背景:前期工作发现自噬相关基因Uvrag缺失导致年龄相关心肌病,但细胞衰老在此过程中的作用尚不清楚。目的:研究Uvrag基因缺失对心脏细胞衰老的影响。方法:从Uvrag基因缺失纯合子小鼠和野生型小鼠中各取14只雄性小鼠分两批分别饲养到3月...背景:前期工作发现自噬相关基因Uvrag缺失导致年龄相关心肌病,但细胞衰老在此过程中的作用尚不清楚。目的:研究Uvrag基因缺失对心脏细胞衰老的影响。方法:从Uvrag基因缺失纯合子小鼠和野生型小鼠中各取14只雄性小鼠分两批分别饲养到3月龄和8月龄。采用real time RT-PCR检测小鼠心脏组织衰老相关分泌表型相关因子mRNA表达变化,苏木精-伊红染色、天狼猩红染色、衰老相关β-半乳糖苷酶染色进行心肌组织学观察,透射电镜观察小鼠心肌细胞超微结构的变化,蛋白免疫印迹检测小鼠心脏组织p53蛋白表达量。实验方案经上海交通大学动物实验伦理委员会批准。结果与结论:①苏木精-伊红染色和天狼猩红染色结果显示,Uvrag基因缺失导致单个心肌细胞显著增大及心脏纤维化;②透射电镜显示,Uvrag基因缺失小鼠心肌细胞线粒体形态异常、排列紊乱,肌浆网肿胀;③衰老相关β-半乳糖苷酶染色发现,Uvrag基因缺失小鼠心脏衰老细胞显著增多;④real time RT-PCR结果表明,Uvrag基因缺失小鼠心脏衰老相关分泌表型因子表达显著上调;⑤Western blot结果显示Uvrag基因缺失导致心脏组织衰老调控关键蛋白p53表达显著升高;⑥结果说明,自噬相关基因Uvrag缺失促进小鼠心脏细胞衰老;Uvrag基因是潜在的延缓心脏衰老及抗衰老相关心脏疾病的靶分子。展开更多
Objective: Rubicon is an inhibitory interacting protein of the autophagy-related protein Uvrag. We previously showed thatRubicon deficiency promotes autophagic fluxin vivo and that autophagy can degrade lipid droplets...Objective: Rubicon is an inhibitory interacting protein of the autophagy-related protein Uvrag. We previously showed thatRubicon deficiency promotes autophagic fluxin vivo and that autophagy can degrade lipid droplets. This study aimed to investigate the effects of Rubicon deficiency on fasting-induced hepatic steatosis.Methods: Two-month-old wild-type (WT) andRubicon-deficient mice were subjected to feeding or fasting for 24 hours to induce hepatic steatosis. The distribution of liver lipid droplets was revealed by oil red O staining. Hepatic and plasma triglyceride, non-esterified fatty acid (NEFA), and cholesterol levels were detected using commercially available kits. Real-time reverse transcriptasepolymerase chain reaction was performed to analyze the mRNA expression of genes related to lipid metabolism in the liver. Western blot was conducted to assess autophagy-related protein levels in the liver. The animal experiments were approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University, China.Results: We showed that under fasting conditions,Rubicon-deficient mice had more lipid droplets in the liver than WT controls. Consistent with these results, the hepatic triglyceride, NEFA, and cholesterol levels in fastedRubicon-deficient mice were significantly higher than those of fasted WT controls. The levels ofSREBP-1, a key regulator of lipid synthesis, were significantly lower in livers from fasted WT mice than those of fed WT mice. However, the decrease inSREBP-1 in fasted mice was attenuated byRubicon deficiency. Western blot analysis demonstrated that the fasting-induced increase in autophagic flux was amplified byRubicon deficiency. Finally, we showed thatRubicon deficiency in mice led to elevated plasma triglyceride and NEFA acid levels under fasting conditions.Conclusion: Rubicon deficiency exacerbates fasting-induced hepatic steatosis in mice.展开更多
文摘背景:前期工作发现自噬相关基因Uvrag缺失导致年龄相关心肌病,但细胞衰老在此过程中的作用尚不清楚。目的:研究Uvrag基因缺失对心脏细胞衰老的影响。方法:从Uvrag基因缺失纯合子小鼠和野生型小鼠中各取14只雄性小鼠分两批分别饲养到3月龄和8月龄。采用real time RT-PCR检测小鼠心脏组织衰老相关分泌表型相关因子mRNA表达变化,苏木精-伊红染色、天狼猩红染色、衰老相关β-半乳糖苷酶染色进行心肌组织学观察,透射电镜观察小鼠心肌细胞超微结构的变化,蛋白免疫印迹检测小鼠心脏组织p53蛋白表达量。实验方案经上海交通大学动物实验伦理委员会批准。结果与结论:①苏木精-伊红染色和天狼猩红染色结果显示,Uvrag基因缺失导致单个心肌细胞显著增大及心脏纤维化;②透射电镜显示,Uvrag基因缺失小鼠心肌细胞线粒体形态异常、排列紊乱,肌浆网肿胀;③衰老相关β-半乳糖苷酶染色发现,Uvrag基因缺失小鼠心脏衰老细胞显著增多;④real time RT-PCR结果表明,Uvrag基因缺失小鼠心脏衰老相关分泌表型因子表达显著上调;⑤Western blot结果显示Uvrag基因缺失导致心脏组织衰老调控关键蛋白p53表达显著升高;⑥结果说明,自噬相关基因Uvrag缺失促进小鼠心脏细胞衰老;Uvrag基因是潜在的延缓心脏衰老及抗衰老相关心脏疾病的靶分子。
基金This study was supported by the National Natural Science Foundation of China(Nos.81974024 and 82171567)the Shanghai Natural Science Foundation(No.16ZR1418200).
文摘Objective: Rubicon is an inhibitory interacting protein of the autophagy-related protein Uvrag. We previously showed thatRubicon deficiency promotes autophagic fluxin vivo and that autophagy can degrade lipid droplets. This study aimed to investigate the effects of Rubicon deficiency on fasting-induced hepatic steatosis.Methods: Two-month-old wild-type (WT) andRubicon-deficient mice were subjected to feeding or fasting for 24 hours to induce hepatic steatosis. The distribution of liver lipid droplets was revealed by oil red O staining. Hepatic and plasma triglyceride, non-esterified fatty acid (NEFA), and cholesterol levels were detected using commercially available kits. Real-time reverse transcriptasepolymerase chain reaction was performed to analyze the mRNA expression of genes related to lipid metabolism in the liver. Western blot was conducted to assess autophagy-related protein levels in the liver. The animal experiments were approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University, China.Results: We showed that under fasting conditions,Rubicon-deficient mice had more lipid droplets in the liver than WT controls. Consistent with these results, the hepatic triglyceride, NEFA, and cholesterol levels in fastedRubicon-deficient mice were significantly higher than those of fasted WT controls. The levels ofSREBP-1, a key regulator of lipid synthesis, were significantly lower in livers from fasted WT mice than those of fed WT mice. However, the decrease inSREBP-1 in fasted mice was attenuated byRubicon deficiency. Western blot analysis demonstrated that the fasting-induced increase in autophagic flux was amplified byRubicon deficiency. Finally, we showed thatRubicon deficiency in mice led to elevated plasma triglyceride and NEFA acid levels under fasting conditions.Conclusion: Rubicon deficiency exacerbates fasting-induced hepatic steatosis in mice.
