Participation of autophagy in the degeneration process of rat hepatocytes after transplantation following prolonged cold preservation

Cold ischemia-warm reperfusion injury of liver grafts has been investigated thoroughly, but its underlying mechanism remains poorly understood. Here we show that autophagy is involved not only during cold preservation but also during warm reperfusion following transplantation. Immunohistochemistry using an antibody against LC3, a microtubule associated protein 1 light chain 3 and a marker of autophagosomes, showed dot-like weak staining in hepatocytes of rat liver grafts during cold preservation. Since University of Wisconsin solution for graft preservation lacks amino acids, the induction of autophagy in hepatocytes was similar to that under starvation conditions. Intense immunopositive punctate structures were detected abundantly in the hepatocytes 30 min after the beginning of reperfusion. LC3-positive granules were often co-localized in ED2-positive Kupffer cells at 60 min of the reperfusion phase. The molecular form of LC3 was mainly LC3-II, a membrane-bound form, during reperfusion, especially at 30 min of the phase. Electron microscopic examination demonstrated numerous vacuolar structures in hepatocytes at 30 min of the reperfusion period, while some hepatocytes with such vacuolar structures were present in the sinusoidal lumen. At the late stage of the reperfusion period, Kupffer cells contained phagocytosed cells that possessed numerous autophagic vacuoles/autolysosomes and nuclei with condensed chromatin. Our results showing the presence of autophagic vacuoles/autolysosomes in hepatocytes of liver grafts after the start of reperfusion suggest that warm reperfusion acted as a stress stimulus to hepatocytes. Moreover, the stress response of hepatocytes may be involved in their degeneration process.     

Cholesterol accumulation is associated with lysosomal dysfunction and autophagic stress in Npc1 -/- mouse brain

Niemann-Pick type C (NPC) disease is an autosomal recessive disorder caused by mutations of NPC1 and NPC2 genes. Progressive neurodegeneration that accompanies NPC is fatal, but the underlying mechanisms are still poorly understood. In the present study, we characterized the association of autophagic-lysosomal dysfunction with cholesterol accumulation in Npc1(-/-) mice during postnatal development. Brain levels of lysosomal cathepsin D were significantly higher in mutant than in wild-type mice. Increases in cathepsin D occurred first in neurons and later in astrocytes and microglia and were both spatially and temporally associated with intracellular cholesterol accumulation and neurodegeneration. Furthermore, levels of ubiquitinated proteins were higher in endosomal/lysosomal fractions of brains from Npc1(-/-) mice than from wild-type mice. Immunoblotting results showed that levels of LC3-II were significantly higher in brains of mutant than wild-type mice. Combined LC3 immunofluorescence and filipin staining showed that LC3 accumulated within filipin-labeled cholesterol clusters inside Purkinje cells. Electron microscopic examination revealed the existence of autophagic vacuole-like structures and multivesicles in brains from Npc1(-/-) mice. These results provide strong evidence that cholesterol accumulation-induced changes in autophagy-lysosome function are closely associated with neurodegeneration in NPC.     

Exacerbation of ischemia-induced amyloid-β generation by diabetes is associated with autophagy activation in mice brain

To evaluate effect of diabetes on transient ischemia-induced brain damage and autophagy activity, streptozotocin (STZ)-induced diabetic mellitus (DM) mice were subjected to transient common carotid artery occlusion (CCAO) operation. After the operation, immunohistochemistry and transmission electron microscopy (EM) were performed to investigate the astrocytes activation, amyloid-β protein (Aβ) expression and accumulation of autophagy-like vacuoles containing electron-dense material (avd); and hallmarks of autophagy, the microtubule-associated protein light chain 3 (LC3)-II, was detected by western blot analysis. The results showed that DM amplified stroke-induced astrocytes activation and Aβ generation. Western blot analysis showed that LC3-II conjugate was drastically up-regulated at early stages post ischemia and it last for at least 72 h in DM mice brain. DM mice demonstrated increased baseline level of LC3-II as comparing to normal mice; DM also amplified stroke-induced LC3-II level. Under EM, avd was most markedly accumulated in neurons of DM mice brain after ischemia. Immunofluorescence double-staining showed that most Aβ and autophagosomes co-localized. Therefore, our results suggested that exacerbation of ischemia-induced Aβ generation by diabetes might be associated with autophagy activation in mice brain, and modulating neuronal autophagy might be a new therapeutic strategy to depress the risk of development of dementia in diabetic patients with stroke.     

