Xiong Xiaoyang, Liu Liang, Xu Feng, Wu Xiaofeng, Yin Zifei, Dong Yi, Qian Pingkang
Department of Joint and orthopedics, Traditional Chinese Medicine Hospital of Kunshan, Suzhou, Jiangsu 215300, China.
ACS Omega. 2021 Mar 12;6(11):7638-7645. doi: 10.1021/acsomega.0c06212. eCollection 2021 Mar 23.
: Arthritis is a cartilage degenerative disease that is mainly induced by the degradation of the cartilage extracellular matrix (ECM), which is found to be regulated by the expression level of a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMT-5), an enzyme degrading Aggrecans in the ECM. Feprazone is a classic nonsteroidal anti-inflammatory drug with promising efficacy in arthritis. The present study aims to investigate the protective effect of Feprazone on the degraded Aggrecan in the human chondrocytes induced with tumor necrosis factor-α (TNF-α) and to clarify the underlying mechanism. : To investigate the effect of Feprazone, the CHON-001 chondrocytes were stimulated with TNF-α (10 ng/mL) in the presence or absence of Feprazone (3, 6 μM) for 24 h. Mitochondrial membrane potential was evaluated using the Rhodamine 123 assay. The gene expressions of interleukin-1β (IL-1β), interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and ADAMTS-5 in the treated chondrocytes were detected using real-time quantitative polymerase chain reaction (qRT-PCR), and the protein levels of these targets were determined using enzyme-linked immunosorbent assay (ELISA). SOX-4 was knocked down by transfecting the siRNA into the chondrocytes. Western blot analysis was utilized to evaluate the expression levels of SOX-4, Aggrecan, and protein kinase C (PKCα). : First, the reduced mitochondrial membrane potential (ΔΨ) and secretion of proinflammatory factors (IL-1β, IL-8, and MCP-1) induced by TNF-α were significantly reversed by treatment with Feprazone. Second, the expression of Aggrecan was significantly decreased by stimulation with TNF-α via upregulation of ADAMTS-5 but was dramatically reversed by the introduction of Feprazone. Third, we found that TNF-α elevated the expression of ADAMTS-5 by upregulating SOX-4, which was observed to be related to the activation of PKCα. Lastly, the elevated expression of SOX-4 induced by TNF-α was significantly reversed by Feprazone. : Feprazone might ameliorate TNF-α-induced loss of Aggrecan via the inhibition of the SOX-4/ADAMTS-5 signaling pathway.
关节炎是一种软骨退行性疾病，主要由软骨细胞外基质（ECM）降解引起，研究发现其受含血小板反应蛋白基序的解聚素和金属蛋白酶5（ADAMT - 5）的表达水平调节，ADAMT - 5是一种可降解ECM中聚集蛋白聚糖的酶。非普拉宗是一种经典的非甾体抗炎药，对关节炎有显著疗效。本研究旨在探讨非普拉宗对肿瘤坏死因子-α（TNF-α）诱导的人软骨细胞中聚集蛋白聚糖降解的保护作用，并阐明其潜在机制。
为研究非普拉宗的作用，在有或无非普拉宗（3、6 μM）存在的情况下，用TNF-α（10 ng/mL）刺激CHON - 001软骨细胞24小时。使用罗丹明123检测法评估线粒体膜电位。用实时定量聚合酶链反应（qRT-PCR）检测处理后软骨细胞中白细胞介素-1β（IL-1β）、白细胞介素-8（IL-8）、单核细胞趋化蛋白1（MCP-1）和ADAMTS-5的基因表达，并用酶联免疫吸附测定（ELISA）法测定这些靶点的蛋白水平。通过将小干扰RNA（siRNA）转染到软骨细胞中来敲低SOX-4。利用蛋白质印迹分析评估SOX-4、聚集蛋白聚糖和蛋白激酶C（PKCα）的表达水平。
首先，非普拉宗治疗可显著逆转TNF-α诱导的线粒体膜电位降低（ΔΨ）和促炎因子（IL-1β、IL-8和MCP-1）分泌。其次，TNF-α刺激通过上调ADAMTS-5显著降低聚集蛋白聚糖的表达，但非普拉宗的加入可显著逆转这一现象。第三，我们发现TNF-α通过上调SOX-4来提高ADAMTS-5的表达，这与PKCα的激活有关。最后，TNF-α诱导的SOX-4表达升高被非普拉宗显著逆转。
非普拉宗可能通过抑制SOX-4/ADAMTS-5信号通路改善TNF-α诱导的聚集蛋白聚糖丢失。
