MOE Joint International Research Laboratory of Animal Health and Food safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
Biomolecules. 2019 Nov 1;9(11):682. doi: 10.3390/biom9110682.
Many studies have focused on how autophagy plays an important role in intestinal homeostasis under pathological conditions. However, its role in the intestine during hibernation remains unclear. In the current study, we characterized in vivo up-regulation of autophagy in enterocytes of the small intestine of Chinese soft-shelled turtles during hibernation. Autophagy-specific markers were used to confirm the existence of autophagy in enterocytes through immunohistochemistry (IHC), immunofluorescence (IF), and immunoblotting. IHC staining indicated strong, positive immunoreactivity of the autophagy-related gene (ATG7), microtubule-associated protein light chain (LC3), and lysosomal-associated membrane protein 1 (LAMP1) within the mucosal surface during hibernation and poor expression during nonhibernation. IF staining results showed the opposite tendency for ATG7, LC3, and sequestosome 1 (p62). During hibernation ATG7 and LC3 showed strong, positive immunosignaling within the mucosal surface, while p62 showed strong, positive immunosignaling during nonhibernation. Similar findings were confirmed by immunoblotting. Moreover, the ultrastructural components of autophagy in enterocytes were revealed by transmission electron microscopy (TEM). During hibernation, the cumulative formation of phagophores and autophagosomes were closely associated with well-developed rough endoplasmic reticulum in enterocytes. These autophagosomes overlapped with lysosomes, multivesicular bodies, and degraded mitochondria to facilitate the formation of autophagolysosome, amphisomes, and mitophagy in enterocytes. Immunoblotting showed the expression level of PTEN-induced kinase 1 (PINK1), and adenosine monophosphate-activated protein kinase (AMPK) was enhanced during hibernation. Furthermore, the exosome secretion pathway of early-late endosomes and multivesicular bodies were closely linked with autophagosomes in enterocytes during hibernation. These findings suggest that the entrance into hibernation is a main challenge for reptiles to maintain homeostasis and cellular quality control in the intestine.
许多研究集中于自噬在病理条件下如何在肠道中发挥重要作用。然而，其在冬眠期间肠道中的作用尚不清楚。在本研究中，我们通过免疫组织化学（IHC）、免疫荧光（IF）和免疫印迹分析，鉴定了中华鳖在冬眠期间小肠上皮细胞中自噬的体内上调。自噬特异性标志物用于通过 IHC、IF 和免疫印迹确认上皮细胞中自噬的存在。IHC 染色显示，在冬眠期间，黏膜表面上自噬相关基因（ATG7）、微管相关蛋白轻链（LC3）和溶酶体相关膜蛋白 1（LAMP1）的阳性免疫反应强烈，而非冬眠时表达较弱。IF 染色结果显示 ATG7、LC3 和自噬体相关蛋白 1（p62）呈相反趋势。在冬眠期间，黏膜表面上 ATG7 和 LC3 呈强烈的阳性免疫信号，而 p62 在非冬眠时呈强烈的阳性免疫信号。免疫印迹也证实了这一发现。此外，透射电子显微镜（TEM）揭示了上皮细胞中自噬的超微结构成分。在冬眠期间，噬菌斑和自噬体的累积形成与上皮细胞中发达的粗面内质网密切相关。这些自噬体与溶酶体、多泡体和降解的线粒体重叠，有利于自噬溶酶体、双联体和噬线粒体在肠上皮细胞中的形成。免疫印迹显示，PTEN 诱导激酶 1（PINK1）和腺苷单磷酸激活蛋白激酶（AMPK）的表达水平在冬眠期间增强。此外，早期-晚期内体和多泡体的外泌体分泌途径在冬眠期间与上皮细胞中的自噬体密切相关。这些发现表明，进入冬眠是爬行动物在肠道中维持体内平衡和细胞质量控制的主要挑战。
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