Autophagy is involved in the ischemic preconditioning

Autophagy is a key pathway for the clearance of damaged organelles. Ischemic preconditioning (IPC) and autophagy are enhanced by mild hypoxic insults, but the association between autophagy and IPC remains unclear. We investigated the existence and role of autophagy in IPC. In an in vitro PC12 cell model, IPC increased generation and degradation of autophagosomes, as revealed by increased LC3-II bands, cathepsin D positive cells, lysosomal activity and autophagic vacuoles on electron microscopy. Autophagic activity was blocked using 3-methyladenine during IPC, and cell viabilities were measured using FASC and WST-1 assays. Inhibition of autophagy, especially during reperfusion or lethal oxygen-glucose deprivation periods ameliorated the neuroprotective effects of IPC. Moreover, inhibiting autophagy also attenuated Hsp70 upregulation induced by IPC. These findings imply that autophagy participates in IPC-induced neuroprotection, and that autophagy might provide a means of neuroprotection against cerebral ischemia.     

Autophagy induced by infectious hematopoietic necrosis virus inhibits intracellular viral replication and extracellular viral yields in epithelioma papulosum cyprini cell line

Infectious hematopoietic necrosis virus (IHNV) is a common pathogen that causes severe disease in the salmonid aquaculture industry. Recent work demonstrated that autophagy plays an important role in pathogen invasion by activating innate and adaptive immunity. This study investigated the relationship between IHNV and autophagy in epithelioma papulosum cyprini cells. The electron microscopy results show that IHNV infection can induce typical autophagosomes which are representative structures of autophagy activation. The punctate accumulation of green fluorescence-tagged microtubule-associate protein 1 light chain 3 (LC3) and the protein conversion from LC3-I to LC3-II were respectively confirmed by confocal fluorescence microscopy and western blotting. Furthermore, the effects of autophagy on IHNV replication were also clarified by altering the autophagy pathway. The results showed that rapamycin induced autophagy can inhibit both intracellular viral replication and extracellular viral yields, while autophagy inhibitor produced the opposite results. These findings demonstrated that autophagy plays an antiviral role during IHNV infection.     

Autophagic cell death and its execution by lysosomal cathepsins

In the last decade, the molecular mechanisms of apoptosis, a major type of active cell death (type I cell death) have largely been clarified in mammalian cells. Particularly, the caspase family of proteinases has been shown to play crucial roles in the execution of apoptosis. Differing from apoptosis, type II cell death is known to be associated with autophagosomes/autolysosomes and appear in the developing nervous system (CLARKE, 1990). We have previously shown that delayed neuronal death occurring in the CA1 pyramidal layer of the gerbil hippocampus after brief forebrain ischemia is apoptotic in nature and autophagosomes/autolysosomes abundantly appear in the neurons before DNA fragmentation. To further understand the roles of autophagosomes/autolysosomes in active cell death, we examined the apoptosis of PC12 cells using morphological and biochemical techniques. PC12 cells are known to undergo apoptosis when cultured in the absence of serum. In such an environment, the mitochondrial pathway of apoptosis is activated; cytochrome c is released from mitochondria, and caspase-9/caspase-3 are activated. We have first examined morphological features of PC12 cells during the apoptotic process following serum deprivation, and found that autophagy is induced from the early stage of the process in the cells before typical nuclear changes. When autophagy is inhibited in the cells by 3-methyladenine, an autophagy inhibitor, they are largely protected from apoptosis. In relation to the induction of autophagy in PC12 cells following serum deprivation, immunoreactivity, protein amounts, and the proteolytic activity of lysosomal proteinases, particularly cathepsins B and D, are all greatly altered; those of cathepsin B drastically decrease in the cells from the early stage of serum-deprived cultures, whereas those of cathepsin D increase. Moreover, PC12 cells overexpressing cathepsin D undergo apoptosis more rapidly in serum-deprived cultures than wild-type cells, whereas those overexpressing cathepsin B increase the viability. These lines of evidence suggest that autophagy is involved in PC12 cell death following serum deprivation, this type of cell death being regulated by lysosomal proteinases, cathepsins B and D, downstream autophagy.     

