Blue LED causes autophagic cell death in human osteosarcoma by increasing ROS generation and dephosphorylating EGFR

Osteosarcoma (OS) is the most common primary malignant bone tumour in adolescence. Lately, light-emitting diodes (LED)-based therapy has emerged as a new promising approach for several diseases. However, it remains unknown in human OS. Here, we found that the blue LED irradiation significantly suppressed the proliferation, migration and invasion of human OS cells, while we observed blue LED irradiation increased ROS production through increased NADPH oxidase enzymes NOX2 and NOX4, as well as decreased Catalase (CAT) expression levels. Furthermore, we revealed blue LED irradiation-induced autophagy characterized by alterations in autophagy protein markers including Beclin-1, LC3-II/LC3-I and P62. Moreover, we demonstrated an enhanced autophagic flux. The blockage of autophagy displayed a remarkable attenuation of anti-tumour activities of blue LED irradiation. Next, ROS scavenger N-acetyl-L-cysteine (NAC) and NOX inhibitor diphenyleneiodonium (DPI) blocked suppression of OS cell growth, indicating that ROS accumulation might play an essential role in blue LED-induced autophagic OS cell death. Additionally, we observed blue LED irradiation decreased EGFR activation (phosphorylation), which in turn led to Beclin-1 release and subsequent autophagy activation in OS cells. Analysis of EGFR colocalization with Beclin-1 and EGFR-immunoprecipitation (IP) assay further revealed the decreased interaction of EGFR and Beclin-1 upon blue LED irradiation in OS cells. In addition, Beclin-1 down-regulation abolished the effects of blue LED irradiation on OS cells. Collectively, we concluded that blue LED irradiation exhibited anti-tumour effects on OS by triggering ROS and EGFR/Beclin-1-mediated autophagy signalling pathway, representing a potential approach for human OS treatment.     

Necrostatin-1 Suppresses Autophagy and Apoptosis in Mice Traumatic Brain Injury Model

Traumatic brain injury (TBI) results in neuronal apoptosis, autophagic cell death and necroptosis. Necroptosis is a newly discovered caspases-independent programmed necrosis pathway which can be triggered by activation of death receptor. Previous works identified that necrostatin-1 (NEC-1), a specific necroptosis inhibitor, could reduce tissue damage and functional impairment through inhibiting of necroptosis process following TBI. However, the role of NEC-1 on apoptosis and autophagy after TBI is still not very clear. In this study, the amount of TBI-induced neural cell deaths were counted by PI labeling method as previously described. The expression of autophagic pathway associated proteins (Beclin-1, LC3-II, and P62) and apoptotic pathway associated proteins (Bcl-2 and caspase-3) were also respectively assessed by immunoblotting. The data showed that mice pretreated with NEC-1 reduced the amount of PI-positive cells from 12 to 48?h after TBI. Immunoblotting results showed that NEC-1 suppressed TBI-induced Beclin-1 and LC3-II activation which maintained p62 at high level. NEC-1 pretreatment also reversed TBI-induced Bcl-2 expression and caspase-3 activation, as well as the ratio of Beclin-1/Bcl-2. Both 3-MA and NEC-1 suppressed TBI-induced caspase-3 activation and LC3-II formation, Z-VAD only inhibited caspase-3 activation but increased LC3-II expression at 24?h post-TBI. All these results revealed that multiple cell death pathways participated in the development of TBI, and NEC-1 inhibited apoptosis and autophagy simultaneously. These coactions may further explain how can NEC-1 reduce TBI-induced tissue damage and functional deficits and reflect the interrelationship among necrosis, apoptosis and autophagy.     

The attenuation of Th1 and Th17 responses via autophagy protects against methicillin-resistant Staphylococcus aureus-induced sepsis

Whether autophagy affects methicillin-resistant Staphylococcus aureus (MRSA)-induced sepsis and the associated mechanisms are largely unknown. This study investigated the role of autophagy in MRSA-induced sepsis. The levels of microtubule-associated protein light chain 3 (LC3)-II/I, Beclin-1 and p62 after USA300 infection were examined by Western blotting and immunohistochemical staining. Bacterial burden analysis, hematoxylin-eosin staining, and Kaplan–Meier analysis were performed to evaluate the effect of autophagy on MRSA-induced sepsis. IFN-γ and IL-17 were analyzed by ELISA, and CD4⁺ T cell differentiation was assessed by flow cytometry. Our results showed that LC3-II/I and Beclin-1 were increased, while p62 was decreased after infection. Survival rates were decreased in the LC3B^−/− and Beclin-1^+/− groups, accompanied by worsened organ injuries and increased IFN-γ and IL-17 levels, whereas rapamycin alleviated organ damage, decreased IFN-γ and IL-17 levels, and improved the survival rate. However, there was no significant difference in bacterial burden. Flow cytometric analysis showed that rapamycin treatment decreased the frequencies of Th1 and Th17 cells, whereas these cells were upregulated in the LC3B^−/− and Beclin-1^+/− groups. Therefore, autophagy plays a protective role in MRSA-induced sepsis, which may be partly associated with the alleviation of organ injuries via the downregulation of Th1 and Th17 responses. These results provide a nonantibiotic treatment strategy for sepsis.     

