Raised intracellular glucose concentrations reduce aggregation and cell death caused by mutant huntingtin exon 1 by decreasing mTOR phosphorylation and inducing autophagy

Huntington's disease is caused by a CAG trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract. This gain-of-function mutation is associated with huntingtin aggregation and cell death. Autophagy is an important clearance route for mutant huntingtin exon 1. While mammalian target of rapamycin (mTOR) is a key regulator of autophagy, the upstream modifiers of this process are poorly understood. Our previous expression profiling studies in HD cell models observed changes in four genes associated with glucose metabolism, including the GLUT1 glucose transporter. A role for intracellular glucose as a modulator for polyglutamine toxicity was suggested as cell death was reduced by GLUT1 overexpression. Here we show that the protective effect of GLUT1 is associated with decreased huntingtin exon 1 aggregation in cell models. Consistent with this result, we also observed reduced aggregation and enhanced clearance of mutant huntingtin when cells were cultured in raised glucose concentrations (8 g/l). These effects were mimicked by 8 g/l 2-deoxyglucose (2DOG) (transported, phosphorylated but not metabolized further), but not with 8 g/l 3-O-methyl glucose (transported but not metabolized further). Thus, this phenomenon is probably mediated by glucose-6-phosphate. Increased clearance of mutant huntingtin by raised glucose (8 g/l) and 2DOG correlated with increased autophagy and reduced phosphorylation of mTOR, S6K1 and Akt. Thus, raised intracellular glucose/glucose 6-phosphate levels reduce mutant huntingtin toxicity by increasing autophagy via mTOR and possibly Akt. As mTOR and Akt regulate a diversity of crucial cellular processes, our data also suggest a major new set of targets for intracellular glucose signalling.     

Rapamycin alleviates toxicity of different aggregate-prone proteins

Many neurodegenerative diseases are caused by intracellular, aggregate-prone proteins, including polyglutamine-expanded huntingtin in Huntington's disease (HD) and mutant tau in fronto-temporal dementia/tauopathy. Previously, we showed that rapamycin, an autophagy inducer, enhances mutant huntingtin fragment clearance and attenuated toxicity. Here we show much wider applications for this approach. Rapamycin enhances the autophagic clearance of different proteins with long polyglutamines and a polyalanine-expanded protein, and reduces their toxicity. Rapamycin also reduces toxicity in Drosophila expressing wild-type or mutant forms of tau and these effects can be accounted for by reductions in insoluble tau. Thus, our studies suggest that the scope for rapamycin as a potential therapeutic in aggregate diseases may be much broader than HD or even polyglutamine diseases.     

Accelerating the clearance of mutant huntingtin protein aggregates through autophagy induction by europium hydroxide nanorods

Autophagy is one of the well-known pathways to accelerate the clearance of protein aggregates, which contributes to the therapy of neurodegenerative diseases. Although there are numerous reports that demonstrate the induction of autophagy with small molecules including rapamycin, trehalose and lithium, however, there are few reports mentioning the clearance of aggregate-prone proteins through autophagy induction by nanoparticles. In the present article, we have demonstrated that europium hydroxide [Eu^III(OH)₃] nanorods can reduce huntingtin protein aggregation (EGFP-tagged huntingtin protein with 74 polyQ repeats), responsible for neurodegenerative diseases. Again, we have found that these nanorods induce authentic autophagy flux in different cell lines (Neuro 2a, PC12 and HeLa cells) through the expression of higher levels of characteristic autophagy marker protein LC3-II and degradation of selective autophagy substrate/cargo receptor p62/SQSTM1. Furthermore, depression of protein aggregation clearance through the autophagy blockade has also been observed by using specific inhibitors (wortmannin and chloroquine), indicating that autophagy is involved in the degradation of huntingtin protein aggregation. Since [Eu^III(OH)₃] nanorods can enhance the degradation of huntingtin protein aggregation via autophagy induction, we strongly believe that these nanorods would be useful for the development of therapeutic treatment strategies for various neurodegenerative diseases in near future using nanomedicine approach.     