Class A scavenger receptor activation inhibits endoplasmic reticulum stress-induced autophagy in macrophage

Macrophage death in advanced atherosclerosis promotes plaque necrosis and destabilization. Involvement of autophagy in bulk degradation of cellular components has been recognized recently as an important mechanism for cell survival under endoplasmic reticulum (ER) stress. We previously found that the engagement of class A scavenger receptor (SR-A) triggered JNK-dependent apoptosis in ER-stressed macrophages. However, pro-apoptotic mechanisms mediated by SR-A are not fully understood. Therefore, we sought to see if SR-A mediated apoptosis was associated with autophagy in macrophages. Here, we showed that fucoidan inhibited microtubule-associated protein light chain 3-phospholipid conjugates (LC3-II) formation as well as the number of autophagosomes under ER stress. The inhibition of LC3-II formation was paralleled by the activation of the mTOR pathway, and the inhibition of mTOR allowed LC3-II induction in macrophages treated with thapsigargin plus fucoidan. Furthermore, apoptosis induced by fucoidan was prevented under ER stress by the mTOR inhibitor. We propose that fucoidan, a SR-A agonist, may contribute to macrophage apoptosis during ER stress by inhibiting autophagy.     

自噬在晚期糖基化终产物诱导的内皮细胞凋亡中的作用

目的: 研究晚期糖基化终产物(AGEs)对人脐静脉内皮细胞(HUVECs)自噬水平的影响并探讨自噬在AGEs诱导的内皮细胞凋亡中的作用。方法: 用AGEs处理HUVECs,相同条件牛血清白蛋白处理为对照组,Western blotting检测相应蛋白表达的变化,电镜观察细胞自噬体的出现,流式细胞术检测细胞凋亡,MTT比色法测定细胞活性。结果: AGEs处理HUVECs后,自噬相关蛋白LC3-Ⅱ的表达显著上调并呈时间和浓度依赖性,电镜观察到细胞胞浆内自噬体数量增加;与对照组比较,AGEs处理组内皮细胞凋亡率增加,活性下降,自噬抑制剂3-甲基腺嘌呤预处理的AGEs组细胞活性较AGEs组进一步下降,凋亡率继续增加。AGEs处理HUVECs后,蛋白激酶B(Akt)和哺乳动物雷帕霉素靶蛋白(mTOR)的磷酸化水平也明显下调, Akt激活剂胰岛素样生长因子1预处理后,Akt的磷酸化水平增加,自噬相关蛋白LC3-Ⅱ的增高表达被抑制。结论: AGEs通过PI3K/Akt/mTOR介导的信号通路诱导HUVECs自噬水平升高。自噬在AGEs诱导的内皮细胞凋亡中对细胞起保护作用。     

DICER activates autophagy and promotes cisplatin resistance in non-small cell lung cancer by binding with let-7i-5p

ObjectiveDrug resistance is the main obstacle in the treatment of non-small cell lung cancer (NSCLC). This study aimed to explore the mechanism of DICER in NSCLC resistance and its downstream signaling pathways.MethodsThe A549 cisplatin (DDP)-resistant strain A549/DDP was established. A549/DDP cells were transfected with DICER- and let-7i-5p-related vectors, and treated with autophagy activator rapamycin. The cell viability and apoptosis were tested by CCK-8 assay and flow cytometry, respectively. The formation of autophagosomes was observed with a transmission electron microscopy. RT-qPCR and Western blot assay were conducted to detect expression levels of DICER, let-7i-5p, autophagy-related proteins, and the PI3K/AKT/mTOR pathway-related proteins. The dual luciferase reporter gene assay was implemented to confirm the targeted binding of DICER and let-7i-5p.ResultsDICER was highly expressed in DDP-resistant NSCLC tissues and cells, and DICER could target and negatively regulate the expression of let-7i-5p. DDP treatment could inhibit the viability and promote cell apoptosis of A549/DDP cells. Downregulation of DICER in A549/DDP cells exhibited a decrease of cell viability, a decreased ratio of LC3-II/LC3-I and autophagosomes, together with an elevation of cell apoptosis rate and the phosphorylation levels of PI3K/AKT/mTOR. Treatment of rapamycin and let-7i-5p inhibitor reversed the effects of downregulated DICER in cell viability, ratio of LC3-II/LC3-I, autophagosomes, cell apoptosis rate and the phosphorylation levels of PI3K/AKT/mTOR in A549/DDP cells.ConclusionOur research suggests that DICER promotes autophagy and DDP resistance in NSCLC through downregulating let-7i-5p, and inhibits the activation of PI3K/AKT/mTOR pathway.     