Redox regulation of mitophagy in the lung during murine Staphylococcus aureus sepsis

Oxidative mitochondrial damage is closely linked to inflammation and cell death, but low levels of reactive oxygen and nitrogen species serve as signals that involve mitochondrial repair and resolution of inflammation. More specifically, cytoprotection relies on the elimination of damaged mitochondria by selective autophagy (mitophagy) during mitochondrial quality control. This aim of this study was to identify and localize mitophagy in the mouse lung as a potentially upregulatable redox response to Staphylococcus aureus sepsis. Fibrin clots loaded with S. aureus (1×10⁷ CFU) were implanted abdominally into anesthetized C57BL/6 and B6.129X1-Nfe2l2tm1Ywk/J (Nrf2^−/−) mice. At the time of implantation, mice were given vancomycin (6 mg/kg) and fluid resuscitation. Mouse lungs were harvested at 0, 6, 24, and 48 h for bronchoalveolar lavage (BAL), Western blot analysis, and qRT-PCR. To localize mitochondria with autophagy protein LC3, we used lung immunofluorescence staining in LC3–GFP transgenic mice. In C57BL/6 mice, sepsis-induced pulmonary inflammation was detected by significant increases in mRNA for the inflammatory markers IL-1β and TNF-α at 6 and 24 h, respectively. BAL cell count and protein also increased. Sepsis suppressed lung Beclin-1 protein, but not mRNA, suggesting activation of canonical autophagy. Notably sepsis also increased the LC3-II autophagosome marker, as well as the lung׳s noncanonical autophagy pathway as evidenced by loss of p62, a redox-regulated scaffolding protein of the autophagosome. In LC3–GFP mouse lungs, immunofluorescence staining showed colocalization of LC3-II to mitochondria, mainly in type 2 epithelium and alveolar macrophages. In contrast, marked accumulation of p62, as well as attenuation of LC3-II in Nrf2-knockout mice supported an overall decrease in autophagic turnover. The downregulation of canonical autophagy during sepsis may contribute to lung inflammation, whereas the switch to noncanonical autophagy selectively removes damaged mitochondria and accompanies tissue repair and cell survival. Furthermore, mitophagy in the alveolar region appears to depend on activation of Nrf2. Thus, efforts to promote mitophagy may be a useful therapeutic adjunct for acute lung injury in sepsis.     

The role of autophagy in the pathogenesis of exposure keratitis

Incomplete tear film spreading and eyelid closure can cause defective renewal of the ocular surface and air exposure‐induced epithelial keratopathy (EK). In this study, we characterized the role of autophagy in mediating the ocular surface changes leading to EK. Human corneal epithelial cells (HCECs) and C57BL/6 mice were employed as EK models, respectively. Transmission electron microscopy (TEM) evaluated changes in HCECs after air exposure. Each of these models was treated with either an autophagy inhibitor [chloroquine (CQ) or 3‐methyladenine (3‐MA)] or activator [Rapamycin (Rapa)]. Immunohistochemistry assessed autophagy‐related proteins, LC3 and p62 expression levels. Western blotting confirmed the expression levels of the autophagy‐related proteins [Beclin1 and mammalian target of rapamycin (mTOR)], the endoplasmic reticulum (ER) stress‐related proteins (PERK, eIF2α and CHOP) and the PI3K/Akt/mTOR signalling pathway‐related proteins. Real‐time quantitative PCR (qRT‐PCR) determined IL‐1β, IL‐6 and MMP9 gene expression levels. The TUNEL assay detected apoptotic cells. TEM identified autophagic vacuoles in both EK models. Increased LC3 puncta formation and decreased p62 immunofluorescent staining and Western blotting confirmed autophagy induction. CQ treatment increased TUNEL positive staining in HCECs, while Rapa had an opposite effect. Similarly, CQ injection enhanced air exposure‐induced apoptosis and inflammation in the mouse corneal epithelium, which was inhibited by Rapa treatment. Furthermore, the phosphorylation status of PERK and eIF2α and CHOP expression increased in both EK models indicating that ER stress‐induced autophagy promoted cell survival. Taken together, air exposure‐induced autophagy is indispensable for the maintenance of corneal epithelial physiology and cell survival.     

