Universidade Federal de São Paulo - UNIFESP, Escola Paulista de Medicina - EPM, Departamento de Morfologia e Genética, Disciplina de Histologia e Biologia Estrutural, São Paulo, SP, Brazil.
Universidade Federal de São Paulo - UNIFESP, Escola Paulista de Medicina - EPM, Departmento de Ginecologia, São Paulo, SP, Brazil.
Biomed Pharmacother. 2018 Feb;98:406-415. doi: 10.1016/j.biopha.2017.12.089. Epub 2017 Dec 27.
Estrogen maintains osteocyte viability, whereas its deficiency induces osteocyte apoptosis. As autophagy is important for osteocyte viability, we hypothesized whether the anti-apoptotic effect of estrogen is related to autophagy in osteocytes. Thirty adult female rats were sham-operated (SHAM) or ovariectomized (OVX). After three weeks, twelve rats of SHAM and OVX groups were killed before treatment (basal period), whereas the remaining rats received estrogen (OVXE) or vehicle (OVX) for 45 days. Fragments of maxilla containing alveolar process of the first molars were embedded in paraffin or Araldite. Paraffin-sections were stained with hematoxylin/eosin for histomorphometry, or subjected to the silver impregnation method for morphological analysis of osteocyte cytoplasmic processes. Autophagy was analyzed by immunohistochemical detections of beclin-1, MAP-LC3α and p62, whereas apoptosis was evaluated by immunohistochemical detections of cleaved caspase-3 and BAX, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) method and by ultrastructural analysis. Araldite-semithin sections were subjected to the Sudan-black method for detection of lipids. OVX-basal group showed high frequency of caspase-3-, TUNEL- and p62-positive osteocytes accompanied with low frequency of beclin-1- and MAP-LC3α-positive osteocytes. At 45 days, OVXE group exhibited higher number of osteocytes, higher frequency of beclin-1- and MAP-LC3α-positive osteocytes, and lower frequency of caspase-3, BAX-, TUNEL- and p62-positive osteocytes than OVX group. Significant reduction in bone area was observed in the OVX compared to OVXE and SHAM groups. The highest frequency of Sudan-Black-positive osteocytes and osteocytes with scarce cytoplasmic processes, or showing apoptotic features were mainly observed in OVX groups. Our results indicate that estrogen deficiency decreases autophagy and increases apoptosis, whereas estrogen replacement enhances osteocyte viability by inhibiting apoptosis and maintaining autophagy in alveolar process osteocytes. These results suggest that the anti-apoptotic effect of estrogen may be, at least in part, related to autophagy regulation in osteocytes.
雌激素维持破骨细胞的活力，而其缺乏则诱导破骨细胞凋亡。由于自噬对破骨细胞的活力很重要，我们假设雌激素的抗凋亡作用是否与破骨细胞中的自噬有关。30 只成年雌性大鼠行假手术（SHAM）或卵巢切除术（OVX）。3 周后，12 只 SHAM 和 OVX 组大鼠在治疗前（基础期）被处死，而其余大鼠接受雌激素（OVXE）或载体（OVX）治疗 45 天。含有第一磨牙牙槽突的上颌骨片段包埋在石蜡或芳醛中。石蜡切片用苏木精/伊红染色进行组织形态计量学分析，或用银浸法进行破骨细胞细胞质突起的形态学分析。通过免疫组化检测 beclin-1、MAP-LC3α 和 p62 分析自噬，通过免疫组化检测 cleaved caspase-3 和 BAX、TUNEL（末端脱氧核苷酸转移酶 dUTP 缺口末端标记）法和超微结构分析评估凋亡。芳醛半薄切片用苏丹黑法检测脂质。OVX 基础组显示 caspase-3、TUNEL 和 p62 阳性破骨细胞的频率较高，而 beclin-1 和 MAP-LC3α 阳性破骨细胞的频率较低。45 天时，OVXE 组破骨细胞数量较多，beclin-1 和 MAP-LC3α 阳性破骨细胞频率较高，caspase-3、BAX、TUNEL 和 p62 阳性破骨细胞频率较低，与 OVX 组相比。与 OVXE 和 SHAM 组相比，OVX 组的骨面积明显减少。苏丹黑阳性破骨细胞和细胞质突起稀少或表现出凋亡特征的破骨细胞的频率最高，主要见于 OVX 组。我们的结果表明，雌激素缺乏会降低自噬并增加凋亡，而雌激素替代通过抑制凋亡和维持破骨细胞中的自噬来增强破骨细胞的活力。这些结果表明，雌激素的抗凋亡作用可能至少部分与破骨细胞中的自噬调节有关。
