Connecting chaperone-mediated autophagy dysfunction to cellular senescence

Chaperone-mediated autophagy (CMA) is one of the main pathways of the lysosome-autophagy proteolytic system. It regulates different cellular process through the selective degradation of cytosolic proteins. In ageing, the function of CMA is impaired causing an inefficient stress response and the accumulation of damaged, oxidized or misfolded proteins, which is associated with numerous age-related diseases. Deficient protein degradation alters cellular proteostasis and activates signaling pathways that culminate in the induction of cellular senescence, whose accumulation is a typical feature of ageing. However, the relationship between CMA activity and cellular senescence has been poorly studied. Here, we review and integrate evidence showing that CMA dysfunction correlates with the acquisition of many hallmarks of cellular senescence and propose that loss of CMA function during aging promotes cellular senescence.     

Angiotensin II desensitization and Ca2+ and Na+ fluxes in vascular smooth muscle cells

The role of ion fluxes in angiotensin II (AII) desensitization (tachyphylaxis) was investigated by studying Na⁺ and Ca²⁺ translocation in cultured vascular smooth muscle cells from the rat aorta. The effects of AII were compared to those of [1-sarcosine]-AII (Sar¹-AII), an analogue which also induces tachyphylaxis, and [2-lysine]-AII (Lys²-AII), an analogue that does not show this property. Maximally effective concentrations of the three peptides induced a rapid and transient increase in ⁴⁵Ca²⁺ efflux, a rapid and sustained decrease in total cell Ca²⁺ and an increased Na⁺ permeability. Repeated treatments, at short intervals, with either of the three peptides abolished the effect on Ca²⁺ efflux, and this desensitization was slowly reversible. A 30-min rest period was sufficient for full recovery of the response of cells that were desensitized by Lys²AII, whereas the recovery from AII or Sar¹AII-desensitization was still not complete after 60 min. Our results suggest that the difference in the behaviour of the tachyphylactic AII and Sar¹-AII and the non-tachyphylactic Lys²-AII lays not in the production of different signals upon binding to the receptor, but in a difference in the hormone-receptor interaction itself.     

Up-regulation of autophagy and apoptosis of cochlear hair cells in mouse models for deafness

IntroductionHearing loss is one of the most common sensory disorders. Recent findings have shown that the apoptotic program and autophagy are related to hearing loss. The aim of the study was to explore the effects of noise and cisplatin exposure on apoptosis and autophagy in the hair cells of the cochleae.Material and methodsC57BL/6 mice were randomly divided into 3 groups (n = 10 for each): the control group, the noise model group and the cisplatin model group. Auditory brainstem response (ABR) measurements were used to detect the hearing thresholds. TUNEL assay was used to evaluate cell apoptosis. Western blot and immunofluorescence were performed to examine the apoptosis- and autophagy-related proteins.ResultsThe mice exhibited substantial hearing loss after noise and cisplatin exposure. Additionally, more TUNEL positive cells were observed in the mice after noise and cisplatin exposure compared with the control group. Moreover, the protein expression levels of Beclin-1, LC3-II, Bax and cleaved caspase-3 were significantly increased, while the expression of Bcl-2 was notably decreased in the cochlea after noise (p = 0.0278, 0.0075, 0.0142, 0.0158, 0.0131 respectively) and cisplatin (p = 0.0220, 0.0075, 0.0024, 0.0161, 0.0452 respectively) exposure compared with the control group. Besides, the ratio of LC3-II/LC3-I was substantially higher in the mice treated by cisplatin (p = 0.0046) and noise (p = 0.0220) compared with the control group.ConclusionsOur findings demonstrated for the first time that noise and cisplatin exposure promoted apoptosis and autophagy in the hair cells of the cochleae. This study provides new insights into the mechanisms of noise- or cisplatin-induced hearing loss.     