Differential ERK activation during autophagy induced by europium hydroxide nanorods and trehalose: Maximum clearance of huntingtin aggregates through combined treatment

Accelerating the clearance of intracellular protein aggregates through elevation of autophagy represents a viable approach for the treatment of neurodegenerative diseases. In our earlier report, we have demonstrated the enhanced degradation of mutant huntingtin protein aggregates through autophagy process induced by europium hydroxide nanorods [EHNs: Eu^III(OH)₃], but the underlying molecular mechanism of EHNs mediated autophagy was unclear. The present report reveals that EHNs induced autophagy does not follow the classical AKT-mTOR and AMPK signaling pathways. The inhibition of ERK1/2 phosphorylation using the specific MEK inhibitor U0126 partially abrogates the autophagy as well as the clearance of mutant huntingtin protein aggregates mediated by EHNs suggesting that nanorods stimulate the activation of MEK/ERK1/2 signaling pathway during autophagy process. In contrast, another mTOR-independent autophagy inducer trehalose has been found to induce autophagy without activating ERK1/2 signaling pathway. Interestingly, the combined treatment of EHNs and trehalose leads to more degradation of mutant huntingtin protein aggregates than that obtained with single treatment of either nanorods or trehalose. Our results demonstrate the rational that further enhanced clearance of intracellular protein aggregates, needed for diverse neurodegenerative diseases, may be achieved through the combined treatment of two or more autophagy inducers, which stimulate autophagy through different signaling pathways.     

Role of autophagy and proteasome degradation pathways in apoptosis of PC12 cells overexpressing human α-synuclein

Parkinson's disease is a common neurodegenerative disease in the elderly. Its causes and mechanisms are not clearly understood. To explore the specific role of autophagy and the ubiquitin-proteasome pathway in apoptosis, a specific proteasome inhibitor and macroautophagy inhibitor and stimulator were selected to investigate pheochromocytoma (PC12) cell lines transfected with human mutant (A30P) and wild-type (WT) α-synuclein. The apoptosis ratio was assessed by flow cytometry. LC3, heat shock protein 70 (hsp70) and caspase-3 expression in cell culture were determined by Western blot. The hallmarks of apoptosis and autophagy were assessed with transmission electron microscopy. Compared to the control group or the rapamycin (autophagy stimulator) group, the apoptosis ratio in A30P and WT cells was significantly higher after treatment with inhibitors of the proteasome and macroautophagy. The results of Western blots for caspase-3 expression were similar to those of flow cytometry; hsp70 protein was significantly higher in the proteasome inhibitor group than in control, but in the autophagy inhibitor and stimulator groups, hsp70 was similar to control. These findings show that inhibition of the proteasome and autophagy promotes apoptosis, and the macroautophagy stimulator rapamycin reduces the apoptosis ratio. And inhibiting or stimulating autophagy has less impact on hsp70 than the proteasome pathway.     

Induction of autophagy in Cx3cr1+ mononuclear cells limits IL-23/IL-22 axis-mediated intestinal fibrosis

Intestinal fibrosis is an excessive proliferation of myofibroblasts and deposition of collagen, a condition frequently seen in Crohn's disease (CD). The mechanism underlying myofibroblast hyper-proliferation in CD needs to be better understood. In this report, we found that mTOR inhibitor rapamycin or mTOR deletion in CX3Cr1⁺ mononuclear phagocytes inhibits expression of interleukin (IL)−23, accompanied by reduced intestinal production of IL-22 and ameliorated fibrosis in the TNBS-induced fibrosis mouse model. This inhibition of IL-23 expression is associated with elevated autophagy activity. Ablating the autophagy gene Atg7 increases the expression of IL-23, leading to increased expression of IL-22 and increased fibrosis. Both induction of IL-22 and intestinal fibrosis occurred in RAG^−/− mice and depletion of innate lymphoid cells (ILCs) attenuates the fibrotic reaction, suggesting that the pro-fibrotic process is independent of T and B cells. Moreover, IL-22 facilitates the transformation of fibroblasts into myofibroblasts. Finally, the fibrotic reaction was attenuated upon neutralization of either IL-23 or IL-22. Altogether, this study elucidated a signaling cascade underlying intestinal fibrosis in which altered mTOR/autophagy in CX3Cr1⁺ mononuclear phagocytes up-regulates the IL-23/IL-22 axis, leading to an excessive fibrotic response. Thus, our findings suggest that this cascade could be a therapeutic target for alleviation of CD fibrosis.     

