Zhang C, Lin S, Li T, Jiang Y, Huang Z, Wen J, Cheng W, Li H
Department of Orthodontics, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, China.
Department of Biostatistics, Robert Stempel College of Public Health and Social Work, Florida International University, USA.
Osteoarthritis Cartilage. 2017 Aug;25(8):1324-1334. doi: 10.1016/j.joca.2017.03.018. Epub 2017 Apr 7.
This study aimed to identify the mechanisms underlying mandibular chondrocyte cell death and cartilage thinning in response to mechanical force.
An in vivo model (compressive mechanical force) and an in vitro model (TNF-α+cycloheximide) were used to induce mandibular chondrocyte necroptosis. Hematoxylin and eosin staining and transmission electron microscopy were used to assess histological and subcellular changes in mandibular chondrocyte. Immunohistochemistry, western blotting, and real-time PCR were performed to evaluate changes in necroptotic protein markers. Cell activity, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were examined in vitro.
The expression of RIP1, RIP3 and Caspase-8 in mandibular chondrocytes significantly increased after 4 days of compressive mechanical force. Furthermore, the inhibition of necroptosis by Necrostatin-1 (Nec-1) or the inhibition of apoptosis by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD) partially restored mechanical force-mediated mandibular cartilage thinning and chondrocyte death. Moreover, a synergistic effect on cell death inhibition and mandibular cartilage thickness restoration were found when treated with Nec-1+Z-VAD. The results of the in vitro model were in line with the in vivo ones, indicating that the changes in MMP and ROS generation contributed to mandibular chondrocyte apoptosis and necroptosis.
In addition to apoptosis, necroptosis also plays critical roles in pathological changes in mandibular cartilage after compressive mechanical force stimulation, implying RIP1, a master protein that mediates both necroptosis and apoptosis, as a potential therapeutic target in temporal mandibular osteoarthritis.
本研究旨在确定下颌软骨细胞因机械力而发生细胞死亡和软骨变薄的潜在机制。
采用体内模型（压缩机械力）和体外模型（TNF-α+环己酰亚胺）诱导下颌软骨细胞坏死性凋亡。苏木精-伊红染色和透射电子显微镜用于评估下颌软骨细胞的组织学和亚细胞变化。进行免疫组织化学、蛋白质印迹和实时聚合酶链反应以评估坏死性凋亡蛋白标志物的变化。在体外检测细胞活性、线粒体膜电位（MMP）和活性氧（ROS）。
压缩机械力作用4天后，下颌软骨细胞中RIP1、RIP3和Caspase-8的表达显著增加。此外，坏死性凋亡抑制剂Necrostatin-1（Nec-1）或凋亡抑制剂N-苄氧羰基-Val-Ala-Asp-氟甲基酮（Z-VAD）部分恢复了机械力介导的下颌软骨变薄和软骨细胞死亡。此外，用Nec-1+Z-VAD处理时，发现对细胞死亡抑制和下颌软骨厚度恢复有协同作用。体外模型的结果与体内模型一致，表明MMP和ROS生成的变化促成了下颌软骨细胞凋亡和坏死性凋亡。
除凋亡外，坏死性凋亡在压缩机械力刺激后下颌软骨的病理变化中也起关键作用，这意味着RIP1作为介导坏死性凋亡和凋亡的主要蛋白，是颞下颌骨关节炎的潜在治疗靶点。
