Transmembrane protein 208 affects autophagy and mitochondrial function in chondrocytes

doi:10.3969/j.issn.2095-4344.1228

Abstract

Abstract:

BACKGROUND: Decreased level of autophagy in chondrocytes is an important mechanism of osteoarthritis. The autophagy inhibitory gene transmembrane protein 208 is elevated in late-stage osteoarthritis cartilage, and this gene may promote the development of osteoarthritis by inhibiting autophagy of chondrocytes.
OBJECTIVE: To investigate the effects of chondrocyte transmembrane protein 208 gene on autophagy and mitochondrial function in osteoarthritis model in vitro.
METHODS: The cartilage tissue was from the discarded cartilage of patients undergoing knee arthroplasty. The mild lesion cartilage on non-weight-bearing face (Outerbridge I-II grade) was as early-term group, and the severe lesion cartilage on heavy-weight surface (Outerbridge III-IV grade) was as the late-term group. The content of transmembrane protein 208 in different cartilage tissues was detected. The study was in accordance with the ethical criteria of Peking University First Hospital, and the cartilage donors and their family members signed an informed consent form. Passage 4 normal human chondrocytes were obtained and then stimulated by interleukin-1β to establish the in vitro osteoarthritis. The chondrocytes were pretreated by autophagy activator rapamycin or autophagy inhibitor 3MA, followed by interleukin-1β added for 12, 24 and 48 hours to observe the morphology of chondrocytes. The content of transmembrane protein 208 gene was detected by real-time PCR. Western blot assay was used to detect autophagy-associated protein light chain proteins 3B and P62 to evaluate autophagy levels. The apoptosis of chondrocytes was detected by Annexin V-FITC method. Mitochondrial damage was assessed by fluorescent detection with MitoSOX Red dye and JC-1 dye.
RESULTS AND CONCLUSION: (1) The expression of transmembrane protein 208 was significantly increased in severe osteoarthritis cartilage. (2) After stimulation with interleukin-1β for 12, 24 and 48 hours, the expression level of transmembrane protein 208 in chondrocytes decreased initially, and then increased. The level of autophagy rose initially and then fell. The apoptotic level gradually increased, and mitochondrial damage gradually became severe. (3) Rapamycin could significantly reduce the expression of transmembrane protein 208, increase the level of autophagy, and reduce the apoptosis and mitochondrial damage in chondrocytes after interleukin-1β stimulation. (4) To conclude, transmembrane protein 208 inhibits autophagy in chondrocytes, and increased expression of this gene may lead to increased damage of chondrocytes under stress.

Key words: transmembrane protein 208, autophagy, osteoarthritis, apoptosis, mitochondrial damage, interleukin-1β, rapamycin, the Natural Science Foundation of Beijing

CLC Number:

R446

Li Xiang, Meng Zhichao, Jiao Yang, Yu Bingxiao, Talatibaike•Maimaitijuma, Cao Yongping . Transmembrane protein 208 affects autophagy and mitochondrial function in chondrocytes[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(23): 3636-3642.

Figures/Tables 2

2.1 跨膜蛋白208在重度病变骨关节炎软骨组织中表达增加膝关节骨关节炎患者中，轻度病变软骨组织(Outerbridge0-Ⅰ级)跨膜蛋白208的表达水平较低；重度病变软骨组织(OuterbridgeⅣ级)跨膜蛋白208的表达水平明显增高，见图1。 2.2 白细胞介素1β可改变软骨细胞自噬水平改变、细胞凋亡升高及线粒体损伤 2.2.1 软骨细胞自噬水平利用白细胞介素1β刺激软骨细胞建立骨关节炎体外模型，分别选取12，24，48 h 3个时间节点观察软骨细胞状态变化。白细胞介素1β刺激软骨细胞后，直接光镜观察发现软骨细胞的浓度下降、部分细胞形态发生改变，见图2。软骨细胞自噬水平在白细胞介素1β刺激早期(12 h)轻度升高，随刺激时间延长(24，48 h)，细胞自噬水平逐渐下降。用Western Blot方法检测轻链蛋白3BⅡ/轻链蛋白3BⅠ相对表达量及P62蛋白表达量，评估软骨细胞的自噬水平。白细胞介素1β刺激12 h后，软骨细胞轻链蛋白3BⅡ/轻链蛋白3BⅠ较对照组显著升高，而细胞内P62水平较对照组显著降低，说明白细胞介素1β刺激12 h后，软骨细胞自噬水平有明显提升。白细胞介素1β刺激24，48 h后，软骨细胞轻链蛋白3BⅡ/轻链蛋白3BⅠ较12 h时显著降低，细胞内P62水平显著升高，说明更长时间的刺激(24，48 h)会导致软骨细胞自噬水平逐渐下降。见图3。 2.2.2 细胞凋亡水平利用AnnexinV-FITC法(流式细胞法)评估软骨细胞的凋亡水平，软骨细胞在应激状态下，凋亡水平升高，见图4。白细胞介素1β刺激时间越长，凋亡水平越高，雷帕霉素可以缓解白细胞介素1β刺激造成的细胞凋亡水平升高，而3-MA预处理会加重白细胞介素1β刺激对软骨细胞的伤害。 2.2.3 线粒体损伤情况利用JC-1染料检测细胞内线粒体膜电位，评估线粒体损伤情况。白细胞介素1β刺激可加重线粒体膜电位降低，见图5，且随白细胞介素1β刺激时间延长线粒体损伤加重，雷帕霉素预处理可缓解白细胞介素1β诱导的线粒体损伤，3-MA预处理可加重白细胞介素1β诱导的线粒体损伤。 2.3 白细胞介素1β应激导致软骨细胞跨膜蛋白208表达量随自噬水平变化白细胞介素1β刺激软骨细胞早期 (12 h)，跨膜蛋白208表达降低，随着刺激时间延长(24，48 h)，跨膜蛋白208表达逐渐增高，跨膜蛋白208表达量变化和自噬水平呈相反趋势。见图6。 2.4 自噬水平的改变可影响跨膜蛋白208的表达雷帕霉素刺激软骨细胞后，轻链蛋白3B表达增加，P62表达减少，说明雷帕霉素可明显提高软骨细胞的自噬水平，3-MA可明显降低自噬水平。雷帕霉素预处理后再进行应激状态诱导，软骨细胞跨膜蛋白208表达水平整体降低，3-MA预处理后，软骨细胞跨膜蛋白208表达水平升高，预处理虽然改变细胞的自噬水平，但跨膜蛋白208的表达量仍受白细胞介素1β刺激出现变化，呈现早期下降后期升高的趋势，见图7。"

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