Autophagy induced by Alexander disease-mutant GFAP accumulation is regulated by p38/MAPK and mTOR signaling pathways

Glial fibrillary acidic protein (GFAP) is the principle intermediate filament (IF) protein in astrocytes. Mutations in the GFAP gene lead to Alexander disease (AxD), a rare, fatal neurological disorder characterized by the presence of abnormal astrocytes that contain GFAP protein aggregates, termed Rosenthal fibers (RFs), and the loss of myelin. All GFAP mutations cause the same histopathological defect, i.e. RFs, though little is known how the mutations affect protein accumulation as well as astrocyte function. In this study, we found that GFAP accumulation induces macroautophagy, a key clearance mechanism for prevention of aggregated proteins. This autophagic response is negatively regulated by mammalian target of rapamycin (mTOR). The activation of p38 MAPK by GFAP accumulation is in part responsible for the down-regulation of phosphorylated-mTOR and the subsequent activation of autophagy. Our study suggests that AxD mutant GFAP accumulation stimulates autophagy, in a manner regulated by p38 MAPK and mTOR signaling pathways. Autophagy, in turn, serves as a mechanism to reduce GFAP levels.     

IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux

Huntington's disease (HD), an inherited neurodegenerative disorder, is caused by an expansion of cytosine-adenine-guanine repeats in the huntingtin gene. The aggregation of mutant huntingtin (mtHTT) and striatal cell loss are representative features to cause uncontrolled movement and cognitive defect in HD. However, underlying mechanism of mtHTT aggregation and cell toxicity remains still elusive. Here, to find new genes modulating mtHTT aggregation, we performed cell-based functional screening using the cDNA expression library and isolated IRE1 gene, one of endoplasmic reticulum (ER) stress sensors. Ectopic expression of IRE1 led to its self-activation and accumulated detergent-resistant mtHTT aggregates. Treatment of neuronal cells with ER stress insults, tunicamycin and thapsigargin, increased mtHTT aggregation via IRE1 activation. The kinase activity of IRE1, but not the endoribonuclease activity, was necessary to stimulate mtHTT aggregation and increased death of neuronal cells, including SH-SY5Y and STHdhQ111/111 huntingtin knock-in striatal cells. Interestingly, ER stress impaired autophagy flux via IRE1-TRAF2 pathway, thus enhancing cellular accumulation of mtHTT. Atg5 deficiency in M5-7 cells increased mtHTT aggregation but blocked ER stress-induced mtHTT aggregation. Further, ER stress markers including p-IRE1 and autophagy markers such as p62 were up-regulated exclusively in the striatal tissues of HD mouse models and in HD patients. Moreover, down-regulation of IRE1 expression rescues the rough-eye phenotype by mtHTT in a HD fly model. These results suggest that IRE1 plays an essential role in ER stress-mediated aggregation of mtHTT via the inhibition of autophagy flux and thus neuronal toxicity of mtHTT aggregates in HD.     

α-Synuclein levels modulate Huntington's disease in mice

α-Synuclein and mutant huntingtin are the major constituents of the intracellular aggregates that characterize the pathology of Parkinson's disease (PD) and Huntington's disease (HD), respectively. α-Synuclein is likely to be a major contributor to PD, since overexpression of this protein resulting from genetic triplication is sufficient to cause human forms of PD. We have previously demonstrated that wild-type α-synuclein overexpression impairs macroautophagy in mammalian cells and in transgenic mice. Overexpression of human wild-type α-synuclein in cells and Drosophila models of HD worsens the disease phenotype. Here, we examined whether α-synuclein overexpression also worsens the HD phenotype in a mammalian system using two widely used N-terminal HD mouse models (R6/1 and N171-82Q). We also tested the effects of α-synuclein deletion in the same N-terminal HD mouse models, as well as assessed the effects of α-synuclein deletion on macroautophagy in mouse brains. We show that overexpression of wild-type α-synuclein in both mouse models of HD enhances the onset of tremors and has some influence on the rate of weight loss. On the other hand, α-synuclein deletion in both HD models increases autophagosome numbers and this is associated with a delayed onset of tremors and weight loss, two of the most prominent endophenotypes of the HD-like disease in mice. We have therefore established a functional link between these two aggregate-prone proteins in mammals and provide further support for the model that wild-type α-synuclein negatively regulates autophagy even at physiological levels.     