低强度超声联合微泡通过提高ROS水平促进甲状腺癌细胞自噬性死亡

目的:探讨低强度超声联合微泡造影剂对甲状腺癌细胞自噬性死亡的作用,并分析自噬的激活机制及其对细胞活力的影响。方法:采用频率20 k Hz、功率80 m W的低强度超声联合微泡造影剂处理人甲状腺癌TPC1细胞,处理细胞60、120和240 s后,采用Live/Dead实验和CCK-8实验分析细胞死亡和活力;Western blot分析微管相关蛋白1轻链3Ⅱ(microtubule-associated protein 1 light chain 3-Ⅱ,LC3-Ⅱ)、自噬相关蛋白5(autophagy-related protein 5,ATG5)和SQSTM1/P62的蛋白水平变化;单丹磺酰戊二胺(monodansylcadaverine,MDC)染色、绿色荧光蛋白(green fluorescent protein,GFP)-LC3转染和透射电镜观察细胞内自噬体的数量;2',7'-二氯二氢荧光素二乙酸脂(DCFDA)染色分析细胞活性氧簇(reactive oxygen species,ROS)的水平,用N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)抑制氧化应激水平,分析ROS在自噬激活中的作用;ATG5 siRNA转染抑制自噬水平并分析自噬性死亡的作用。结果:低强度超声联合微泡显著促进TPC1细胞死亡,抑制TPC1细胞活力(P0.05),并与处理时间显著相关。相对于单纯低强度超声组和微泡组,超声联合微泡显著升高LC3-Ⅱ和ATG5蛋白水平,抑制P62蛋白水平(P0.05)。MDC染色、GFP-LC3转染和透射电镜观察发现,超声联合微泡明显增加TPC1细胞中自噬体的数量。超声联合微泡与单纯低强度超声组和微泡组相比,提高了细胞的ROS水平,而NAC显著降低超声联合微泡提高的LC3-Ⅱ蛋白水平(P0.05)。ATG5 siRNA抑制自噬并显著增加细胞活力(P0.05)。结论:本研究说明低强度超声联合微泡可能通过提高甲状腺癌细胞中的ROS水平促进细胞的自噬性死亡,从而引起甲状腺癌细胞死亡。     

DHRSX,A Novel Non-Classical Secretory Protein Associated With Starvation Induced Autophagy

Dehydrogenase/reductase (SDR family) X-linked (DHRSX) is a novel human gene without any substantial functional annotation and was initially cloned and identified in our laboratory. In this study, we present evidence that it encodes a non-classical secretory protein and promotes starvation induced autophagy. Using the Baf.A1 assay and N-terminal sequencing, we showed that DHRSX is secreted in a non-classical form. We expressed and purified a recombinant human GST-DHRSX fusion protein. Functional studies revealed that HeLa and U2OS cells overexpressing DHRSX or treated with the GST-DHRSX fusion protein exhibited higher levels of starvation-induced autophagy, resulting in increased endogenous LC3-II levels, a punctate GFP-LC3 distribution, and structures associated with autophagy, with a lower accumulation of autophagy substrates such as p62 and polyQ80. Accordingly, knockdown of endogenous DHRSX through specific siRNAs reduced LC3-II levels obviously in U2OS cells induced by starvation. Collectively, these results demonstrate that DHRSX is a novel non-classical secretory protein involved in the positive regulation of starvation induced autophagy and provide a new avenue for research on this protein family and autophagy regulation.     