TFDP3对前列腺癌LNCaP细胞自噬及凋亡调控作用的初步研究

目的:研究TFDP3对前列腺癌LNCaP细胞自噬及凋亡作用的影响,以及探讨TFDP3与E2F1相互作用后对前列腺癌LNCaP细胞自噬及凋亡的调控作用。方法:采用重组质粒pcDNA3.1-TFDP3,pCMV-E2F1-HA分别转染LNCaP细胞,并设空载体对照组。转染24 h后提取细胞总RNA和蛋白,以实时定量RT-PCR检测TFDP3、E2F1、以及LC3B基因表达的变化,Western blot方法检测自噬相关基因(LC3B)蛋白表达水平的变化。并采用流式细胞仪检测细胞凋亡的变化。结果:TFDP3可以诱导LNCaP细胞中自噬基因LC3B的表达,并且这种作用可以受到E2F1的抑制;TFDP3可以抑制E2F1诱导的细胞凋亡。结论:TFDP3可以诱导LNCaP细胞中自噬基因LC3B的表达,可以抑制E2F1诱导的细胞凋亡,提示TFDP3在前列腺癌细胞中发挥着重要的调控作用。     

C5a induces caspase‐dependent apoptosis in brain vascular endothelial cells in experimental lupus

Blood–brain barrier (BBB) dysfunction complicates central nervous system lupus, an important aspect of systemic lupus erythematosus. To gain insight into the underlying mechanism, vascular corrosion casts of brain were generated from the lupus mouse model, MRL/lpr mice and the MRL/MpJ congenic controls. Scanning electron microscopy of the casts showed loss of vascular endothelial cells in lupus mice compared with controls. Immunostaining revealed a significant increase in caspase 3 expression in the brain vascular endothelial cells, which suggests that apoptosis could be an important mechanism causing cell loss, and thereby loss of BBB integrity. Complement activation occurs in lupus resulting in increased generation of circulating C5a, which caused the endothelial layer to become ‘leaky’. In this study, we show that C5a and lupus serum induced apoptosis in cultured human brain microvascular endothelial cells (HBMVECs), whereas selective C5a receptor 1 (C5aR1) antagonist reduced apoptosis in these cells, demonstrating C5a/C5aR1‐dependence. Gene expression of initiator caspases, caspase 1 and caspase 8, and pro‐apoptotic proteins death‐associated protein kinase 1, Fas‐associated protein (FADD), cell death‐inducing DNA fragmentation factor 45 000 MW subunit A‐like effector B (CIDEB) and BCL2‐associated X protein were increased in HBMVECs treated with lupus serum or C5a, indicating that both the intrinsic and extrinsic apoptotic pathways could be critical mediators of brain endothelial cell apoptosis in this setting. Overall, our findings suggest that C5a/C5aR1 signalling induces apoptosis through activation of FADD, caspase 8/3 and CIDEB in brain endothelial cells in lupus. Further elucidation of the underlying apoptotic mechanisms mediating the reduced endothelial cell number is important in establishing the potential therapeutic effectiveness of C5aR1 inhibition that could prevent and/or reduce BBB alterations and preserve the physiological function of BBB in central nervous system lupus.     

Induction of similar features of apoptosis in human and bovine vascular endothelial cells treated by 7-ketocholesterol

Cholesterol oxides have numerous cytotoxic effects and those oxidized in the C7 position have been shown to induce apoptosis in bovine aortic endothelial cells (BAEC). The aim of the present study was to determine whether apoptosis also occurs in human vascular endothelial cells (HUVEC) treated with 7-ketocholesterol. To this end, cultured BAEC and HUVEC were incubated for 48 h with 7-ketocholesterol (concentration range 5–80 μg/ml) and the characteristics of cell death were assessed by various methods: counting of adherent and non-adherent cells; analysis of DNA fragmentation pattern; and morphological study by light, fluorescence, and electron microscopy. The 7-ketocholesterol treatment was accompanied by a decrease in the number of adherent cells and an increase in the number of non-adherent cells. Apoptotic cells, recognized by fragmented and/or condensed nuclei after staining with Hoechst 33342 or Giemsa, were mainly detected among non-adherent cells, and agarose gel electrophoresis revealed a typical internucleosomal DNA fragmentation among 7-ketocholesterol-treated cells. The DNA fragmentation was no longer detected when HUVEC and BAEC were simultaneously incubated with 0·5 mmol/l zinc chloride, which is known to inhibit Ca²⁺/Mg²⁺-dependent endonucleases. Finally, the ultrastructural abnormalities observed by electron microscopy in both 7-ketocholesterol-treated HUVEC and BAEC were remarkably similar and were mainly characterized by condensed chromatin, altered mitochondria, disturbed organization of the cytoskeleton, and vacuoles containing myelin figures and/or cell debris; apoptotic bodies were also frequently detected. It is concluded that 7-ketocholesterol constitutes a potent inducer of apoptosis in endothelial vascular cells of both bovine and human origin, suggesting that cholesterol oxides may be involved in the early steps of the atherosclerotic process in humans. © 1997 John Wiley & Sons, Ltd.     