Autophagy activation: a novel mechanism of atorvastatin to protect mesenchymal stem cells from hypoxia and serum deprivation via AMP-activated protein kinase/mammalian target of rapamycin pathway

Autophagy is a complex "self-eating" process and could be utilized for cell survival under stresses. Statins, which could reduce apoptosis in mesenchymal stem cells (MSCs) during both ischemia and hypoxia/serum deprivation (H/SD), have been proved to induce autophagy in some cell lines. We have previously shown that atorvastatin (ATV) could regulate AMP-activated protein kinase (AMPK), a positive modulator of autophagy, in MSCs. Thus, we hypothesized that autophagy activation through AMPK and its downstream molecule mammalian target of rapamycin (mTOR) may be a novel mechanism of ATV to protect MSCs from apoptosis during H/SD. Here, we demonstrated that H/SD induced autophagy in MSCs significantly as identified by increasing acidic vesicular organelle-positive cells, type II of light chain 3 (LC3-II) expression, and autophagosome formation. The levels of H/SD-induced apoptosis were increased by autophagy inhibitor 3-methyladenine (3-MA) while decreased by rapamycin, an autophagic inducer. ATV further enhanced the autophagic activity observed in MSCs exposed to H/SD. Treatment with 3-MA attenuated ATV-induced autophagy and abrogated the protective effects of ATV on MSC apoptosis, while rapamycin failed to cause additional effects on either autophagy or apoptosis compared with ATV alone. The phosphorylation of AMPK was upregulated whereas the phosphorylation of mTOR was downregulated in ATV-treated MSCs, which were both attenuated by AMPK inhibitor compound C. Further, treatment with compound C reduced the ATV-induced autophagy in MSCs under H/SD. These data suggest that autophagy plays a protective role in H/SD-induced apoptosis of MSCs, and ATV could effectively activate autophagy via AMPK/mTOR pathway to enhance MSC survival during H/SD.     

Temsirolimus,the mTOR inhibitor,induces autophagy in adenoid cystic carcinoma: In vitro and in vivo

Temsirolimus acts as a mammalian target of rapamycin (mTOR)-dependent autophagic inhibitor. In order to clarify its effects and mechanisms on human salivary adenoid cystic carcinoma (ACC), we examined whether temsirolimus induced autophagy as the mTOR inhibitor in ACC, both in vitro and in vivo. In this study, MTT assay showed that the inhibition effect of temsirolimus assumed an obvious dose–response relationship on ACC-M cells, and the 50% inhibitory concentration (IC₅₀) approached 20 μmol/l; numerous autophagosomes were observed by the transmission electron microscopy (TEM) in temsirolimus treatment groups; notably, expression of LC3 and Beclin1 was significantly up-regulated by temsirolimus. More importantly, the xenograft model provided further evidence of temsirolimus-induced autophagy in vivo by inhibiting mTOR activation as well as up-regulation the expression of Beclin1. These results suggest that temsirolimus could act as an mTOR inhibitor to induce autophagy in adenoid cystic carcinoma both in vitro and in vivo.     

Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6K pathway

Robust production of type I interferon (IFN-alpha/beta) in plasmacytoid dendritic cells (pDCs) is crucial for antiviral immunity. Here we show involvement of the mammalian target of rapamycin (mTOR) pathway in regulating interferon production by pDCs. Inhibition of mTOR or its 'downstream' mediators, the p70 ribosomal S6 protein kinases p70S6K1 and p70S6K2, during pDC activation by Toll-like receptor 9 (TLR9) blocked the interaction of TLR9 with the adaptor MyD88 and subsequent activation of the interferon-regulatory factor IRF7, which resulted in impaired IFN-alpha/beta production. Microarray analysis confirmed that inhibition of mTOR by the immunosuppressive drug rapamycin suppressed antiviral and anti-inflammatory gene expression. Consistent with this, targeting rapamycin-encapsulated microparticles to antigen-presenting cells in vivo resulted in less IFN-alpha/beta production in response to CpG DNA or the yellow fever vaccine virus strain 17D. Thus, mTOR signaling is crucial in TLR-mediated IFN-alpha/beta responses by pDCs.     

EGCG通过mTOR通路拮抗低氧诱导的PC12细胞的凋亡与自噬

 目的:氯化钴（cobalt chloride, CoCl2）诱导的低氧具有神经毒性,可以诱导神经细胞的凋亡和自噬。茶多酚活性成分表没食子儿茶素没食子酸酯（epigallocatechin gallate, EGCG）具有一定的抗细胞凋亡和自噬的作用,但作用机理尚未完全阐明。近年来有研究报道,雷帕霉素靶蛋白（mammalian target of rapamycin,mTOR）通路参与了多种神经功能的调节,如神经细胞的分化成熟、抗氧化应激等。为此,我们用CoCl2诱导低氧引起细胞的凋亡与自噬,从mTOR通路探讨EGCG拮抗CoCl2诱导低氧引起细胞凋亡与自噬的作用机制。方法:研究使用Wes-tern blotting方法测定CoCl2以及EGCG处理后mTOR和自噬蛋白beclin-1的表达,使用ELISA法检测细胞caspase-3表达,CCK-8检测细胞活力,免疫荧光法观察LC-3在胞核内的表达。结果:CoCl2诱导低氧引起了细胞凋亡与自噬,而EGCG通过mTOR通路拮抗了CoCl2诱导低氧引起的细胞凋亡与自噬,阻断mTOR通路则逆转了EGCG对神经细胞的保护作用。结论: EGCG通过mTOR通路拮抗了CoCl2诱导的低氧引起的PC12细胞的凋亡与自噬。     

Mammalian target of rapamycin in spinal cord neurons mediates hypersensitivity induced by peripheral inflammation

mTOR, the mammalian target of rapamycin, is a serine–threonine kinase known to regulate cell proliferation and growth. mTOR has also been implicated in neuronal synaptic plasticity as well as in pain transmission in models of chemically induced and neuropathic pain. To date, the role of mTOR as a modulator of inflammatory pain has not been examined. In this study, we investigated the role of mTOR in Sprague–Dawley rats using the carrageenan model of inflammatory pain. mRNA of Ras homolog enriched in brain (Rheb), a GTPase that positively regulates mTOR activation, was significantly increased 2 h following carrageenan injection. Four hours after induction of inflammation phosphorylation (p) of p70S6 kinase (S6K), ribosomal protein S6 (S6) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) was increased, indicating mTOR activation. Inhibition of spinal mTOR with intrathecal (i.t.) injection of rapamycin (0.1–3 μg) led to a dose-dependent decrease in carrageenan-induced thermal hyperalgesia and a reduction of mechanical allodynia. In vitro studies confirmed rapamycin inhibition of the mTOR pathway. Carrageenan-induced activation of the mTOR pathway in rats was localized predominantly to dorsal horn neurons in the superficial lamina. Taken together, these data show that the mTOR pathway is activated in dorsal horn neurons during inflammatory pain, and that inhibition of spinal mTOR attenuates inflammation-induced thermal and tactile hypersensitivity. Hence, our study indicates that spinal mTOR is an important regulator of spinal sensitization and suggests that targeting mTOR may provide a new avenue for pain therapy.     