氧化低密度脂蛋白经AMPK/mTOR信号通路诱导HUVECs自噬

氧化低密度脂蛋白（oxygenized low density lipoprotein, ox-LDL）诱导人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）损伤有助于动脉粥样硬化（atherosclerosis, AS）的发展。但ox-LDL对HUVECs自噬的影响及机制尚不清楚。为探究其机制,采用体外培养HUVECs,建立ox-LDL损伤模型。透射电子显微镜观察HUVECs中自噬体的变化;Western印迹法检测p-AMPK、AMPK、p-mTOR、mTOR及Beclin1、LC3-II、P62的表达。结果显示,与对照组比较,透射电子显微镜下观察到ox-LDL组的自噬体明显增多。Western印迹结果显示,与对照组比较,ox-LDL组Beclin1(0.81±0.04 vs. 1.83±0.11,P＜0.01)、LC3-II(0.80±0.06 vs. 1.61±0.06, P＜0.01)和P62(0.65±0.10 vs. 1.64±0.17, P＜0.01)表达显著增高。ox-LDL和BafilomycinA1共同干预组Beclin-1(3.15±0.15 vs. 3.17±0.13, P>0.05)、LC3-II(2.95±0.12 vs. 2.96±0.12, P >0.05)和P62(3.26±0.15 vs. 3.19±0.15, P>0.05)表达与BafilomycinA1组无显著差异,ox-LDL未使自噬起始增加,可能是降解受损导致自噬体的积累。与对照组比较,ox-LDL增加p-AMPK (0.47±0.03 vs. 0.96±0.03, P＜0.01)表达,并降低p-mTOR(0.86±0.04 vs. 0.25±0.05, P＜0.01)表达。单独阻断mTOR时, Beclin-1(0.81±0.05 vs. 2.19±0.17, P＜0.01)、LC3-II(0.76±0.13 vs 2.00±0.05, P＜0.01)和P62(0.74±0.12 vs. 1.94±0.11, P＜0.01)表达显著增加。亮氨酸（Leucine）可增加p-mTOR(0.87±0.11 vs. 1.67±0.07, P＜0.01)表达,并降低Beclin-1(0.81±0.05 vs. 0.37±0.03, P＜0.01)、LC3-II（0.76±0.13 vs. 0.41±0.02, P＜0.01）和P62（0.76±0.10 vs. 0.44±0.04, P＜0.01）表达,但ox-LDL可使Leucine预处理后的p-mTOR（1.67±0.11 vs. 0.82±0.02, P＜0.01）表达显著降低,并且Beclin-1（0.37±0.03 vs. 0.78±0.04, P＜0.01）、LC3-II（0.41±0.02 vs. 0.78±0.02, P＜0.01）和P62（0.44±0.04 vs. 0.74±0.04, P＜0.01）表达显著增加。说明mTOR参与ox-LDL诱导的自噬。与ox-LDL组相比,ox-LDL和Si-AMPK共同处理组p-mTOR(0.25±0.05 vs. 0.46±0.03, P＜0.01)表达增加以及Beclin-1(1.97±0.04 vs. 1.26±0.12, P＜0.01)、LC3-II(1.42±0.10 vs. 0.95±0.05, P＜0.01)和P62(1.58±0.09 vs. 0.98±0.11, P＜0.01)表达降低。以上结果表明,ox-LDL通过AMPK/mTOR途径诱导HUVECs发生自噬,并且导致自噬体的积累。     

雷公藤甲素诱导肝细胞选择性自噬的形态学观察

摘要 目的：观察雷公藤甲素诱导肝细胞选择性自噬的水平。方法：在雷公藤甲素给药处理小鼠的尾静脉高压注射GFP-LC3质粒，制备肝细胞自噬示踪模型，观察雷公藤甲素在动物体内诱导肝细胞自噬的水平。在GFP-LC3稳定表达的L02细胞株中转入RFP-P62质粒，用活细胞工作站和荧光显微镜动态观察雷公藤甲素诱导L02细胞选择性自噬的轮廓，同时也观察细胞自噬复合体LC3-P62的变化。结果：动物实验结果表明，雷公藤甲素可显著诱导肝细胞自噬体的形成，Western blot结果显示P62蛋白和LC3II蛋白的表达趋势一致。活细胞动态观察及免疫荧光双标记实验结果表明LC3-P62存在共定位，提示雷公藤甲素可诱导肝细胞自噬流的形成。结论：雷公藤甲素可诱导肝细胞选择自噬，其生物学意义可能与肝细胞损伤后修复相关。     

