补体激活与自噬在年龄相关性黄斑变性中的研究进展

年龄相关性黄斑变性(age-related macular degeneration,ARMD)是一种随年龄增长而发病率逐渐上升的黄斑部疾病,目前认为其发病因素与患者的年龄、遗传、吸烟、氧化应激、免疫炎症反应、RPE细胞老化和代谢异常等有关。补体系统在机体防御感染、免疫调节和炎症应答中扮演重要角色,补体系统异常激活引起免疫炎症近年来被认为是ARMD发病的重要原因。而自噬过程也参与了ARMD的发病。正常的自噬是细胞自我保护以及维持稳态的一个重要途径,当自噬被阻断时,可加剧氧化应激损伤,导致ARMD的发展。补体激活与自噬过程的均衡调节是控制ARMD发展的重要手段。     

Expression and role of autophagy related protein p62 and LC3 in the retina in a rat model of acute ocular hypertension

AIM: To investigate the expression and possible role of the autophagy related protein p62 and LC3 in the retina based on a rat model of acute ocular hypertension.METHODS: Fifty rats were randomized into five groups: control group A, B, C, and D. Groups A to D all received normal saline perfusion into the anterior chamber with pressure of 80 mm Hg for one hour, and retina tissue was obtained at 6, 12, 24 and 48 h after perfusion respectively, to investigate the activation of autophagy following ischemiareperfusion. The distribution and semi-quantification of autophagy related protein p62 and LC3 in the retina were detected using immunohistochemistry technique. The expression level of these two proteins was evaluated using Western blot.RESULTS: The number of retinal ganglion cells(RGCs) decreased with increasing reperfusion time, and significant reduction in the retinal thickness was observed 48 h after perfusion. In normal adult rats, LC3 protein was mainly expressed in the ganglion cell layer(GCL), and p62 protein was expressed in the nerve fiber layer(NFL), GCL, inner plexiform layer(IPL), inner nuclear layer(INL) and outer plexiform layer(OPL). In comparison to the control group, the expression level of LC3-II was higher in all the experimental groups(P<0.05), with the peak expression at 12 h after reperfusion. Additionally, the expression level of p62 was higher in all the experimental groups than the control(P<0.05, except for group A), with the peak level occurred 24 h after reperfusion. CONCLUSION: Both p62 and LC3 show low level and uneven expression in the retina of normal adult rats. Acute ocular hypertension can lead to upregulation of LC3-II and p62 expression in the retina. Autophagy flux is damaged 12 h after reperfusion, potentially resulting in further loss of RGCs.     

调节自噬提高人肝癌Bel-7402/FU细胞株对5-氟尿嘧啶的敏感性

目的：探讨细胞自噬对肝癌Bel-7402／FU细胞5-氟尿嘧啶敏感性的影响。方法：选取Bel-7402、Bel-7402／FU细胞株,分为对照组、5-氟尿嘧啶组、自噬抑制剂3-甲基腺苷组以及5-氟尿嘧啶＋3-甲基腺苷组,MTT法了解抑制细胞自噬对肝癌5-氟尿嘧啶IC50值的影响;流式细胞术检测抑制细胞自噬对细胞凋亡的影响;GFP-LC3质粒转染观察细胞浆中GFP-LC3分布情况。结果：Bel-7402细胞株与Bel-7402／FU细胞株5-氟尿嘧啶IC50值分别为4．66、68．14μg／mL,凋亡率分别为13．809／6、1.09％。3-甲基腺苷联合5-氟尿嘧啶作用后Bel-7402细胞株与Bel-7402／FU细胞株的氟尿嘧啶IC50值分别为4．31、29．44μg／mL,凋亡率分别为13．82％、6．86％。在3-甲基腺苷作用下,Bel-7402／5-FU细胞株5-氟尿嘧啶诱导的点状样GFP-LC3的细胞数量减少。结论：抑制细胞自噬可降低Bel-7402／FU对5-氟尿嘧啶IC50值,诱导细胞凋亡,从而有效地逆转Bel-7402／FU对5-氟尿嘧啶耐药。     

Aliskiren ameliorates pressure overload-induced heart hypertrophy and fibrosis in mice

Aim:

Aliskiren (ALK) is a renin inhibitor that has been used in the treatment of hypertension. The aim of this study was to determine whether ALK could ameliorate pressure overload-induced heart hypertrophy and fibrosis, and to elucidate the mechanisms of action.

Methods:

Transverse aortic constriction (TAC) was performed in mice to induce heart pressure overload. ALK (150 mg·kg⁻¹·d⁻¹, po), the autophagy inhibitor 3-methyladenine (10 mg·kg⁻¹ per week, ip) or the PKCβI inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY333531","term_id":"1257370768","term_text":"LY333531"}}LY333531 (1 mg·kg⁻¹·d-¹, po) was administered to the mice for 4 weeks. Heart hypertrophy, fibrosis and function were evaluated based on echocardiography, histological and biochemical measurements. Mechanically stretched cardiomyocytes of rats were used for in vitro experiments. The levels of signaling proteins were measured using Western blotting, while the expression of the relevant genes was analyzed using real-time QRT-PCR.

