【作者】 吴萍；

【导师】 刘选明； 郭新红；

【作者基本信息】 湖南大学 ， 化学， 2020， 博士

【摘要】 自噬是溶酶体降解细胞内物质的总称,其作用机制是在粗面内质网和其他一些细胞器的非核糖体附着区的双层膜上形成自噬体,从而形成自噬溶酶体,降解细胞内容物,适应细胞和某些细胞器的代谢需要。自噬在多种不同类型的细胞中均有研究,但它在骨骼肌蛋白质降解中的作用研究报道甚少,特别是在鱼类中。在细胞营养充足的条件下,自噬可以被有效地抑制,而在营养缺乏时自噬被激活。自噬与活性氧(ROS)具有显显著相关性,自噬的激活可以增加ROS的水平,但是当ROS过量或清除被抑制时ROS的产生可触发自噬。关于自噬在斑马鱼肝脏和大脑中表现出昼夜节律性已被证实,但至目前尚未有关于营养胁迫影响鱼类骨骼肌自噬节律变化的研究报道。为探索解析在营养胁迫即饥饿状态下自噬调节骨骼肌代谢稳态的信号传导机制,本文分析研究了短期饥饿对幼年鳜鱼骨骼肌抗氧化能力、活性氧(ROS)含量、自噬相关基因和自噬体形成的影响。同时,开展了罗非鱼幼鱼中自噬基因对饥饿反应的昼夜节律变化的研究,主要研究结果如下:(1)成功克隆了鳜鱼8个自噬基因c DNA全长序列,并对蛋白结构分析发现其均含有自噬基因特征性的UBQ,Atg14和丝氨酸/苏氨酸蛋白酶位点结构域,其进化上与鲈形目鱼类亲缘性最高。组织差异性表达分析发现10个重要自噬基因Atg4b,Atg4c,Atg4d,Atp6v1d,Becn1,Bnip3,Gabarapl1,LC3a,LC3b和Ulk1 m RNA分别在肠道、肝脏和肌肉组织中显著表达。(2)在饥饿条件下,鳜鱼幼鱼肌肉中ROS含量显著升高。与正常组比较,饥饿2天后鳜鱼肌肉抗氧化酶SOD、CAT、GPx、和GST活力以及MDA、ASA和AHRFR含量均显著增加;同时,GPx、GSTA、GST4A、GSTT1、Mn SOD、Zn SOD和CAT等基因表达量显著上升,抗氧化信号分子包括Nrf2、S6K1和m TOR m RNA的表达上调,但Keap1基因m RNA表达则下调,肌肉的抗损伤能力增强。但饥饿5天后,其抗氧化信号因子和抗氧化酶相关基因表达均下降,抗氧化酶活力下降,导致抗氧化能力减弱。(3)在短期饥饿2天的鳜鱼幼鱼肌肉中,自噬基因(Bnip3除外)以及自噬小体变化不显著,LC3-II/LC3-I蛋白水平没有显著差异,自噬水平基本维持不变。当饥饿时间延长至第5天,自噬基因m RNA表达水平显著上升,电镜观察到肌肉自噬体数量也显著增多,LC3-II/LC3-I蛋白水平显著增加,引起自噬增强。(4)预测了一个饥饿条件下ROS介导的氧化应激对鱼类肌肉细胞自噬的作用模式图,ROS通过调控抗氧化信号分子Nrf2-Keap1介导抗氧化酶基因激活,从而抗氧化酶活力增加。而当氧化应激达到极限,细胞自噬发生。(5)利用双荧光素酶报告基因检测技术揭示,miR-181a-5p和miR-194a可以抑制抗氧化基因Nrf2的表达。同时,miR-1、miR-16a和miR-29a则抑制抗氧化基因Keap1的表达。其结果预测在鱼类中miRNA对自噬具有调控作用。(6)采用生物钟余弦分析,发现营养胁迫使得肌肉自噬基因的节律发生改变。同时,作为自噬标记物的LC3(Lc3a、Lc3b和Lc3c)在正常饲喂状态和禁食条件均具节律性,且波形图无变化。比较分析发现,禁食组肌肉中的自噬相关基因如Atg4b、Becn1、Bnip3la、Bnip3lb、Lc3a和Ulk1b等m RNA表达水平较正常组显著上调。电镜分析结果证实饥饿胁迫后,肌肉中自噬小体亦相应增多。(7)通过注射Nr1d1的激动剂GSK4112,导致核心生物钟基因Nr1d1节律紊乱,并导致自噬基因节律性改变。当Nr1d1过表达时,肌肉中的自噬相关基因Atg4c、Atp6v1d、LC3a和Ulk1a表达水平明显下调,说明生物钟对自噬节律具有调控作用。更多还原

