Endoplasmic reticulum stress participates in the process of high glucose-induced apoptosis in nucleus pulposus cells

doi:10.3969/j.issn.2095-4344.0542

Abstract

Abstract:

BACKGROUND: Intervertebral disc degeneration (IDD) is the main cause of low back pain, and nucleus pulposus cell apoptosis is an important risk factor for IDD. However, the underling mechanism at molecular level remains unknown.
OBJECTIVE: To investigate whether endoplasmic reticulum stress participates in the high glucose-induced apoptosis in nucleus pulposus cells.
METHODS: Passage 3 rabbit nucleus pulposus cells were randomized into four groups, and cultured in the normal culture medium (blank control group), culture medium containing 100 mmol/L glucose (high-glucose group), capspase inhibitor Z-ATAD-FMK (inhibitor group) or Z-ATAD-FMK and 100 mmol/L glucose (combination group). After 6 hours of culture, the viability, reactive oxygen species level, and apoptosis rate were examined in nucleus pulposus cells. The protein expression levels of Grp78 and Caspase-12 were assayed.
RESULTS AND CONCLUSION: Compared with the blank control group, high glucose increased the nucleus pulposus cell apoptosis, reactive oxygen species accumulation, as well as expression of Grp78 and Caspase-12. Whereas, Z-ATAD-FMK (the inhibitor of Caspase-12) abrogated the cytotoxic effect caused by high glucose. Therefore, endoplasmic reticulum stress-mediated apoptosis is a new signaling pathway of high-glucose induced apoptosis in nucleus pulposus cells. The generation and accumulation of reactive oxygen species is the main contributor of endoplasmic reticulum stress.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Apoptosis, Endoplasmic Reticulum, Intervertebral Disk, Diabetes Mellitus, Tissue Engineering

CLC Number:

R318

Li Xianzhou, Zhang Cunxin. Endoplasmic reticulum stress participates in the process of high glucose-induced apoptosis in nucleus pulposus cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(36): 5778-5784.

Figures/Tables 2

2.1 髓核细胞形态学观察原代髓核细胞接种后，经倒置显微镜下观察，可见细胞呈球形，大小不等，有的吸附在培养瓶底，有的悬浮在培养液中。培养七八天后，可见有部分髓核细胞贴壁。贴壁后的髓核细胞呈圆形并且体积变大，伸出伪足后呈三角形、多角形或短梭形。细胞核较大，有时可见双核，核仁明显，胞浆中可见分泌颗粒，细胞折光性好。约2周后，髓核细胞完全贴壁，细胞伸出的伪足相互连接成片，细胞周围可见基质样物质沉淀。培养约3周时，原代髓核细胞汇合可达85%以上，见图1。 2.2 髓核细胞鉴定结果 2.2.1 髓核细胞经甲苯胺蓝染色可见细胞内的聚集蛋白多糖被染成天蓝色，越靠近细胞核，着色越深，见图2A。 2.2.2 苏木精-伊红染色可见髓核细胞呈三角形、多角形或短梭形等不规则形状，胞核位于细胞中央或偏向一侧胞壁，染成蓝色，胞浆染成粉红色，越靠近胞核，胞浆着色越深，见图2B。 2.2.3 细胞免疫组织化学染色可见胞质中Ⅱ型胶原蛋白染成黄褐色，越靠近细胞核着色越深，见图2C。 2.2.4 免疫荧光染色可见Ⅱ型胶原蛋白呈绿色荧光，大部分位于胞质中，且靠近胞核处荧光强度越强，胞核呈蓝色荧光，见图2D。 2.3 ATAD-FMK可使髓核细胞免于高葡萄糖引起的细胞毒害作用 CCK-8检测发现高浓度葡萄糖可显著抑制髓核细胞的增殖活性，使用Z-ATAD-FMK预处理2 h发现，Z-ATAD-FMK可显著拮抗高浓度葡萄糖所致的细胞毒性，见图3A。这种变化趋势同样发现在细胞内总活性氧的检测结果中，见图3B。流式细胞术检测发现，高糖组髓核细胞凋亡率显著增加，Z-ATAD-FMK预处理髓核细胞后，与对照组比较，虽然不能显著降低髓核细胞凋亡率，但与高糖组比较，其差异有显著性意义(P < 0.05) ，见图3C，D。 2.4 高浓度葡萄糖引起的内质网应激参与了髓核细胞的凋亡过程 Western blot技术用来分析Caspase-12和Grp78表达变化，使用β-actin作为内参。灰度分析发现，高浓度葡萄糖可显著增加Caspase-12和Grp78的表达，而Z-ATAD-FMK可抑制高糖引起的Caspase-12和Grp78的表达增加，见图4。"

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