Bisretinoid-mediated Complement Activation on Retinal Pigment Epithelial Cells Is Dependent on Complement Factor H Haplotype

Age-related macular degeneration (AMD) is a common central blinding disease of the elderly. Homozygosity for a sequence variant causing Y402H and I62V substitutions in the gene for complement factor H (CFH) is strongly associated with risk of AMD. CFH, secreted by many cell types, including those of the retinal pigment epithelium (RPE), is a regulatory protein that inhibits complement activation. Recessive Stargardt maculopathy is another central blinding disease caused by mutations in the gene for ABCA4, a transporter in photoreceptor outer segments (OS) that clears retinaldehyde and prevents formation of toxic bisretinoids. Photoreceptors daily shed their distal OS, which are phagocytosed by the RPE cells. Here, we investigated the relationship between the CFH haplotype of human RPE (hRPE) cells, exposure to OS containing bisretinoids, and complement activation. We show that hRPE cells of the AMD-predisposing CFH haplotype (HH402/VV62) are attacked by complement following exposure to bisretinoid-containing Abca4^−/− OS. This activation was dependent on factor B, indicating involvement of the alternative pathway. In contrast, hRPE cells of the AMD-protective CFH haplotype (YY402/II62) showed no complement activation following exposure to either Abca4^−/− or wild-type OS. The AMD-protective YY402/II62 hRPE cells were more resistant to the membrane attack complex, whereas HH402/VV62 hRPE cells showed significant membrane attack complex deposition following ingestion of Abca4^−/− OS. These results suggest that bisretinoid accumulation in hRPE cells stimulates activation and dysregulation of complement. Cells with an intact complement negative regulatory system are protected from complement attack, whereas cells with reduced CFH synthesis because of the Y402H and I62V substitutions are vulnerable to disease.     

苦参素复合长春新碱对HCT-8/VCR细胞耐药性的影响及其机制*

目的: 探究苦参素复合长春新碱对HCT-8/VCR细胞耐药性的影响及其机制。方法: HCT-8/VCR体外培养,分为空白组、苦参素单药组,长春新碱单药组、苦参素和长春新碱联合组,每组6个复孔。CCK-8法检测各组HCT-8/VCR细胞的耐药性;MDC法检测细胞内自噬小泡的变化;ELISA法检测IL-6的含量;Western blot检测自噬相关基因与免疫因子TLR4蛋白的表达水平。结果: 苦参素复合长春新碱可降低HCT-8/VCR细胞的耐药性,逆转倍数为3.23;与空白组比较,苦参素组和联合组自噬体含量降低、IL-6含量也明显降低(P＜0.01),长春新碱组自噬体含量升高,IL-6含量也明显升高(P＜0.01);联合组与苦参素组比较,自噬体含量升高,而IL-6含量降低(P＜ 0.01);Western blot实验表明,与空白组比较,苦参素组P62的表达降低(P＜0.05),LC3-Ⅱ/LC3-Ⅰ、Beclin-1、TLR4均升高(P＜0.05),长春新碱组和联合组P62的表达升高(P＜0.05),LC3-Ⅱ/LC3-Ⅰ、Beclin-1、TLR4均降低(P＜ 0.05);联合组与长春新碱组比较,P62的表达升高(P＜0.05),LC3-Ⅱ/LC3-Ⅰ、Beclin-1、TLR4均降低(P＜0.05)。结论: 苦参素与长春新碱联合可显著降低HCT-8/VCR的耐药性,其机制与抑制自噬活动和TLR4信号活化有关。     

Protection against Experimental Stroke by Ganglioside GM1 Is Associated with the Inhibition of Autophagy