Regulation of vascular smooth muscle cell autophagy by DNA nanotube-conjugated mTOR siRNA

The efficient delivery of short interfering RNA (siRNA) is an enormous challenge in the field of gene therapy. Herein, we report a delivery nanosystem based on programmed DNA self-assembly mammalian target of rapamycin (mTOR) siRNA-loaded DNA nanotubes (DNA-NTs). We demonstrate that these siRNA-DNA-NTs can be effectively transfected into pulmonary arterial smooth muscle cells (PASMCs) via endocytosis; and that the loaded mTOR siRNA can induce obvious autophagy and inhibit cell growth under both normal and hypoxic conditions. Moreover, we found that mTOR siRNA can control the autophagy and proliferation of PASMCs under hypoxic condition, suggesting a potential therapeutic application for mTOR siRNA in diseases involving abnormal autophagy in PASMCs.     

Mammalian target of rapamycin is required for thrombopoietin-induced proliferation of megakaryocyte progenitors

Thrombopoietin (TPO) is a potent regulator of megakaryopoiesis and stimulates megakaryocyte (MK) progenitor expansion and MK differentiation. In this study, we show that TPO induces activation of the mammalian target of rapamycin (mTOR) signaling pathway, which plays a central role in translational regulation and is required for proliferation of MO7e cells and primary human MK progenitors. Treatment of MO7e cells, human CD34+, and primary MK cells with the mTOR inhibitor rapamycin inhibits TPO-induced cell cycling by reducing cells in S phase and blocking cells in G0/G1. Rapamycin markedly inhibits the clonogenic growth of MK progenitors with high proliferative capacity but does not reduce the formation of small MK colonies. Addition of rapamycin to MK suspension cultures reduces the number of MK cells, but inhibition of mTOR does not significantly affect expression of glycoproteins IIb/IIIa (CD41) and glycoprotein Ib (CD42), nuclear polyploidization levels, cell size, or cell survival. The downstream effectors of mTOR, p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), are phosphorylated by TPO in a rapamycin- and LY294002-sensitive manner. Part of the effect of the phosphatidyl inositol 3-kinase pathway in regulating megakaryopoiesis may be mediated by the mTOR/S6K/4E-BP1 pathway. In conclusion, these data demonstrate that the mTOR pathway is activated by TPO and plays a critical role in regulating proliferation of MK progenitors, without affecting differentiation or cell survival.     

Inositol monophosphatase regulates localization of synaptic components and behavior in the mature nervous system of C. elegans

Although recent studies have provided significant molecular insights into the establishment of neuronal polarity in vitro, evidence is lacking on the corresponding phenomena in vivo, including correct localization of synaptic components and the importance of this process for function of the nervous system as a whole. RIA interneurons act as a pivotal component of the neural circuit for thermotaxis behavior in the nematode Caenorhabditis elegans and provide a suitable model to investigate these issues, having a neurite clearly divided into pre- and post-synaptic regions. In a screen for thermotaxis mutants, we identified the gene ttx-7, which encodes myo-inositol monophosphatase (IMPase), an inositol-producing enzyme regarded as a bipolar disorder-relevant molecule for its lithium sensitivity. Here we show that mutations in ttx-7 cause defects in thermotaxis behavior and localization of synaptic proteins in RIA neurons in vivo. Both behavioral and localization defects in ttx-7 mutants were rescued by expression of IMPase in adults and by inositol application, and the same defects were mimicked by lithium treatment in wild-type animals. These results suggest that IMPase is required in central interneurons of the mature nervous system for correct localization of synaptic components and thus for normal behavior.     

Avian reovirus triggers autophagy in primary chicken fibroblast cells and Vero cells to promote virus production

Avian reovirus (ARV) is an important cause of disease in poultry. Although ARV is known to induce apoptosis in infected cells, the interaction between ARV and its target cells requires further elucidation. In this report, we show that the ARV isolate strain GX/2010/1 induces autophagy in both Vero and primary chicken embryonic fibroblast (CEF) cells based on the appearance of an increased number of double-membrane vesicles, the presence of GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) dot formation, and the elevated production of LC3II. We further demonstrate that the class I phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway contributes to autophagic induction by ARV infection. Moreover, treatment of ARV-infected cells with the autophagy inducer rapamycin increased viral yields, while inhibition of the autophagosomal pathway using chloroquine led to a decrease in virus production. Altogether, our studies strongly suggest that autophagy may play a critical role in determining viral yield during ARV infection.     

