05). Cell apoptosis: Compared with Con group, the apoptosis rate of renal tubular epithelial cells in CI-AKI group increased (vs CI-AKI group, P 0.05), while rapamycin pretreatment significantly and dose-dependent decreased the apoptosis rate of renal tubular epithelial cells induced by contrast medium (Rap 1/2 vs CI-AKI group, P 0.05; Rap 1 vs Rap 2 group, P 0.05). Intraperitoneal injection of contrast agent can successfully establish a new animal model of CI-AKI. 2. Contrast agent can induce mitochondrial damage and activate autophagy and mitochondrial autophagy. However, activated autophagy and mitochondrial autophagy are insufficient to remove damaged mitochondria. Overaccumulating damaged mitochondria will result in the release of large amounts of ROS and Cytc, leading to the release of renal tissue. Oxidative stress injury and apoptosis of renal tubular epithelial cells. 3. Rapamycin pretreatment can enhance the levels of autophagy and mitochondrial autophagy in renal tissue of CI-AKI rats. Enhanced levels of autophagy and mitochondrial autophagy can alleviate contrast-induced mitochondrial injury, oxidative stress injury and tubular epithelial cell apoptosis, thereby delaying CI-AKI rats. The degree of renal injury in AKI rats.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692
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