Ganglioside GM1, which is particularly abundant in the central nervous system (CNS), is closely associated with the protection against several CNS disorders. However, controversial findings have been reported on the role of GM1 following ischemic stroke. In the present study, using a rat middle cerebral artery occlusion (MCAO) model, we investigated whether GM1 can protect against ischemic brain injury and whether it targets the autophagy pathway. GM1 was delivered to Sprague-Dawley male rats at 3 doses (25 mg/kg, 50 mg/kg, 100 mg/kg) by intraperitoneal injection soon after reperfusion and then once daily for 2 days. The same volume of saline was given as a control. Tat–Beclin-1, a specific autophagy inducer, was administered by intraperitoneal injection at 24 and 48 hours post-MCAO. Infarction volume, mortality and neurological function were assessed at 72 hours after ischemic insult. Immunofluorescence and Western blotting were performed to determine the expression of autophagy-related proteins P62, LC3 and Beclin-1 in the penumbra area. No significant changes in mortality and physiological variables (heart rate, blood glucose levels and arterial blood gases) were observed between the different groups. However, MCAO resulted in enhanced conversion of LC3-I into LC3-II, P62 degradation, high levels of Beclin-1, a large area infarction (26.3±3.6%) and serious neurobehavioral deficits. GM1 (50 mg/kg) treatment significantly reduced the autophagy activation, neurobehavioral dysfunctions, and infarction volume (from 26.3% to 19.5%) without causing significant adverse side effects. However, this biological function could be abolished by Tat–Beclin-1. In conclusion: GM1 demonstrated safe and robust neuroprotective effects that are associated with the inhibition of autophagy following experimental stroke.     

脊髓自噬功能激活与大鼠2型糖尿病神经病理性疼痛的关系

目的：探讨脊髓自噬功能与大鼠2型糖尿病神经病理性疼痛（DNP）的关系。方法：雄性SD大鼠（42只）高糖高脂饲养8周,腹腔单次注射链脲佐菌素（STZ）制备大鼠2型糖尿病模型。两周后检测机械缩足阈值（MWT）和热缩足潜伏期（TWL）,降至基础值80%以下者为2型糖尿病神经病理性疼痛大鼠,记为DNP组（24只）;未降至基础值80%以下者为2型糖尿病无神经病理性疼痛大鼠,记为DA组（18只）。另取18只大鼠为对照（control,C）组,普通饲料喂养。于确定DA与DNP分组后的第3、7和14天,测定机械缩足阈值（MWT）和热缩足潜伏期（TWL）,并在行为学检测结束后各组随机取6只大鼠处死,取L4~L6脊髓膨大,采用Western blot法检测自噬特异性蛋白微管相关蛋白1（Beclin-1）、微管相关蛋白1轻链3（LC3）和P62的表达。另取6只7 d DNP组大鼠采用免疫荧光双染法检测脊髓背角P62与小胶质细胞、星形胶质细胞、神经元的共表达情况。结果：连续8周喂养高糖高脂饲料的SD大鼠的血浆胰岛素水平升高,胰岛素敏感指数下调,表明出现胰岛素抵抗;在腹腔注射STZ后,血糖升高达到2型糖尿病诊断标准（≥16.7 mmol/L）;与C组、DA组比较,DNP组大鼠在第3、7和14天时MWT降低,TWL缩短,并且脊髓背角LC3-Ⅱ、Beclin-1表达上调,P62表达下降（P<0.05）。免疫荧光双染色显示,P62在脊髓背角表达,主要与神经元共存,少量与小胶质细胞共存,几乎不与星形胶质细胞共表达。结论：2型糖尿病神经病理性疼痛大鼠脊髓LC3-Ⅱ、Beclin-1和P62表达的改变提示脊髓自噬功能激活;脊髓背角中神经元自噬激活在2型糖尿病大鼠DNP的发生和发展起着关键作用。     

Deletion of autophagy inducer RB1CC1 results in degeneration of the retinal pigment epithelium

Autophagy regulates cellular homeostasis and response to environmental stress. Within the retinal pigment epithelium (RPE) of the eye, the level of autophagy can change with both age and disease. The purpose of this study is to determine the relationship between reduced autophagy and age-related degeneration of the RPE. The gene encoding RB1CC1/FIP200 (RB1-inducible coiled-coil 1), a protein essential for induction of autophagy, was selectively knocked out in the RPE by crossing Best1-Cre mice with mice in which the Rb1cc1 gene was flanked with Lox-P sites (Rb1cc1^flox/flox). Ex vivo and in vivo analyses, including western blot, immunohistochemistry, transmission electron microscopy, fundus photography, optical coherence tomography, fluorescein angiography, and electroretinography were performed to assess the structure and function of the retina as a function of age. Deletion of Rb1cc1 resulted in multiple autophagy defects within the RPE including decreased conversion of LC3-I to LC3-II, accumulation of autophagy-targeted precursors, and increased numbers of mitochondria. Age-dependent degeneration of the RPE occurred, with formation of atrophic patches, subretinal migration of activated microglial cells, subRPE deposition of inflammatory and oxidatively damaged proteins, subretinal drusenoid deposits, and occasional foci of choroidal neovascularization. There was secondary loss of photoreceptors overlying the degenerated RPE and reduction in the electroretinogram. These observations are consistent with a critical role of autophagy in the maintenance of normal homeostasis in the aging RPE, and indicate that disruption of autophagy leads to retinal phenotypes associated with age-related degeneration.     

