Effect of endoplasmic reticulum stress on chondrocytes in a rat model of knee osteoarthritis

doi:10.3969/j.issn.2095-4344.2017.16.008

Abstract

Abstract:

BACKGROUND: Endoplasmic reticulum (ER) stress has been proved to be related to the occurrence of diabetes, dilated cardiomyopathy and neurodegenerative diseases. Indeed, it is closely associated with osteoarthritis.
OBJECTIVE: To explore the effect of ER stress on the chondrocyte viability as well as the occurrence and development of osteoarthritis in rats.
METHODS: Rat chondrocytes were isolated and cultured, and the ER stress in the rat chondrocytes was by 10 mg/L tunicamycin. The expression levels of ER stress markers C/EBP-homologous protein and 78 kDa glucose-regulated protein were detected by western blot assay, and the proliferation and apoptosis of chondrocytes were detected by cell counting kit-8 assay and AnnexinV-FITC flow cytometry, respectively. In the in vivo experiment, 15 Sprague-Dawley rats were selected and subjected to anterior cruciate ligament transection and medial meniscectomy to establish an animal model of osteoarthritis. Tunicamycin, tauroursodeoxycholic acid and PBS (blank control group) were respectively injected into the articular cavity, and then the progression of osteoarthritis was assessed by hematoxylin-eosin staining at 4 weeks after treatment.
RESULTS AND CONCLUSION: After addition of tunicamycin, the expression levels of C/EBP-homologous protein and 78 kDa glucose-regulated protein were significantly upregulated, the viability of chondrocytes was decreased gradually, while the apoptotic rate was increased significantly. Results from gross observation and hematoxylin-eosin staining suggested that tunicamycin promoted the progression of osteoarthritis and tauroursodeoxycholic acid delayed the deterioration of cartilage in the rats. These findings indicate that ER stress results in the decreased chondrocyte viability and increased apoptosis, which may be an important pathogenesis of osteoarthritis. Additionally, tauroursodeoxycholic acid can effectively alleviate osteoarthritis induced by ER 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: Endoplasmic Reticulum, Chondrocytes, Osteoarthritis, Tunicamycin, Deoxycholic Acid, Tissue Engineering

CLC Number:

R318

Wu Hao, Meng Zhi-chao, Cao Yong-ping, Pan Li-ping, Zhou Xing-tong, Yang Xin, Liu Heng, Wang Rui, Cui Yun-peng, Li Xiang, Li Zhuo-yang. Effect of endoplasmic reticulum stress on chondrocytes in a rat model of knee osteoarthritis[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(16): 2502-2508.

Figures/Tables 2

2.1 大鼠关节软骨细胞形态及鉴定大鼠原代软骨细胞在贴壁前为圆球形，呈悬浮状态，培养9-12 h部分贴壁，绝大部分在24 h时贴壁。贴壁后的细胞变为梭状，后逐渐伸展为多角形。大鼠软骨细胞胞浆丰富，细胞核清晰呈类圆形，多数为1个核仁(见图1A)。随着细胞密度的不断增加，单层生长的软骨细胞出现局灶性立体生长集落。培养六七天后，单层软骨细胞铺满整个培养瓶底部。经过传代后，细胞生长速度变快，第3代大鼠关节软骨细胞4 d左右可以汇合至90%以上(见图1B)。 2.2 衣霉素作用下大鼠关节软骨细胞生长曲线在正常条件下，第3代大鼠关节软骨细胞在接种后，经过2天的潜伏期后开始进入对数生长期，对数生长期可维持2天，第6天开始进入平台期。在第4天加入10 mg/L衣霉素处理后，处理组细胞增殖活性在加药后24，48，72 h较正常细胞均明显下降(P < 0.05)，且下降程度随时间的延长而显著增加(见图2)。 2.3 衣霉素作用下大鼠关节软骨细胞的凋亡大鼠软骨细胞在受到衣霉素作用后，细胞凋亡量随着作用时间的延长逐渐增多。在10 mg/L衣霉素作用下，自加药6 h起，细胞凋亡率较0 h(对照组)即显著增加(P < 0.05)，并随着衣霉素作用时间的延长而继续增高(见图3)。 2.4 衣霉素作用下大鼠软骨细胞GRP78、CHOP的表达变化 GRP78的表达：在正常细胞内，GRP78的表达量较低。在衣霉素作用下，至药物作用12 h，大鼠软骨细胞GRP78的表达较正常细胞均明显增加(P < 0.01)，并随着药物作用时间的延长继续增加(见图4A，图4B)。 CHOP的表达：药物作用6 h起，CHOP的表达量即较前显著增加(6 h 0.298±0.068 vs. 0 h 0.029±0.016，P < 0.05)；至药物作用12 h，CHOP表达量达到峰值(0.764±0.061)，至24-72 h，CHOP表达逐渐下降(见图4A，图4C)。 2.5 内质网应激反应对大鼠骨关节炎进展的影响 15只大鼠随机均分为3组，实验过程无脱失，全部进入结果分析。在骨关节炎模型建立4周后，各组大鼠膝关节股骨滑车均可见不同程度磨损。其中关节腔注射衣霉素的膝关节股骨滑车磨损最为明显，注射牛磺熊去氧胆酸者软骨损伤较轻，与注射PBS者较接近(见图5A-C)；3组大鼠膝关节股骨后髁软骨均磨损轻微(见图5D-F)。苏木精-伊红染色可见，注射衣霉素的膝关节软骨层破坏最明显，而注射牛磺熊去氧胆酸者关节软骨层结构较完整，注射PBS者介于2者之间(见图6A-C)；与大体标本结果类似，苏木精-伊红染色下观察3组大鼠膝关节股骨后髁软骨均磨损轻微，3组间差异不明显(见图6D-F)。"

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