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.     

Angiotensin II and fluid ingestion in old rats

Fluid ingestion was studied in Fischer 344/Brown Norway F1 rats aged 3, 12, 20, and 24 months of age. There was an age-related decrease in fluid ingestion when fluid intake was measured over 24 h. After water deprivation, 24- and 20-month-old rats drank less than 3- and 12-month-old rats. Twelve, 20-, and 24-month-old rats had less fluid intake associated with food deprivation than did 3-month-old rats. Three month old rats drank more fluid after angiotensin II than did 12-, 20-, and 24-month-old rats when expressed as fluid intake per kg body weight. These studies confirm that the rat is a reasonable model to study age-related hypodipsia.     

组蛋白去乙酰化酶抑制剂TSA诱导人乳腺癌细胞凋亡及自噬

目的 探讨组蛋白去乙酰化酶抑制剂曲古霉素(TSA)对人乳腺癌细胞凋亡及自噬的作用及其可能的分子机制。方法采用MTT法检测T47D细胞的增殖能力;流式细胞术检测细胞凋亡的变化;Western blot法检测凋亡及自噬相关蛋白的表达。结果 TSA对人乳腺癌细胞T47D具有增殖抑制作用(P0.05);TSA诱导T47D细胞发生凋亡,下调BCL-2/Bax比值,上调Caspase-3的表达(P0.05);同时自噬相关蛋白LC3B及Beclin-1的表达也明显增加(P0.05,P0.01)。结论 TSA体外能抑制乳腺癌细胞的增殖,其机制与诱导细胞凋亡和自噬作用有关。     

血管紧张素Ⅱ 1型受体自身抗体通过上调自噬诱导INS-1胰岛β细胞凋亡

目的:本研究旨在探究血管紧张素Ⅱ1型受体自身抗体(AT1-AA)能否引起胰岛β细胞的凋亡,自噬是否参与其中以及其所发挥的作用。方法:10-6mol/L AT1-AA处理INS-1细胞24 h后,用流式细胞术、Western blot及Hoechst 33258染色检测细胞的凋亡水平;Western blot检测自噬相关蛋白LC3和beclin 1的蛋白水平。使用血管紧张素Ⅱ1型受体拮抗剂替米沙坦以及经典的自噬抑制剂3-甲基腺嘌呤(3-MA)预处理细胞1 h之后再加入AT1-AA处理24 h,检测细胞凋亡、自噬及存活率的变化情况。结果:10-6mol/L AT1-AA处理INS-1细胞24 h可明显降低细胞存活率(P0.05)。与阴性Ig G对照组相比,AT1-AA分别处理细胞12 h、24 h和36 h后细胞凋亡水平明显增高(P0.05);此外,LC3及beclin 1的蛋白水平也随处理时间的延长逐渐升高,且凋亡和自噬水平的升高均可被替米沙坦所阻滞。使用自噬抑制剂3-MA预处理之后,细胞的凋亡率较单独给予AT1-AA处理组明显降低(P0.05)。结论:AT1-AA可通过血管紧张素Ⅱ1型受体上调自噬来诱导INS-1胰岛β细胞凋亡。     