IGF-1-Mediated Survival from Induced Death of Human Primary Cultured Retinal Pigment Epithelial Cells Is Mediated by an Akt-Dependent Signaling Pathway

Degeneration of the human retinal pigmented epithelium (hRPE) is involved in several eye disorders such as age-related macular degeneration (AMD). In this study, we investigated the protective effect of IGF-1 on human primary cultured RPE cells and its underlying mechanism. IGF-1 dose- and time-dependently promoted the survival of RPE cells from serum deprivation. Western blot showed that IGF-1 stimulated the activation of the PI3K/Akt and MAPK pathways in hRPE. Inhibition of the PI3K/Akt pathway by the PI3K-specific inhibitor, LY294002 or inhibition of Akt by Akt-specific inhibitors Akt inhibitor VIII or SN-38, or downregulation Akt with siRNA specific for Akt blocked the effect of IGF-1 on hRPE. In contrast, blockade of the MAPK pathway with a specific inhibitor PD98059 had no effect. Interestingly, vitreous IGF-1 injection reversed the inhibitory effect of light exposure (a dry AMD model) on both a wave and b wave. Immunocytochemistry showed that vitreous IGF-1 injections promoted the survival of RPE cells in rat retina and the expression of RPE65 in RPE cells from light injury. These results indicate that IGF-1 is able to protect hRPE cell from different insults in vivo and in vitro. Further detailed studies may lead the way to a therapeutic intervention for retinal diseases in which cell death is an underlying contributory mechanism.     

A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates

Phagocytosis and autophagy are typically dedicated to degradation of substrates of extrinsic and intrinsic origins respectively. Although overlaps between phagocytosis and autophagy were reported, the use of autophagy for ingested substrate degradation by nonprofessional phagocytes has not been described. Blood-separated tissues use their tissue-specific nonprofessional phagocytes for homeostatic phagocytosis. In the testis, Sertoli cells phagocytose spermatid residual bodies produced during germ cell differentiation. In the retina, pigmented epithelium phagocytoses shed photoreceptor tips produced during photoreceptor renewal. Spermatid residual bodies and shed photoreceptor tips are phosphatidylserine-exposing substrates. Activation of the tyrosine kinase receptor MERTK, which is implicated in phagocytosis of phosphatidylserine-exposing substrates, is a common feature of Sertoli and retinal pigmented epithelial cell phagocytosis. The major aim of our study was to investigate to what extent phagocytosis by Sertoli cells may be tissue specific. We analyzed in Sertoli cell cultures that were exposed to either spermatid residual bodies (legitimate substrates) or retina photoreceptor outer segments (illegitimate substrates) the course of the main phagocytosis stages. We show that whereas substrate binding and ingestion stages occur similarly for legitimate or illegitimate substrates, the degradation of illegitimate but not of legitimate substrates triggers autophagy as evidenced by the formation of double-membrane wrapping, MAP1LC3A-II/LC3-II clustering, SQSTM1/p62 degradation, and by marked changes in ATG5, ATG9 and BECN1/Beclin 1 protein expression profiles. The recruitment by nonprofessional phagocytes of autophagy for the degradation of ingested cell-derived substrates is a novel feature that may be of major importance for fundamentals of both apoptotic substrate clearance and tissue homeostasis.     

Calpain-mediated cleavage of Beclin-1 and autophagy deregulation following retinal ischemic injury in vivo

