27-Hydroxycholesterol Contributes to Lysosomal Membrane Permeabilization-Mediated Pyroptosis in Co-cultured SH-SY5Y Cells and C6 Cells
27-Hydroxycholesterol Contributes to Lysosomal Membrane Permeabilization-Mediated Pyroptosis in Co-cultured SH-SY5Y Cells and C6 Cells
复制标题
27-羟基胆固醇有助于共培养的 SH-SY5Y 细胞和 C6 细胞中溶酶体膜透化介导的细胞焦亡
DOI：
10.3389/fnmol.2019.00014
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发表时间：
2019-03-01
影响因子：
4.8
通讯作者：
Xiao, Rong
中科院分区：
文献类型：
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作者：
Chen, Si;Zhou, Cui;Xiao, Rong
目的：新的证据表明，27-羟基胆固醇（27-OHC）通过诱导细胞毒性和胆固醇代谢紊乱引起神经退行性疾病。本研究的目的是确定27-OHC对神经退行性疾病发展中神经元中溶酶体膜透化（LMP）和焦亡的影响。研究方法：本研究采用体外共培养SH-SY 5 Y细胞和C6细胞，观察27-OHC对神经元溶酶体功能、LMP及细胞内凋亡相关因子的影响。用LTR法检测溶酶体pH值、体积和数量的变化。吖啶橙子（AO）染色法检测神经元内LMP的表达。扫描电镜观察细胞形态学变化。Western blot检测溶酶体功能相关蛋白[包括组织蛋白酶B（CTSB）、组织蛋白酶D（CTSD）、溶酶体相关膜蛋白-1（LAMP-1）、LAMP-2]和细胞凋亡相关蛋白[包括凋亡样受体P3（NLRP 3）、Gasdermin D（GSDMD）、caspase-1和白细胞介素（IL）-1β]的含量。结果如下：结果显示，27-OHC处理组的溶酶体功能相关蛋白如CTSB（p <0.05）、CTSD（p < 0.05）、LAMP-1（p < 0.01）、LAMP-2（p < 0.01）的水平高于对照组。AO染色和LTR染色显示27-OHC可诱导LMP引起溶酶体功能障碍。27-OHC处理组神经元中GSDMD（p < 0.01）、NLRP 3（p < 0.001）、caspase-1（p < 0.01）和IL-1β（p < 0.01）等凋亡相关因子蛋白含量增加。此外，CTSB通过LMP泄漏到细胞质中并诱导细胞凋亡。本研究的结果还表明，CTSB参与了焦亡的激活。结论：27-OHC通过诱导LMP和神经元凋亡参与了细胞死亡的病理过程。
Purpose: Emerging evidence suggests that 27-Hydroxycholesterol (27-OHC) causes neurodegenerative diseases through the induction of cytotoxicity and cholesterol metabolism disorder. The objective of this study is to determine the impacts of 27-OHC on lysosomal membrane permeabilization (LMP) and pyroptosis in neurons in the development of neural degenerative diseases. Methods: In this study, SH-SY5Y cells and C6 cells were co-cultured in vitro to investigate the influence of 27-OHC on the function of lysosome, LMP and pyroptosis related factors in neuron. Lyso Tracker Red (LTR) was used to detect the changes of lysosome pH, volume and number. Acridine orange (AO) staining was also used to detect the LMP in neurons. Then the morphological changes of cells were observed by a scanning electron microscope (SEM). The content of lysosome function associated proteins [including Cathepsin B (CTSB), Cathepsin D (CTSD), lysosomal-associated membraneprotein-1 (LAMP-1), LAMP-2] and the pyroptosis associated proteins [including nod-like recepto P3 (NLRP3), gasdermin D (GSDMD), caspase-1 and interleukin (IL)-1β] were detected through Western blot. Results: Results showed higher levels of lysosome function associated proteins, such as CTSB (p < 0.05), CTSD (p < 0.05), LAMP-1 (p < 0.01), LAMP-2; p < 0.01) in 27-OHC treated group than that in the control group. AO staining and LTR staining showed that 27-OHC induced lysosome dysfunction with LMP. Content of pyroptosis related factor proteins, such as GSDMD (p < 0.01), NLRP3 (p < 0.001), caspase-1 (p < 0.01) and IL-1β (p < 0.01) were increased in 27-OHC treated neurons. Additionally, CTSB was leaked through LMP into the cytosol and induced pyroptosis. Results from the present study also suggested that the CTSB is involved in activation of pyroptosis. Conclusion: Our data indicate that 27-OHC contributes to the pathogenesis of cell death by inducing LMP and pyroptosis in neurons.