血管紧张素Ⅱ升高血管内皮细胞中ROS水平并激活自噬通路

目的: 研究血管紧张素Ⅱ(AngⅡ)对血管内皮细胞自噬的影响及其可能机制。方法: 以EA.hy926血管内皮细胞为研究对象,荧光酶标仪检测AngⅡ(10^-7mol/L)、AngⅡ联合N-乙酰半胱氨酸(NAC,50 μmol/L)作用24 h后细胞中活性氧簇(ROS)水平;用不同浓度(10^-8、10^-7、10^-6mol/L)AngⅡ作用24 h或10^-7mol/L AngⅡ作用不同时间(0 h、6 h、12 h、24 h、36 h)后,通过Western blotting分析微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)蛋白变化、吖啶橙染色结合荧光显微镜观察自噬体形成情况来判断细胞中自噬发生情况;AngⅡ(10^-7mol/L)联合自噬抑制剂3-甲基腺嘌呤(3-MA,2 mmol/L)或NAC(50 μmol/L)处理24 h后用同样方法检测细胞中自噬的发生情况。结果: 细胞经AngⅡ刺激后细胞内ROS的水平显著升高(P<0.05),LC3-Ⅱ蛋白表达明显增强(P<0.05),自噬体形成明显增加;3-MA或NAC可显著抑制内皮细胞中AngⅡ诱导的LC3-Ⅱ蛋白表达(P<0.05)与自噬体形成。结论: AngⅡ可以通过升高血管内皮细胞内ROS的水平从而诱导自噬的发生。     

The development of an immunohistochemical method to detect the autophagy-associated protein LC3-II in human tumor xenografts

Autophagy is believed to be an important process during tumorgenesis, and in recent years it has been shown to be modulated in response to a number of conventional anticancer agents. Furthermore, the development of targeted small molecule inhibitors, such as those to the PI3K-AKT-mTOR pathway, has presented a molecular link between the disruption of this signalling cascade and the process of autophagy. The cellular consequence of stimulating or inhibiting autophagy in cancer cells is not completely understood, so it is important that this process be monitored, along with antiproliferative and apoptotic biomarkers, in the preclinical setting. The field of autophagy is still evolving, and there is a constantly changing set of criteria for the assessment of the process in cells, tissues, and organs. The gold standard technique for analyzing autophagy in mammalian cells remains transmission electron microscopy, which has many limitations and is often difficult to perform on in vivo tissue including human tumor xenografts. In order to monitor autophagy in human tumor xenogaft tissue, we have taken the approach to develop an immunohistochemical (IHC) method for the detection of the autophagosome-associated protein, microtubule-associated protein 1 light chain 3 (LC3), in human tumor xenografts. After synthesis, LC3 is cleaved to form LC3-I, and upon induction of autophagy, LC3-I is conjugated to the lipid phosphatidylethanolamine to form LC3-II, which is tightly bound to the membrane of the autophagosome. It is thought that detection of endogenous LC3-II by IHC could be difficult because of the relatively low level of expression of the protein. Here we present the validation of an IHC method to detect LC3 in human tumor xenografts that we believe is able to distinguish LC3-I from LC3-II. It is hoped that this assay can become a useful tool for the detection of autophagy in preclinical xenograft models and determine the effects of anticancer therapies on the autophagic process.     

磷酸肌酸减少大鼠缺血再灌注心肌细胞凋亡及自噬泡的数量

目的 研究磷酸肌酸减少大鼠缺血再灌注心肌细胞凋亡及白噬的能力.方法 雄性SD大鼠24只,体质量200 ～ 250 g,随机均分为假手术(sham)组、缺血/再灌注(ischemia-reperfusion,I/R)组和磷酸肌酸钠(phosphocreatine,CP)干预组.其中CP组按4 mg/kg磷酸肌酸钠剂量于再灌注前经右股静脉注射.用TUNEL法检测心肌细胞凋亡;电子显微镜观察心肌细胞自噬泡的发生和线粒体的形态学改变;Western blot检测微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)蛋白的表达.结果 I/R组与sham组相比,线粒体超微结构损伤加重,自噬泡数量增多(P＜0.01),心肌细胞凋亡率明显增加(P＜0.01);CP干预组可减轻I/R组线粒体超微结构损伤,自噬泡数量减少(P＜0.05),降低心肌细胞凋亡率(P ＜0.05);LC3-Ⅱ作为评价自噬强度的指标,I/R组与sham组相比,LC3-Ⅱ蛋白的表达明显上调(P＜0.01);而CP与I/R组相比,该蛋白表达明显下调(P＜0.05).结论 磷酸肌酸通过减少大鼠缺血再灌注心肌细胞凋亡及自噬泡的数量,从而减轻心肌缺血再灌注损伤.     