PI3K/Akt/mTOR信号通路在巨噬细胞自噬及动脉粥样硬化斑块不稳定中的作用

 目的：探讨磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路在巨噬细胞自体吞噬以及动脉粥样硬化斑块不稳定中的作用。方法：利用Akt抑制剂康士得（20 μmol/L）、mTOR抑制剂雷帕霉素（10 nmol/L）及mTOR-siRNA（30 nmol/L）体外处理小鼠RAW 264.7 巨噬细胞株 48 h后,透射电镜观察巨噬细胞自噬体的变化,细胞免疫荧光法及Western blotting法检测微管相关蛋白LC3-II表达,实时荧光定量qRT-PCR和Western blotting法检测Akt、mTOR及自噬相关蛋白Beclin 1的表达,ELISA检测巨噬细胞分泌炎症因子水平。体内实验中, 24只雄性新西兰兔给予球囊损伤+ 1%胆固醇喂养8周,然后随机分为对照组、康士得（1.0 mg·kg-1·d-1）组和雷帕霉素（0.5 mg·kg-1·d-1）组,每组8只,干预4周。血管内超声（IVUS）检测斑块的影像学特征,透射电镜观察斑块中巨噬细胞超微结构的改变,免疫荧光法检测微管相关蛋白LC3-II表达,免疫组织化学法检测巨噬细胞Akt和mTOR的蛋白表达。 结果：与对照组比较,康士得、雷帕霉素及mTOR-siRNA干预巨噬细胞后,透射电镜下观察到自噬体明显增多,微管相关蛋白LC3-II和自噬相关蛋白Beclin 1的表达水平明显上调,而Akt及mTOR 的mRNA及蛋白表达水平明显减少,巨噬细胞分泌的IL-10明显降低,而IFN-γ的分泌显著增加。体内实验： IVUS显示,与对照组比较,康士得组及雷帕霉素组的外弹性膜面积（EEMA）、斑块面积（PA）及斑块负荷（PB）明显减少,透射电镜下观察到巨噬细胞中自噬体增加,组织免疫荧光法示LC3-II明显增加,HE染色显示斑块纤维帽的厚度明显增加,内、中膜厚度显著减低,组织免疫组化染色显示巨噬细胞RAM-11及p-mTOR染色显著减少。结论：选择性抑制PI3K/Akt/mTOR信号通路能诱导巨噬细胞自噬,减少斑块巨噬细胞的浸润, 抑制炎症反应进而稳定动脉粥样硬化易损斑块。     

雷帕霉素激活自噬改善嘌呤霉素氨基核苷诱导的足细胞损伤

目的:通过观察雷帕霉素对PAN 诱导的足细胞损伤及自噬相关蛋白表达的影响,探讨自噬在雷帕霉素保护PAN 诱导的足细胞损伤中的作用及可能机制。方法:构建PAN 诱导的足细胞损伤模型,将足细胞分成对照组(Control 组),PAN 组(加入50 μg/ ml PAN),雷帕霉素组(RAP 组:分别加入100、200、300 ng/ ml 雷帕霉素),PAN+雷帕霉素组(PAN+RAP组:细胞在用含PAN 的培养液培养前1 h,分别用100、200、300 ng/ ml 雷帕霉素进行预处理1 h)。采用Annexin V/ PI 双染法检测细胞凋亡,透射电镜观察自噬小体,Western blot 检测LC3、p62、4EBP1、P70S6K、mTOR 蛋白表达。结果:与对照组比较,PAN组足细胞凋亡增加,自噬体减少,LC3域蛋白表达下调,p62 上调,mTOR、4EBP1、P70S6K 磷酸化水平上调;与PAN 组比较,PAN+RAP 组足细胞凋亡率下降,自噬体增加,LC3域蛋白表达上调,p62 下调,mTOR、4EBP1、P70S6K 磷酸化水平下调。结论:PAN 可以抑制足细胞自噬,促进足细胞凋亡;雷帕霉素可通过激活自噬改善PAN 诱导的足细胞损伤,这种作用可能与雷帕霉素抑制mTOR/4EBP1、P70S6K 信号通路有关。