Ou Dijun, Liu Sheng, Tong Changjun, Tan Hezhong, Yang Yadong, He Chunlei
Department of Orthopedics, Huazhong University of Science and Technology Union Shenzhen Hospital, Shenzhen, Guangdong 518000, P.R. China.
Department of Orthopedics, The First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi 341000, P.R. China.
Exp Ther Med. 2022 Jan;23(1):61. doi: 10.3892/etm.2021.10983. Epub 2021 Nov 21.
Osteoarthritis (OA) is a common degenerative disease that is associated with the degradation of articular cartilage. Accumulating evidence has confirmed that LIM mineralization protein-1 (LMP-1) is an important agent of bone formation and has been shown to be osteoinductive in various types of disease. However, the underlying mechanisms of LMP-1 in the pathogenesis of OA remain unknown. The present study aimed to evaluate the role and potential mechanism of LMP-1 in IL-1β-stimulated OA chondrocytes. CHON-001 cells were transfected with pcDNA3.1-LMP-1, pcDNA3.1, negative control-small interfering (si)RNA or LMP-1 siRNA for 24 h and then induced by IL-1β for 12 h to establish an OA model . Cell viability, apoptosis and inflammatory cytokine (IL-6, IL-8 and TNF-α) release were assessed using MTT assay, flow cytometry and ELISA, respectively. The expression levels of LMP-1, cleaved-caspase 3, phosphorylated (p)-p65, p65, p-JNK and JNK were analyzed using reverse transcription-quantitative PCR and western blotting. Overexpression of LMP-1 notably alleviated the IL-1β-induced inflammatory response in CHON-001 cells, as shown by increased cell viability, decreased apoptosis, suppressed expression of cleaved-caspase 3 and a decreased cleaved-caspase 3/caspase 3 ratio. Moreover, IL-1β-induced secretion of IL-6, IL-8 and TNF-α in CHON-001 cells; this was reversed by pcDNA3.1-LMP-1. However, knocking down LMP-1 expression exert opposite effects on the IL-1β-induced inflammatory response in CHON-001 cells, as evidenced by the decreased cell viability, increased apoptosis, enhanced expression of cleaved-caspase 3 and cleaved-caspase 3/caspase 3 ratio and enhanced secretion of IL-6, IL-8 and TNF-α observed. The present data demonstrated that LMP-1 siRNA notably inhibited LMP-1 expression, suppressed cell viability, promoted apoptosis and enhanced cleaved-caspase 3 expression and cleaved-caspase 3/caspase 3 ratio. In addition, LMP-1 siRNA promoted the release of inflammatory factors in CHON-001 cells. It was also found that pcDNA3.1-LMP-1 inhibited p-p65 and p-JNK expression, as well as decreasing the p-p65/p65 and p-JNK/JNK ratio. Nevertheless, there was no significant difference in the mRNA expression levels of p65 and JNK between the groups. Taken together, these findings indicated that overexpression of LMP-1 alleviated IL-1β-induced chondrocytes injury by regulating the NF-κB and MAPK/JNK pathways, suggesting that LMP-1 may be a valuable therapeutic agent for OA treatment.
骨关节炎（OA）是一种常见的退行性疾病，与关节软骨的降解有关。越来越多的证据证实，LIM矿化蛋白-1（LMP-1）是骨形成的重要因子，并且已被证明在各种类型的疾病中具有骨诱导作用。然而，LMP-1在OA发病机制中的潜在机制仍不清楚。本研究旨在评估LMP-1在白细胞介素-1β（IL-1β）刺激的OA软骨细胞中的作用和潜在机制。将CHON-001细胞用pcDNA3.1-LMP-1、pcDNA3.1、阴性对照小干扰（si）RNA或LMP-1 siRNA转染24小时，然后用IL-1β诱导12小时以建立OA模型。分别使用MTT法、流式细胞术和酶联免疫吸附测定（ELISA）评估细胞活力、细胞凋亡和炎性细胞因子（IL-6、IL-8和肿瘤坏死因子-α（TNF-α））释放。使用逆转录定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法分析LMP-1、裂解的半胱天冬酶-3、磷酸化（p）-p65、p65、p-应激活化蛋白激酶（JNK）和JNK的表达水平。LMP-1的过表达显著减轻了IL-1β诱导的CHON-001细胞中的炎症反应，表现为细胞活力增加、细胞凋亡减少、裂解的半胱天冬酶-3表达受到抑制以及裂解的半胱天冬酶-3/半胱天冬酶-3比值降低。此外，IL-1β诱导CHON-001细胞中IL-6、IL-8和TNF-α的分泌；这被pcDNA3.1-LMP-1逆转。然而，敲低LMP-1表达对IL-1β诱导的CHON-001细胞中的炎症反应产生相反的作用，这通过观察到的细胞活力降低、细胞凋亡增加、裂解的半胱天冬酶-3表达增强和裂解的半胱天冬酶-3/半胱天冬酶-3比值升高以及IL-6、IL-8和TNF-α分泌增强得到证明。目前的数据表明，LMP-1 siRNA显著抑制LMP-1表达，抑制细胞活力，促进细胞凋亡并增强裂解的半胱天冬酶-3表达和裂解的半胱天冬酶-3/半胱天冬酶-3比值。此外，LMP-1 siRNA促进CHON-001细胞中炎性因子的释放。还发现pcDNA3.1-LMP-1抑制p-p65和p-JNK表达，以及降低p-p65/p65和p-JNK/JNK比值。然而，各组之间p65和JNK的mRNA表达水平没有显著差异。综上所述，这些发现表明LMP-1的过表达通过调节核因子-κB（NF-κB）和丝裂原活化蛋白激酶/应激活化蛋白激酶（MAPK/JNK）途径减轻了IL-1β诱导的软骨细胞损伤，表明LMP-1可能是OA治疗的一种有价值的治疗剂。