免疫相关GTP酶Irgm1对小鼠暂时性大脑中动脉缺血再灌注模型脑神经元自噬发生的影响

目的 探讨免疫相关GTP酶1(Irgm1)在小鼠暂时性大脑中动脉缺血再灌注模型(tMCAO)中的表达及其对tMCAO小鼠大脑神经元内自噬发生的影响.方法 采用western blotting和免疫荧光染色,检测Irgm1和自噬相关基因LC3I/Ⅱ及P62在Irgm1+/+小鼠tMCAO脑组织内的表达水平及细胞定位;进一步比较Irgm1+/+及Irgm1-/-小鼠tMCAO大脑神经元内自噬发生的差异.结果 相比于假手术(sham)组,Irgm1在Irgrm1+/+小鼠tMCAO脑组织中表达明显增高,且主要分布于损伤侧皮层神经元内;同时我们也证实在小鼠tMCAO脑组织中LC3-Ⅱ表达增加(t=-16.448,P＜0.01),面P62表达明显减低(t=3.975,P＜0.05).我们进一步实验发现Irgm1-/-小鼠tMCAO脑组织内LC3-Ⅱ表达量低于Irgm1+/+小鼠tMCAO(t=4.073,P＜0.05),而P62表达量高于Irgm1+/+小鼠tMCAO(t=-3.383,P＜0.05).结论 在小鼠tMCAO模型中,Irgm1参与调节大脑神经元的自噬活动.     

脑缺血再灌注损伤程度与自噬相关基因LC3、mTOR表达的相关性研究

目的　探讨局灶性脑缺血再灌注损伤（cerebral ischemia reperfusion, CIR）程度与自噬微管相关蛋白轻链3抗体-Ⅱ（Autophagy microtubule associated protein light chain 3 antibody-Ⅱ,LC3-Ⅱ）、雷帕霉素靶蛋白抗体（Rapamycin target protein antibody,mTOR）表达的相关性。 方法　采用线栓阻断法阻塞大脑中动脉2 h制作大鼠局灶性脑缺血再灌注模型;观察各组间的神经功能缺失评分、TTC梗死体积;应用免疫印迹、免疫荧光法检测大脑顶叶的LC3-Ⅱ、mTOR的表达。 结果　TTC染色显示,梗死体积在缺血再灌24 h达高峰。WB、免疫荧光显示,LC3-Ⅱ在IR中表达增加,且在缺血再灌24 h表达最高;mTOR在缺血再灌24 h、48 h表达最低。相关性分析显示,TTC染色梗死体积与LC3-Ⅱ表达水平高度正相关。 结论　脑缺血再灌注损伤程度可能与LC3、mTOR的表达水平相关。     

Parkinson's disease involves autophagy and abnormal distribution of cathepsin L

Accumulating evidences suggest that the related autophagy-lysosomal mechanism plays a critical role in many neurodegenerative disorders. In this study, we examined postmortem Parkinson's disease (PD) substantia nigra for evidence of cathepsin L by immunofluorescent staining, and found increased expression of cathepsin L in dopamine neurons of PD patients. We confirmed 6-OHDA induced nuclear translocation of cathepsin L in rat substantia nigral neurons as well. Furthermore, we observed autophagic vacuoles and lysosomes were accumulated in the 6-hydroxydopamine (6-OHDA) injured rat substantia nigra neurons with electron microscopy. Immunofluorescent staining showed that LC3 was enriched in dopamine neurons after 6-OHDA treatment. When pretreated with 3-methyladenine (3-MA), dopaminergic neurons were protected from cell death induced by 6-OHDA, associated with the suppression of LC3 and cathepsin L. Our results demonstrate that activation of autophagy and abnormal distribution of cathepsin L may be responsible for dopamine neuron death, involved in the pathogenic cascade event for the development of Parkinson's disease.     

