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| 雌激素缺乏上调TNF-α促发卵巢切除大鼠骨细胞程序性坏死 |
| Estrogen deficiency promotes osteocyte necroptosis in OVX rats by up-regulating TNF-α |
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| DOI：10.3969/j.issn.1006.7108.2017.11.007 |
| 中文关键词: 雌激素 肿瘤坏死因子α 程序性坏死 骨细胞 骨质疏松 大鼠 动物实验 |
| 英文关键词:Tumor necrosis factor-alpha Necroptosis Osteocytes Osteoporosis Rats Animal experimentation |
| 基金项目:海南省自然科学基金项目（817326) |
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| 中文摘要: |
| 目的 探讨雌激素缺乏对去卵巢骨质疏松大鼠骨细胞程序性坏死的影响及其发生机制。方法60只雌性SD大鼠随机分为Control组（12只）和OVX组(48只），分别行假手术或OVX术;再将48只OVX大鼠分为OVX + vehicle、OVX + Nec-l、 OVX + Z-VAD和OVX +E2组，每组12只。Micro-CT扫描评价胫骨近端骨量改变；免疫组化法测定骨细胞TNF-α、RIP1、RIP3 的表达情况，并计算各组阳性细胞百分率;用透射电镜鉴定细胞形态学变化;Western blot检测程序性坏死下游信号通路蛋白 MLKL、Drp1的表达情况。结果 OVX + vehicle组大鼠胫骨近端出现明显的骨量丢失，即OVX + vehicle组Tb. Th、BV/TV、 Tb. N 小于 Control 组(P <0. 01)；而 OVX + vehicle 组 Tb. Sp 大于 Control 组(P <0. 01)。TNF-α、RIP1、RIP3 蛋白在骨细胞胞浆中均有表达;在OVX + vehicle组，TNF-α、RIP1、RIP3阳性骨细胞百分率较Control组显著增多（P < 0. 01)，在OVX + vehicle 组可见大量坏死样结构的骨细胞。应用Nec-1治疗后可有效抑制RIP1蛋白表达，而Z-VAD对RIP1、RIP3蛋白表达无影响， 在OVX + Nec-1组未见到坏死样结构的骨细胞。在OVX + vehicle组，Western blot结果显示MLKL、Drp1蛋白表达较Control组显著增多（P <0.01)，应用Nec-1治疗后可有效抑制MLKL、Drp1蛋白表达，而Z-VAD对MLKL、Drp1蛋白表达无影响。结论 雌激素缺乏可能是通过上调TNF-α表达而促发骨细胞发生程序性坏死的，MLKL、Drp1可能是骨细胞程序性坏死的下游关键信号分子。 |
| 英文摘要: |
| Objective To investigate the effect of estrogen deficiency on the necroptosis of osteocytes in ovariectomized rats and to explore the relevant mechanism. Methods Sixty female Sprague-Dawley rats were randomly assigned to the control group (n = 12) and the ovariectomized (OVX) group (n =48). Rats were subjected to ovariectomy in the OVX group and sham surgery in the control group. Then，forty-eight rats in the OVX group were assigned into the OVX + vehicle group，the OVX + Nec-1 group，the OVX + Z-VAD group，and the OVX + E2 group (n = 12 in each group). Bone mass of proximal tibia was evaluated with micro-CT scan. Immunohistochemical staining and image analysis were used to detect the number of the positive osteocytes of TNF-α，RIP1，RIP3，and the percentage of positive osteocytes in proximal tibia. The morphological feature of the osteocytes was observed with transmission electron microscopy (TEM). The expression of MLKL and Drp1 in osteocytes was detected using Western blotting. Result Significant bone loss was found in the proximal tibia of rats in the OVX + vehicle group. Trabecular bone thickness (TB. Th)，bone volume / total volume (BV/TV)，and trabecular number (TB. N) were all significant lower (P <0. 01) in the OVX + vehicle group than those in the control group. Trabecular bone separation (TB. SP) was significant greater (P <0. 01) in the OVX + vehicle group than that in the control group. TNF-α，RIP1，and RIP3 protein was expressed in the osteocyte cytoplasm.
The percentage of TNF-α, RIP1, and RIP3 positive osteocytes was significantly higher (P <0. 01) in the OVX + vehicle group than that in the control group. In the OVX + vehicle group, a lot of osteocytes showed typical necroptotic morphological features. Nec-1 effectively down-regulated the protein levels of RIP1 and RIP3. However, Z-VAD did not reduce levels of RIP1 and RIP3. Obvious necroptotic cells were not found in the Nec-1 treatment group. Western blotting result revealed that the protein levels of MLKL and Drp1 in osteocytes were higher in the OVX + vehicle group than those in the control group (P < 0. 01). Nec-1 treatment significantly reduced the protein levels of MLKL and Drp1. However, Z-VAD did not reduce levels of MLKL and Drp1. Conclusion Estrogen deficiency induces necroptosis of osteocytes in OVX rats by upregulation of TNF-α expression. MLKL and Drp1 may be the key signaling molecules in the downstream of osteocytes necroptosis. |
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