缺氧诱导因子-1α参与缺氧条件下小胶质细胞的自噬和凋亡

目的:探讨缺氧诱导因子-1α(hypoxia-inducible factor-1 alpha,HIF-1α)在缺氧诱导小胶质细胞自噬和凋亡中的作用。方法:体外培养大鼠小胶质细胞,缺氧处理后利用Iba1免疫组织化学法标记小胶质细胞;用RT-PCR检测缺氧后HIF-1αm RNA的表达;Western Blot检测HIF-1α和自噬标记物LC3的蛋白表达;TUNEL染色法检测细胞凋亡。针对大鼠HIF-1α基因序列构建特异的si RNA并转染小胶质细胞,检测缺氧后LC3表达与细胞凋亡的改变。结果:与对照组相比,缺氧后Iba1表达明显增强、HIF-1α和自噬标记物LC3-II表达上升(P0.05)、衡量细胞自噬水平的LC-II/LC3-I比值增高(P0.05),TUNEL+细胞百分数(35.6±5.9%)较对照组(6.8±1.2%)比较显著增加(P0.05);沉默HIF-1α表达后,LC3-II蛋白和LC-II/LC3-I比值均降低(P0.05)、TUNEL+细胞百分数(23.0±0.8%)较对照组(40.1±4.5%)比较亦显著减少(P0.05)。结论:HIF-1α参与缺氧条件下小胶质细胞的自噬和凋亡。     

Toll样受体4介导的自噬减轻糖基化高密度脂蛋白诱导的血管内皮细胞凋亡

目的:探讨自噬对糖基化高密度脂蛋白(glycosylated high-density lipoprotein,gly-HDL)所致的血管内皮细胞凋亡的影响及其分子机制。方法:体外培养人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),分别与100 mg/L HDL和不同浓度(25、50和100 mg/L)gly-HDL共同孵育24 h;另再培养HUVECs给予1μmol/L自噬诱导剂雷帕霉素或2 mmol/L自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)预处理1 h,或5 mg/L抗Toll样受体4(Toll-like receptor 4,TLR4)单克隆中和抗体预处理30 min,再与gly-HDL(100 mg/L)共同孵育24 h。采用MTT法检测细胞活力,Annexin V-FITC/PI双染法检测细胞凋亡情况,试剂盒测定培养液中乳酸脱氢酶(lactate dehydrogenase,LDH)活性,采用Western blot技术检测自噬标志分子beclin-1和微管相关蛋白1轻链3-Ⅱ(microtubule-associated protein 1 light chain 3-Ⅱ,LC3-Ⅱ)、内质网应激凋亡途径关键分子caspase-12及TLR4的表达变化,采用激光共聚焦显微镜观测细胞内LC3的变化。结果:经gly-HDL处理的HUVECs活力下降,LDH漏出和细胞凋亡显著增加(P<0.01),且caspase-12被激活(P<0.05);雷帕霉素预处理HUVECs后,gly-HDL对细胞的损伤作用和对caspase-12的活化作用减弱(P<0.05);而3-MA预处理HUVECs后,gly-HDL对细胞的损伤作用和对caspase-12的活化作用则进一步加强(P<0.05)。gly-HDL显著上调TLR4的表达,并触发自噬反应,表现为beclin-1和LC3-Ⅱ表达上调及LC3显著颗粒化,且呈浓度依赖性(P<0.05);而抗TLR4单克隆中和抗体预处理可显著抑制gly-HDL所诱导的beclin-1上调和LC3颗粒化(P<0.01)。结论:TLR4介导gly-HDL对HUVECs自噬的诱导作用,而一定程度的自噬可通过抑制caspase-12活化减轻gly-HDL所诱导的HUVECs凋亡。     

p53凋亡刺激蛋白家族对自噬调控作用的研究进展

p53凋亡刺激蛋白家族(apoptosis stimulating proteins of p53 family,ASPPs)由ASPP1、ASPP2和iASPP (inhibitory member of the ASPP family)3个成员组成,该家族可通过与p53家族(p53/p63/p73)结合,调控细胞凋亡。自噬是细胞通过溶酶体溶解并回收利用胞内物质,藉此以维持自我稳态的一种方式。目前认为,自噬功能紊乱与肿瘤、神经退行性病变等多种疾病的发生和发展密切相关。本综述总结了近期关于ASPPs对自噬调控作用的研究进展,这些研究证明ASPPs能调控细胞内的自噬水平,并提示ASPPs可能成为多种疾病的潜在治疗靶点。