MEG3和腺苷对HepG2细胞自噬和增殖的影响

目的:研究母系表达基因3(MEG3)过表达和腺苷对人肝细胞癌HepG2细胞自噬和增殖的影响,并探讨MEG3和腺苷影响HepG2细胞自噬的可能机制和抑制细胞增殖的效果。方法:常规培养HepG2细胞,分为对照组、MEG3组(MEG3慢病毒转染)、腺苷组(1 mmol/L腺苷处理)和MEG3+腺苷组(腺苷组中加入MEG3慢病毒转染)。Western blot检测LC3-Ⅱ、LC3-Ⅰ和哺乳动物雷帕霉素靶蛋白(mTOR)等蛋白的表达,MDC染色观察自噬体数量,CCK-8法观察细胞的活力,细胞计数仪检测活细胞数量。结果:与对照组比较,MEG3组和腺苷组中HepG2细胞内LC3-Ⅱ减少,LC3-Ⅱ/LC3-Ⅰ比值降低,mTOR表达增加,HepG2细胞的活力降低(P0.05),自噬体减少。与MEG3组和腺苷组比较,MEG3+腺苷组中HepG2细胞内LC3-Ⅱ和LC3-Ⅱ/LC3-Ⅰ比值进一步降低,mTOR表达进一步增加,HepG2细胞的活力进一步降低,自噬体减少更加显著(P0.05或P0.01)。MEG3组和腺苷组中HepG2活细胞数在24～72 h较对照组均明显降低(P0.01),在MEG3+腺苷组中降低更为明显(P0.01)。结论:MEG3过表达和低浓度腺苷可激活mTOR通路,抑制HepG2细胞自噬和增殖。MEG3增强了腺苷对HepG2细胞的作用。     

Structural basis of target recognition by Atg8/LC3 during selective autophagy

Autophagy is a non‐selective bulk degradation process in which isolation membranes enclose a portion of cytoplasm to form double‐membrane vesicles, called autophagosomes, and deliver their inner constituents to the lytic compartments. Recent studies have also shed light on another mode of autophagy that selectively degrades various targets. Yeast Atg8 and its mammalian homologue LC3 are ubiquitin‐like modifiers that are localized on isolation membranes and play crucial roles in the formation of autophagosomes. These proteins are also involved in selective incorporation of specific cargo molecules into autophagosomes, in which Atg8 and LC3 interact with Atg19 and p62, receptor proteins for vacuolar enzymes and disease‐related protein aggregates, respectively. Using X‐ray crystallography and NMR, we herein report the structural basis for Atg8–Atg19 and LC3–p62 interactions. Remarkably, Atg8 and LC3 were shown to interact with Atg19 and p62, respectively, in a quite similar manner: they recognized the side‐chains of Trp and Leu in a four‐amino acid motif, WXXL, in Atg19 and p62 using hydrophobic pockets conserved among Atg8 homologues. Together with mutational analyses, our results show the fundamental mechanism that allows Atg8 homologues, in association with WXXL‐containing proteins, to capture specific cargo molecules, thereby endowing isolation membranes and/or their assembly machineries with target selectivity.     

缺血后适应对大鼠局灶性脑缺血再灌注所致自噬的抑制作用

目的:探讨缺血后适应对大鼠局灶性脑缺血再灌注所致自噬的影响。方法:取健康雄性SD大鼠30只,随机分为假手术(sham)组、缺血再灌注(I/R)组、缺血后适应(IPC)组,每组各10只。Sham组仅单纯暴露右侧颈总、颈内和颈外动脉;I/R组采用Longa改良线栓法制备大鼠右侧大脑中动脉缺血2 h、再灌注24 h模型;IPC组大鼠缺血2 h后,同侧颈总动脉再通10 s/闭塞10 s,循环5次,然后全面恢复血流再灌注24 h。采用透射电镜观察脑细胞自噬情况;Western blot法测定各组大鼠脑组织中哺乳动物雷帕霉素靶蛋白(m TOR)、磷酸化m TOR(pm TOR)和微管相关蛋白轻链3(LC3)-Ⅱ的蛋白表达水平;TTC法检测脑梗死面积;干湿称重法测定脑组织含水量;HE染色观察脑组织病理学变化。结果:IPC组m TOR、p-m TOR均显著高于I/R组(P0.05),LC3-Ⅱ显著低于I/R组(P0.01)。IPC组脑梗死面积、脑组织含水量均显著低于I/R组(P0.01)。HE染色显示,IPC组神经元变性、坏死较I/R组明显减轻。透射电镜显示IPC组神经元损伤程度明显减轻,自噬泡明显减少。结论:IPC通过减少细胞内自噬而减轻脑缺血再灌注损伤,可能与增强m TOR作用有关。