Results:

TAC induced marked heart hypertrophy and fibrosis, accompanied by high levels of Ang II in plasma and heart, and by PKCβI/α and ERK1/2 phosphorylation in heart. Meanwhile, TAC induced autophagic responses in heart, i.e. increases in autophagic structures, expression of Atg5 and Atg16 L1 mRNAs and LC3-II and Beclin-1 proteins. These pathological alterations in TAC-mice were significantly ameliorated or blocked by ALK administration. In TAC-mice, 3-methyladenine administration also ameliorated heart hypertrophy, fibrosis and dysfunction, while {"type":"entrez-nucleotide","attrs":{"text":"LY333531","term_id":"1257370768","term_text":"LY333531"}}LY333531 administration inhibited ERK phosphorylation and autophagy in heart. In mechanically stretched cardiomyocytes, {"type":"entrez-protein","attrs":{"text":"CGP53353","term_id":"875191971","term_text":"CGP53353"}}CGP53353 (a PKCβI inhibitor) prevented ERK phosphorylation and autophagic responses, while U0126 (an ERK inhibitor) blocked autophagic responses.

Conclusion:

ALK ameliorates heart hypertrophy, fibrosis and dysfunction in the mouse model in setting of chronic pressure overload, via suppressing Ang II-PKCβI-ERK1/2-regulated autophagy.     

A long non‐coding RNA,GAS5, plays a critical role in the regulation of miR‐21 during osteoarthritis

Growth Arrest‐Specific 5 (GAS5) is known to negatively regulate cell survival and is aberrantly expressed in several cancers. The influence of GAS5 on osteoarthritis (OA) has not been determined. To address this, articular chondrocytes were isolated from relatively normal (Non‐OA) and clear OA regions (OA) of cartilage in total knee replacement (TKR) patients and biopsied normal cartilage. We found that GAS5 was up‐regulated in OA chondrocytes compared with Non‐OA and normal chondrocytes. The over‐expression of GAS5 increased the expression levels of several MMPs, such as MMP‐2, MMP‐3, MMP‐9, MMP‐13, and ADAMTS‐4; stimulated apoptosis; and suppressed autophagic responses. Furthermore, we subsequently identified miR‐21 as a regulator of GAS5 during OA pathogenesis. The expression level of miR‐21 was significantly reduced in OA patients, and the ectopic expression of GAS5 is capable of suppressing miR‐21 induction. Consistent with GAS5 experiments, the introduction of miR‐21 stimulated the apoptosis of chondrocytes and inhibited the expression levels of autophagic complexes, including LC‐3B. In vivo, we found that the introduction of miR‐21 into the cartilage of OA mice significantly stimulated cartilage destruction. Together, these results show that GAS5 contributes to the pathogenesis of OA by acting as a negative regulator of miR‐21 and thereby regulating cell survival. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1628–1635, 2014.     

Berberine attenuates adverse left ventricular remodeling and cardiac dysfunction after acute myocardial infarction in rats: Role of autophagy

The present study aimed to test the hypothesis that berberine, a plant‐derived anti‐oxidant, attenuates adverse left ventricular remodelling and improves cardiac function in a rat model of myocardial infarction (MI). Furthermore, the potential mechanisms that mediated the cardioprotective actions of berberine, in particular the effect on autophagy, were also investigated. Acute MI was induced by ligating the left anterior descending coronary artery of Sprague‐Dawley rats. Cardiac function was assessed by transthoracic echocardiography. The protein activity/levels of autophagy related to signalling pathways (e.g. LC‐3B, Beclin‐1) were measured in myocardial tissue by immunohistochemical staining and western blot. Four weeks after MI, berberine significantly prevented cardiac dysfunction and adverse cardiac remodelling. MI rats treated with low dose berberine (10 mg/kg per day) showed higher left ventricular ejection fraction and fractional shortening than those treated with high‐dose berberine (50 mg/kg per day). Both doses reduced interstitial fibrosis and post‐MI adverse cardiac remodelling. The cardioprotective action of berberine was associated with increased LC‐3B II and Beclin‐1 expressions. Furthermore, cardioprotection with berberine was potentially related to p38 MAPK inhibition and phospho‐Akt activation. The present in vivo study showed that berberine is effective in promoting autophagy, and subsequently attenuating left ventricular remodelling and cardiac dysfunction after MI. The potential underlying mechanism is augmentation of autophagy through inhibition of p38 MAPK and activation of phospho‐Akt signalling pathways.     

Fluorescent Protein-Based Autophagy Biosensors

Autophagy is an essential cellular process of self-degradation for dysfunctional or unnecessary cytosolic constituents and organelles. Dysregulation of autophagy is thus involved in various diseases such as neurodegenerative diseases. To investigate the complex process of autophagy, various biochemical, chemical assays, and imaging methods have been developed. Here we introduce various methods to study autophagy, in particular focusing on the review of designs, principles, and limitations of the fluorescent protein (FP)-based autophagy biosensors. Different physicochemical properties of FPs, such as pH-sensitivity, stability, brightness, spectral profile, and fluorescence resonance energy transfer (FRET), are considered to design autophagy biosensors. These FP-based biosensors allow for sensitive detection and real-time monitoring of autophagy progression in live cells with high spatiotemporal resolution. We also discuss future directions utilizing an optobiochemical strategy to investigate the in-depth mechanisms of autophagy. These cutting-edge technologies will further help us to develop the treatment strategies of autophagy-related diseases.     

Herpesvirus Regulation of Selective Autophagy

Selective autophagy has emerged as a key mechanism of quality and quantity control responsible for the autophagic degradation of specific subcellular organelles and materials. In addition, a specific type of selective autophagy (xenophagy) is also activated as a line of defense against invading intracellular pathogens, such as viruses. However, viruses have evolved strategies to counteract the host’s antiviral defense and even to activate some proviral types of selective autophagy, such as mitophagy, for their successful infection and replication. This review discusses the current knowledge on the regulation of selective autophagy by human herpesviruses.