【Abstract】 Autophagy is a general term for lysosomal degradation of intracellular materials and is a relatively conservative mechanism unique to eukaryotic cells.Autophagy refers to the formation of autophagosomes from the bilayer membrane on the non-ribosome attachment area of the rough endoplasmic reticulum and some other cellular organelles,which leads to the formation of autophagic lysosomes to degrade cell contents and to achieve the metabolic needs of cells and some organelles.Autophagy has been observed in m any different types of cells,but its role in skeletal muscle protein degradation has not been thoroughly studied,especially in aquatic species.Under conditions of sufficient cell nutrition,autophagy can be effectively inhibited,while autophagy is acti vated when nutrition is deficient.Autophagy and ROS do have a strong correlation,not through simple activation or inhibition but through complex mechanisms.The activation of autophagy can increase the level of ROS,but the production of ROS can trigger autophagy when ROS excess or clearance is suppressed.The rhythmic research on autophagy has only been reported in zebrafish liver and brain at different nutritional conditions.To date,there is no report focused on the rhythmic changes of fish skeletal m uscle autophagy upon nutritional deprivation.In order to explore the molecular signal transduction mechanism of autophagy regulating the metabolic homeostasis of skeletal muscle under starvation,the effects of short-term starvation on the antioxidant cap acity,ROS content,autophagy genes and autophagosomes formation of skeletal muscle of Siniperca chuatsi were studied,and the circadian rhythm of autophagy gene response to starvation in juvenile tilapia was alsoassayed.The obtained major results are as follows:(1)Eight autophagy genes from the Siniperca chuatsi have been successfully cloned.And their structural domains,UBQ,atg14 and serine/threonine protease,are characterized.The m RNA expression levels of the ten important autophagy genes including Atg4 b,Atg4c,Atg4 d,Atp6v1d,Becn1,Bnip3,Gabarapl1,LC3 a,LC3b and Ulk1 expression levels were significantly expressed in intestine,liver and muscle tissues,respectively.(2)Under the starvation condition,the ROS content in juvenile Siniperca chuatsi muscles increased significantly.Compared with the control group,the activity of antioxidant enzymes,SOD,CAT,GPx,and GST,and the contents of MDA,ASA,and AHRFR all significantly increased after 2 days of starvation.The expression levels of GPx,GSTA,GS T4 A,GS TT1,Mn SOD,Zn S OD,and CAT genes were significantly up-regulated.The expression levels of antioxidant signal molecules Nrf2,S6K1 and m TOR m RNA were also increased,but Keap1 gene m RNA expression level was down-regulated,which improved the muscle’s ability to resist injury.However,upon further starvation for 5 days,the expression level of antioxidant signal factor genes and regulating antioxidant enzyme genes were down-regulated,and the antioxidant enzyme activity we re decreased,thus caused the antioxidant capacity decliling.(3)The autophagy gene expressions(except Bnip3)and autophagosomes did not change significantly in the muscles of Siniperca chuatsi juveniles upon starved for 2days.There was no significant difference in LC3-II/LC3-I protein levels,and the levels of autophagy remained basically unchanged.When the starvation time was prolonged to the 5 days,the expression level of autophagy gene m RNA was significantlyup-regulated,and the number of autophag osomes and the level of LC3-II/LC3-I protein were also apparantly increased by electron microscopy obervation.Thus,autophagy was significantly enhanced in juvenile Siniperca chuatsi muscle.(4)A working pattern of ROS mediated oxidative stress on autoph agy of muscle cells under starvation was constructed.ROS mediates the activation of antioxidant enzyme genes by regulating the antioxidant signal molecule Nrf2-Keap1,thereby increasing the activity of antioxidant enzymes.However,when oxidative stress i s intolerable,muscle autophagy occurs.(5)Using Dual-Luciferase reporter assay,we found that miR-181a-5p and miR-194 a could inhibit the expression of Nrf2.The other miRNAs,such as miR-1,miR-16 a and miR-29 a can also inhibit the expression of Keap1.The results demonstrate that miRNAs have a regulatory effect on autophagy.(6)Using biological cosine analysis,we found that starvation stress also changed the genes involved in maintaining muscle autophagy rhythmicity.At the same time,only LC3(Lc3a,Lc3 b,and Lc3c),as a marker of autophagy,is still rhythmic regardless of the normal feeding or fastingtreatment,and the COSINOR waveforms are similar.By comparing the m RNA expression levels of autophagy genes in the muscles of the fasting and the normal feeding treatment,it was found that Atg4 b,Becn1,Bnip3 la,Bnip3lb,Lc3 a,and Ulk1 b were significantly upregulated compared with the normal group.Electron microscopy analysis also revealed that autophagosome increased significantly after starvation.(7)GSK4112,the activator of Nr1d1,caused the disorder of Nr1d1,which also caused the rhythmic change of autophagy gene.It also showed that when Nr1d1 was significantly overexpressed,the autophagy genes Atg4 c,Atp6v1d,LC3 a and Ulk1 a expression was significantly down-regulated.Thus,the biological clock could involve in regulating the autophagy rhythmicity.更多还原