Xu Ting, Xu Gaoli, Gu Zhiyuan, Wu Huiling
Department of Stomatology, First Affiliated Hospital, Zhejiang University, Hangzhou, China.
School of Stomatology, Zhejiang University, Hangzhou, China.
Mol Cell Biochem. 2017 May;429(1-2):23-31. doi: 10.1007/s11010-016-2933-5. Epub 2017 Feb 11.
Excessive mechanical loads induce chondrocyte apoptosis and irreversible cartilage degeneration, but the underlying molecular mechanism is poorly understood. The aim of this study was to investigate the possible role of endoplasmic reticulum (ER) stress pathway in hydrostatic pressure (HP)-induced apoptosis in rat mandibular condylar chondrocytes. Chondrocytes were isolated from rat mandibular condylar cartilage and subjected to HP. Cell viability and apoptosis were assessed by Cell Counting Kit-8 and flow cytometry assay. Expression of ER stress-associated molecules was detected by quantitative real-time PCR and western blot analysis. In addition, expression of apoptosis-related proteins (bax, bcl-2, and cleaved-caspase-3) was assessed by western blot. To explore ER stress function, chondrocytes were pretreated with salubrinal before exposure to HP. Expression of type II collagen, aggrecan, MMP-13, and ADAMTS-5 was evaluated by real-time PCR. The results indicated that HP reduced cell viability in a magnitude- and time-dependent manner. HP-induced activation of ER stress pathway by increasing expression of GRP78, CHOP, caspase-12, PERK, and peIF2α in chondrocytes. Moreover, the expression of bax and cleaved-caspase-3 was increased, while the expression of bcl-2 was decreased in response to HP as the stress time prolonged. In addition, salubrinal suppressed HP-induced apoptosis, upregulated type II collagen and aggrecan mRNA expression, and downregulated MMP-13 and ADAMTS-5 mRNA expression in response to HP. These results demonstrate that HP induces apoptosis in mandibular condylar chondrocytes through ER stress-mediated apoptotic pathway. Suppression of ER stress by salubrinal prevents chondrocytes from undergoing apoptosis and matrix degradation induced by HP.
过度的机械负荷会诱导软骨细胞凋亡和不可逆的软骨退变，但其潜在的分子机制尚不清楚。本研究的目的是探讨内质网（ER）应激途径在静水压力（HP）诱导的大鼠下颌髁突软骨细胞凋亡中的可能作用。从大鼠下颌髁突软骨中分离软骨细胞并施加HP。通过细胞计数试剂盒-8和流式细胞术检测细胞活力和凋亡情况。通过定量实时PCR和蛋白质印迹分析检测ER应激相关分子的表达。此外，通过蛋白质印迹评估凋亡相关蛋白（bax、bcl-2和裂解的caspase-3）的表达。为了探究ER应激功能，在暴露于HP之前用salubrinal预处理软骨细胞。通过实时PCR评估II型胶原蛋白、聚集蛋白聚糖、MMP-13和ADAMTS-5的表达。结果表明，HP以剂量和时间依赖性方式降低细胞活力。HP通过增加软骨细胞中GRP78、CHOP、caspase-12、PERK和peIF2α的表达诱导ER应激途径的激活。此外，随着应激时间延长，响应HP时bax和裂解的caspase-3的表达增加，而bcl-2的表达降低。另外，salubrinal抑制HP诱导的凋亡，上调II型胶原蛋白和聚集蛋白聚糖mRNA表达，并下调响应HP时MMP-13和ADAMTS-5 mRNA表达。这些结果表明，HP通过ER应激介导的凋亡途径诱导下颌髁突软骨细胞凋亡。Salubrinal对ER应激的抑制可防止软骨细胞发生HP诱导的凋亡和基质降解。