Autophagy is the major intracellular degradation pathway that regulates long-lived proteins and organelles turnover. This process occurs at basal levels in all cells but it is rapidly upregulated in response to starvation and cellular stress. Although being recently implicated in neurodegeneration, it remains still unclear whether autophagy has a detrimental or protective role. In this study, we investigated the dynamics of the autophagic process in retinal tissue that has undergone transient ischemia, an experimental model that recapitulates features of ocular pathologies, including glaucoma, anterior ischemic optic neuropathy and retinal vessels occlusion. Retinal ischemia, induced in adult rats by increasing the intraocular pressure, was characterized by a reduction in the phosphatidylethanolamine-modified form of LC3 (LC3II) and by a significant decrease in Beclin-1. The latter event was associated with a proteolytic cleavage of Beclin-1, leading to the accumulation of a 50-kDa fragment. This event was prevented by intravitreal treatment with the non-competitive N-methyl-D-aspartate antagonist MK801 and calpain inhibitors or by calpain knockdown. Blockade of autophagy by pharmacological inhibition or Beclin-1 silencing in RGC-5 increased cell death, suggesting a pro-survival role of the autophagic process in this neuronal cell type. Altogether, our results provide original evidence for calpain-mediated cleavage of Beclin-1 and deregulation of basal autophagy in the rat retina that has undergone ocular ischemia/reperfusion injury.     

Tribbles homolog 3-mediated targeting the AKT/mTOR axis in mice with retinal degeneration

Various retinal degenerative disorders manifest in alterations of the AKT/mTOR axis. Despite this, consensus on the therapeutic targeting of mTOR in degenerating retinas has not yet been achieved. Therefore, we investigated the role of AKT/mTOR signaling in rd16 retinas, in which we restored the AKT/mTOR axis by genetic ablation of pseudokinase TRB3, known to inhibit phosphorylation of AKT and mTOR. First, we found that TRB3 ablation resulted in preservation of photoreceptor function in degenerating retinas. Then, we learned that the mTOR downstream cellular pathways involved in the homeostasis of photoreceptors were also reprogrammed in rd16 TRB3^−/− retinas. Thus, the level of inactivated translational repressor p-4E-BP1 was significantly increased in these mice along with the restoration of translational rate. Moreover, in rd16 mice manifesting decline in p-mTOR at P15, we found elevated expression of Beclin-1 and ATG5 autophagy genes. Thus, these mice showed impaired autophagy flux measured as an increase in LC3 conversion and p62 accumulation. In addition, the RFP-EGFP-LC3 transgene expression in rd16 retinas resulted in statistically fewer numbers of red puncta in photoreceptors, suggesting impaired late autophagic vacuoles. In contrast, TRIB3 ablation in these mice resulted in improved autophagy flux. The restoration of translation rate and the boost in autophagosome formation occurred concomitantly with an increase in total Ub and rhodopsin protein levels and the elevation of E3 ligase Parkin1. We propose that TRB3 may retard retinal degeneration and be a promising therapeutic target to treat various retinal degenerative disorders.Subject terms: Translation, Diseases     

Polyphyllin VII Induces an Autophagic Cell Death by Activation of the JNK Pathway and Inhibition of PI3K/AKT/mTOR Pathway in HepG2 Cells

Polyphyllin VII (PP7), a pennogenyl saponin isolated from Rhizoma Paridis, exhibited strong anticancer activities in various cancer types. Previous studies found that PP7 induced apoptotic cell death in human hepatoblastoma cancer (HepG2) cells. In the present study, we investigated whether PP7 could induce autophagy and its role in PP7-induced cell death, and elucidated its mechanisms. PP7 induced a robust autophagy in HepG2 cells as demonstrated by the conversion of LC3B-I to LC3B-II, degradation of P62, formation of punctate LC3-positive structures, and autophagic vacuoles tested by western blot analysis or InCell 2000 confocal microscope. Inhibition of autophagy by treating cells with autophagy inhibitor (chloroquine) abolished the cell death caused by PP7, indicating that PP7 induced an autophagic cell death in HepG2 cells. C-Jun N-terminal kinase (JNK) was activated after treatment with PP7 and pretreatment with SP600125, a JNK inhibitor, reversed PP7-induced autophagy and cell death, suggesting that JNK plays a critical role in autophagy caused by PP7. Furthermore, our study demonstrated that PP7 increased the phosphorylation of AMPK and Bcl-2, and inhibited the phosphorylation of PI3K, AKT and mTOR, suggesting their roles in the PP7-induced autophagy. This is the first report that PP7 induces an autophagic cell death in HepG2 cells via inhibition of PI3K/AKT/mTOR, and activation of JNK pathway, which induces phosphorylation of Bcl-2 and dissociation of Beclin-1 from Beclin-1/Bcl-2 complex, leading to induction of autophagy.     

