線粒體自噬在對比劑所致急性腎損傷中的作用及機制研究
發(fā)布時間：2018-09-08 09:11
【摘要】：目的:1.建立新型CI-AKI大鼠動物模型。2.觀察CI-AKI大鼠腎組織中自噬、線粒體自噬水平的變化,以及氧化應激水平、線粒體損傷和腎小管上皮細胞凋亡情況的改變。3.探討雷帕霉素預處理增強自噬、線粒體自噬活性對對比劑所致線粒體損傷、氧化應激損傷及腎小管上皮細胞凋亡情況的影響。方法:1.健康雄性SD大鼠54只,體重210-230 g,采用計算機隨機法將其隨機分成9組(n=6):正常組(A組):予以尾靜脈注射同等劑量的生理鹽水;不同劑量尾靜脈注射組(B1-3組):尾靜脈分別注射碘海醇3.5,5.25,8.75 g I/kg;不同劑量腹腔注射組(C1-3組):腹腔分別注射碘海醇5.25,8.75,12.25 g I/kg;尾靜脈多次注射組(D1-2組):連續(xù)兩天分別予以尾靜脈注射碘海醇5.25,7.0g I/kg。觀察對比劑或生理鹽水注射24 h后對比劑對大鼠腎功能及腎組織病理的影響,探索新型CI-AKI大鼠動物模型。2.健康雄性SD大鼠24只,體重210-230 g,采用計算機隨機法將其隨機分成4組(n=6):正常組(Con組):予以腹腔注射同等劑量的生理鹽水;CI-AKI模型組(CI-AKI組):予以腹腔注射超大劑量的碘海醇(12.25 g I/kg);低劑量雷帕霉素+對比劑組(Rap1組):注射同等劑量對比劑之前予以連續(xù)腹腔注射雷帕霉素(2 mg/kg/d)一周;高劑量雷帕霉素+對比劑組(Rap2組):注射同等劑量對比劑之前予以連續(xù)腹腔注射雷帕霉素(5 mg/kg/d)一周;對比劑或生理鹽水注射24 h后處死大鼠,觀察雷帕霉素對CI-AKI大鼠腎功能及腎組織病理的影響。采用Western-blot檢測腎組織中LC3、Beclin-1、Pink-1、胞漿及線粒體中Cyt c的表達水平;免疫熒光雙染檢測LC3標記的自噬小體和LAMP-2標記的溶酶體或TOMM20標記的線粒體的共表達情況,檢測細胞自噬、線粒體自噬活性;JC-1法檢測線粒體膜電位(Mitochondrial membrane potential,MMP);TUNEL法檢測腎組織中腎小管上皮細胞凋亡程度;ELISA法檢測大鼠腎組織中MDA含量,探討不同劑量雷帕霉素預處理對CI-AKI大鼠的作用及其機制。結(jié)果:1.與A組比較,B1-3組,C1-2組,D1-2組血肌酐輕度升高,而C3組血肌酐明顯升高(A組vs C3組,P0.05)。與A組比較,B1-3組,C1-2組,D1-2組僅見輕度腎小管變性,C3組大鼠腎組織重度損傷,可見嚴重的腎小管上皮細胞變性、壞死脫落,以及大量空泡變性。2.腎功能及組織病理:與Con組比較,CI-AKI組血肌酐明顯升高,腎組織重度損傷(Con組vs CI-AKI組,P0.05),而雷帕霉素預處理顯著且劑量依賴性地減輕對比劑所致腎功能及組織病理損傷(Rap1/2組vs CI-AKI組,P0.05;Rap1組vs Rap2組,P0.05)。3.自噬水平:與Con組比較,CI-AKI組大鼠腎組織中自噬相關(guān)蛋白LC3和Beclin-1的表達水平增加(Con組vs CI-AKI組,P0.05),LC3標記的自噬小體與LAMP-2標記的溶酶體共定位信號表達增加,對比劑誘導自噬激活。而雷帕霉素預處理顯著且劑量依賴性地增強了自噬相關(guān)蛋白的表達水平(Rap1/2組vs CI-AKI組,P0.05;Rap1組vs Rap2組,P0.05),及自噬小體與溶酶體的共定位表達信號,增強自噬活性。4.線粒體自噬水平:與Con組比較,CI-AKI組大鼠腎組織中線粒體自噬相關(guān)蛋白pink-1的表達水平增加(Con組vs CI-AKI組,P0.05),LC3標記的自噬小體與TOMM-20標記的線粒體共定位信號表達增加,對比劑誘導線粒體自噬激活。而雷帕霉素預處理顯著且劑量依賴性地增強了自噬小體與線粒體的共定位表達信號,增強線粒體自噬活性。5.氧化應激水平:與Con組比較,CI-AKI組大鼠腎組織中MDA含量增多(Con組vs CI-AKI組,P0.05),而雷帕霉素預處理顯著且劑量依賴性地降低了腎組織中MDA含量(Rap1/2組vs CI-AKI組,P0.05;Rap1組vs Rap2組,P0.05)。6.線粒體損傷情況:與Con組比較,CI-AKI組大鼠腎組織中胞漿/線粒體Cyt c水平增加(Con組vs CI-AKI組,P0.05),MMP降低(Con組vs CI-AKI組,P0.05),對比劑導致腎組織中線粒體損傷。而雷帕霉素預處理顯著且劑量依賴性地抑制了線粒體中Cyt c的釋放及MMP的降低,延緩了對比劑所致線粒體損傷(Rap1/2組vs CI-AKI組,P0.05;Rap1組vs Rap2組,P0.05)。7.腎小管上皮細胞凋亡情況:與Con組比較,CI-AKI組腎組織中腎小管上皮細胞凋亡率增加(Con組vs CI-AKI組,P0.05),而雷帕霉素預處理明顯且劑量依賴性地降低了對比劑所致的腎小管上皮細胞凋亡率(Rap1/2組vs CI-AKI組,P0.05;Rap1組vs Rap2組,P0.05)。結(jié)論:1.超大劑量腹腔注射對比劑可成功建立新型CI-AKI大鼠動物模型。2.對比劑可導致腎組織線粒體損傷并激活自噬及線粒體自噬活性。但激活的自噬及線粒體自噬不足以清除受損的線粒體,過度累積的受損線粒體將導致大量ROS和Cyt c釋放,導致腎組織氧化應激損傷及腎小管上皮細胞凋亡。3.雷帕霉素預處理可增強CI-AKI大鼠腎組織中自噬及線粒體自噬水平。增強了的自噬及線粒體自噬水平可以減輕對比劑所致線粒體損傷及其所致的氧化應激損傷和腎小管上皮細胞凋亡,從而延緩CI-AKI大鼠腎臟損傷程度。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R692
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