Department of Sports Medicine, Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University, Haikou, Hainan, China.
Department of Orthopedics, Zibo Orthopaedic Hospital Shandong Province, Zibo, Shandong, China.
J Biochem Mol Toxicol. 2023 Sep;37(9):e23404. doi: 10.1002/jbt.23404. Epub 2023 Jun 23.
The role and mechanism of Gremlin-1 in osteoarthritis (OA) were expected to be probed in this study. Firstly, an in vitro OA model was constructed by stimulating human chondrocyte cell line CHON-001 with IL-1β. Next, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were utilized for assessing the effect of IL-1β with different concentrations (5, 10, and 20 ng/mL) on the activity and Gremlin-1 messenger RNA of CHON-001 cells, respectively. Besides, the influence of knocking down/over-expressing Gremlin-1 on the inflammatory factors (IL-6, TNF-α, IL-18 and PGE2), oxidative stress-related substances (malondialdehyde [MDA]; superoxide dismutase [SOD]; lactate dehydrogenase [LDH]), extracellular matrix (ECM) degradation-related proteins, and mitogen-activated protein kinase (MAPK) pathway proteins in IL-1β-stimulated CHON-001 cells were tested by enzyme-linked immunosorbent assay, related kits, qRT-PCR, and western blot, respectively. IL-1β inhibited CHON-001 cell proliferation and upregulated Gremlin-1 expression in a concentration-dependent manner. Overexpression of Gremlin-1 increased the IL-6, TNF-α, IL-18, PGE2, and MDA levels, enhanced the LDH activity, and decreased the SOD activity in IL-1β-induced CHON-001 cells; while the effect of Gremlin-1 knockdown on the above factors was in contrast with that of the overexpression. Furthermore, overexpression of Gremlin-1 upregulated protein expression of matrix metalloproteinase (MMP)-3, MMP-13, and ADAMTS4 while downregulated protein expression of collagen III, aggrecan, and SOX-9 in IL-1β-stimulated CHON-001 cells. Besides, overexpression of Gremlin-1 increased the p-p38/p38 value while decreased the p-JNK/JNK value in L-1β-stimulated CHON-001 cells; however, knockdown of Gremlin-1 reversed the above results. Gremlin-1 may promote IL-1β-stimulated CHON-001 cell inflammation and ECM degradation by activating the MAPK signaling pathway.
本研究旨在探讨 Gremlin-1 在骨关节炎（OA）中的作用和机制。首先，通过用白细胞介素-1β（IL-1β）刺激人软骨细胞系 CHON-001 构建体外 OA 模型。然后，分别采用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）和实时定量逆转录聚合酶链反应（qRT-PCR）评估不同浓度（5、10 和 20ng/mL）的 IL-1β对 CHON-001 细胞活性和 Gremlin-1 信使 RNA 的影响。此外，通过酶联免疫吸附测定、相关试剂盒、qRT-PCR 和 Western blot 分别检测敲低/过表达 Gremlin-1 对 IL-1β刺激的 CHON-001 细胞中炎症因子（IL-6、TNF-α、IL-18 和 PGE2）、氧化应激相关物质（丙二醛[MDA]；超氧化物歧化酶[SOD]；乳酸脱氢酶[LDH]）、细胞外基质（ECM）降解相关蛋白和丝裂原激活蛋白激酶（MAPK）通路蛋白的影响。IL-1β 呈浓度依赖性抑制 CHON-001 细胞增殖并上调 Gremlin-1 表达。过表达 Gremlin-1 增加了 IL-6、TNF-α、IL-18、PGE2 和 MDA 水平，增强了 IL-1β 诱导的 CHON-001 细胞中的 LDH 活性，降低了 SOD 活性；而敲低 Gremlin-1 对上述因子的作用则与过表达相反。此外，过表达 Gremlin-1 上调了基质金属蛋白酶（MMP）-3、MMP-13 和 ADAMTS4 的蛋白表达，下调了 IL-1β 刺激的 CHON-001 细胞中胶原 III、聚集蛋白聚糖和 SOX-9 的蛋白表达。此外，过表达 Gremlin-1 增加了 IL-1β 刺激的 CHON-001 细胞中 p-p38/p38 值，降低了 p-JNK/JNK 值；然而，敲低 Gremlin-1 则逆转了上述结果。Gremlin-1 可能通过激活 MAPK 信号通路促进 IL-1β 刺激的 CHON-001 细胞炎症和 ECM 降解。