基于自噬与凋亡机制研究左归丸对化疗损伤颗粒细胞及膜细胞的影响

目的 探讨左归丸含药血清对化疗损伤性颗粒细胞和膜细胞的影响及作用机制。方法 制备左归丸含药血清,培养大鼠卵巢颗粒细胞和膜细胞,使用磷酰胺氮芥造模分组后给药。CCK-8法测定颗粒细胞和膜细胞存活率,实时荧光定量PCR法（RT-PCR）及蛋白质免疫印迹法（Western blot）分别检测卵巢自噬启动因子Beclin-1、微管结合蛋白轻链3（LC3B）、自噬受体蛋白p62、凋亡蛋白Bax、Caspase3在转录水平和翻译水平上的表达。结果 10%左归丸含药血清对于细胞存活的挽救率最高。Beclin-1、LC3B、Bax、Caspase3在磷酰胺氮芥作用的颗粒细胞和膜细胞中,相对于空白对照组有高表达（P<0.05）,10%左归丸含药血清可下调上述蛋白质在模型组中的表达（P<0.05）;然而受体蛋白p62较空白对照组升高（P<0.05）,10%左归丸含药血清可上调模型组p62的表达（P<0.05）。此外,在颗粒细胞实验组中,激活或抑制自噬途径后,自噬相关蛋白的表达在发生相应改变的同时,凋亡相关蛋白的表达也会发生相应改变。结论 磷酰胺氮芥可通过促进凋亡、激活自噬/溶酶体降解途径的机制损伤颗粒细胞和膜细胞。10%左归丸含药血清能缓解由此带来的损伤,同时影响了颗粒细胞和膜细胞自噬和凋亡过程。在磷酰胺氮芥损伤颗粒细胞的过程和10%左归丸含药血清缓解其损伤过程中均存在自噬与凋亡串流（cross-talk）。     

Light-emitting diodes and cool white fluorescent light similarly suppress pineal gland melatonin and maintain retinal function and morphology in the rat.

BACKGROUND AND PURPOSE: A novel light-emitting diode (LED) light source for use in animal-habitat lighting was evaluated. METHODS: The LED was evaluated by comparing its effectiveness with that of cool white fluorescent light (CWF) in suppressing pineal gland melatonin content and maintaining normal retinal physiology, as evaluated by use of electroretinography (ERG), and morphology. RESULTS: Pineal melatonin concentration was equally suppressed by LED and CWF light at five light illuminances (100, 40, 10, 1, and 0.1 lux). There were no significant differences in melatonin suppression between LED and CWF light, compared with values for unexposed controls. There were no differences in ERG a-wave implicit times and amplitudes or b-wave implicit times and amplitudes between 100-lux LED-exposed rats and 100-lux CWF-exposed rats. Results of retinal histologic examination indicated no differences in retinal thickness, rod outer segment length, and number of rod nuclei between rats exposed to 100-lux LED and 100-lux CWF for 14 days. Furthermore, in all eyes, the retinal pigmented epithelium was intact and not vacuolated, whereas rod outer segments were of normal thickness. CONCLUSION: LED light does not cause retinal damage and can suppress pineal melatonin content at intensities similar to CWF light intensities.     

Beclin-1-mediated autophagy protects spinal cord neurons against mechanical injury-induced apoptosis

Apoptosis has been widely reported to be involved in the pathogenesis associated with spinal cord injury (SCI). Recently, autophagy has also been implicated in various neuronal damage models. However, the role of autophagy in SCI is still controversial and its interrelationship with apoptosis remains unclear. Here, we used an in vitro SCI model to observe a time-dependent induction of autophagy and apoptosis. Mechanical injury induced autophagy markers such as LC3 lipidation, LC3II/LC3I conversion, and Beclin-1expression. Injured neurons showed decreased cell viability and increased apoptosis. To elucidate the effect of autophagy on apoptosis, the mechanically-injured neurons were treated with the mTOR inhibitor rapamycin and 3-methyl adenine (3-MA), which are known to regulate autophagy positively and negatively, respectively. Rapamycin-treated neurons showed the highest level of cell viability and lowest level of apoptosis among the injured neurons and those treated with 3-MA showed the reciprocal effect. Notably, rapamycin-treated neurons exhibited slightly reduced Bax expression and significantly increasedBcl-2 expression. Furthermore, by plasmid transfection, we showed that Beclin-1-overexpressing neuronal cells responded to mechanical injury with greater LC3II/LC3I conversion and cell viability, lower levels of apoptosis, higher Bcl-2 expression, and unaltered Bax expression as compared to vector control cells. Beclin-1-knockdown neurons showed almost the opposite effects. Taken together, our results suggest that autophagy may serve as a protection against apoptosis in mechanically-injured spinal cord neurons. Targeting mTOR and/or enhancing Beclin-1 expression might be alternative therapeutic strategies for SCI.