Gorbunov Nikolai V, Kiang Juliann G
Armed Forces Radiobiology Research Institute, Bethesda, MD 20889-5603, USA.
J Pathol. 2009 Oct;219(2):242-52. doi: 10.1002/path.2591.
Macroautophagy (mAG) is a lysosomal mechanism of degradation of cell self-constituents damaged due to variety of stress factors, including ionizing irradiation. Activation of mAG requires expression of mAG protein Atg8 (LC3) and conversion of its form I (LC3-I) to form II (LC3-II), mediated by redox-sensitive Atg4 protease. We have demonstrated upregulation of this pathway in the innate host defense Paneth cells of the small intestine (SI) due to ionizing irradiation and correlation of this effect with induction of pro-oxidant inducible nitric oxide synthase (iNOS). CD2F1 mice were exposed to 9.25 Gy gamma-ionizing irradiation. Small intestinal specimens were collected during 7 days after ionizing irradiation. Assessment of ionizing irradiation-associated alterations in small intestinal crypt and villus cells and activation of the mAG pathway was conducted using microscopical and biochemical techniques. Analysis of iNOS protein and the associated formation of nitrites and lipid peroxidation products was performed using immunoblotting and biochemical analysis, and revealed increases in iNOS protein, nitrate levels and oxidative stress at day 1 following ionizing irradiation. Increase in immunoreactivity of LC3 protein in the crypt cells was observed at day 7 following ionizing irradiation. This effect predominantly occurred in the CD15-positive Paneth cells and was associated with accumulation of LC3-II isoform. The formation of autophagosomes in Paneth cells was confirmed by transmission electron microscopy (TEM). Up-regulation of LC3 pathway in the irradiated SI was accompanied by a decreased protein-protein interaction between LC3 and chaperone heat shock protein 70. A high-level of LC3-immunoreactivity in vacuole-shaped structures was spatially co-localized with immunoreactivity of 3-nitro-tyrosine. The observed effects were diminished in iNOS knockout B6.129P2-NOS2(tm1Lau)/J mice subjected to the same treatments. We postulate that the observed up-regulation of mAG in the irradiated small intestine is at least in part mediated by the iNOS signalling mechanism.
巨自噬（mAG）是一种溶酶体机制，用于降解因包括电离辐射在内的多种应激因素而受损的细胞自身成分。mAG的激活需要mAG蛋白Atg8（LC3）的表达及其形式I（LC3-I）向形式II（LC3-II）的转化，这由氧化还原敏感的Atg4蛋白酶介导。我们已经证明，由于电离辐射，小肠（SI）的先天性宿主防御潘氏细胞中该途径上调，并且这种效应与促氧化剂诱导型一氧化氮合酶（iNOS）的诱导相关。将CD2F1小鼠暴露于9.25 Gy的γ电离辐射。在电离辐射后的7天内收集小肠标本。使用显微镜和生化技术评估小肠隐窝和绒毛细胞中与电离辐射相关的变化以及mAG途径的激活。使用免疫印迹和生化分析对iNOS蛋白以及亚硝酸盐和脂质过氧化产物的相关形成进行分析，结果显示在电离辐射后的第1天，iNOS蛋白、硝酸盐水平和氧化应激增加。在电离辐射后的第7天，观察到隐窝细胞中LC3蛋白的免疫反应性增加。这种效应主要发生在CD15阳性的潘氏细胞中，并与LC3-II异构体的积累有关。通过透射电子显微镜（TEM）证实了潘氏细胞中自噬体的形成。照射后的SI中LC3途径的上调伴随着LC3与伴侣热休克蛋白70之间蛋白质-蛋白质相互作用的减少。液泡状结构中高水平的LC3免疫反应性与3-硝基酪氨酸的免疫反应性在空间上共定位。在接受相同处理的iNOS基因敲除B6.129P2-NOS2(tm1Lau)/J小鼠中，观察到的效应减弱。我们推测，在照射后的小肠中观察到的mAG上调至少部分由iNOS信号机制介导。
