毛兰素对 IL-1β 诱导软骨细胞损伤的保护作用及机制

Protective effect and mechanism of erianin against IL-1β-induced chondrocyte injury

摘要

目的 探讨石斛天然提取物毛兰素对白细胞介素 1β（IL-1β）诱导的软骨细胞损伤的保护作用及其机制。方法 建立 IL-1β（10 ng/mL）诱导的软骨细胞损伤模型模拟骨关节炎，分为 6 组：对照组（正常培养基培养）、毛兰素浓度梯度（5、10、20、40、80 nmol/L）处理组。通过免疫印迹法检测炎症蛋白诱导型一氧化氮合酶（iNOS）和环氧化酶Ⅱ型（COX2）的表达水平；使用酶联免疫吸附试验（ELISA）测定细胞上清中 IL-6、肿瘤坏死因子 α（TNF-α）、前列腺素 E2（PGE2）和一氧化氮（NO）的分泌量；通过实时荧光定量 PCR（qRT-PCR）分析细胞外基质代谢相关基因[蛋白聚糖（Aggrecan）、Ⅱ型胶原（Collagen Ⅱ）、基质金属蛋白酶 13（MMP13）、血小板反应蛋白解整合素金属肽酶 5（ADAMTS5）]的 mRNA 表达；采用衰老相关 β-半乳糖苷酶（SA-β-gal）染色观察软骨细胞衰老情况；并利用免疫荧光技术检测核因子 κB（NF-κB）信号在细胞核内的转位情况，分析毛兰素对 NF-κB 通路的影响。结果 在 0～40 nmol/L 浓度范围内，毛兰素对软骨细胞未表现明显的毒性，软骨细胞存活率与对照组比较差异无统计学意义（均 P ＞ 0.05），选择 40 nmol/L 为无毒性浓度。与对照组比较，IL-1β 增加了软骨细胞中 iNOS 和 COX2 表达（均 P ＜ 0.01），同时上清液中 PGE2、NO、IL-6 和 TNF-α 水平升高（均 P ＜ 0.01）；而 40 nmol/L 毛兰素处理可逆转上述炎症指标的表达（均 P ＜ 0.01）。此外，毛兰素能够上调细胞外基质相关基因 Aggrecan 和 Collagen Ⅱ的 mRNA 表达，下调 ADAMTS5 和 MMP13 的 mRNA 水平，并降低衰老软骨细胞的比例（均 P ＜ 0.01）。与 IL-1β 组相比，毛兰素处理后细胞核内 NF-κB 信号强度减弱（ P ＜ 0.01）。结论 毛兰素通过抑制 NF-κB 信号通路，有效缓解 IL-1β 所诱导的软骨细胞炎症反应，并减少细胞外基质降解，延缓软骨细胞衰老。

Abstract

Objective To investigate the protective effect of erianin, a natural extract from Dendrobium, against interleukin-1β (IL-1β)-induced chondrocyte injury and its mechanism. Methods An IL-1β (10 ng/mL)-induced chondrocyte injury model was established to simulate osteoarthritis. The cells were divided into six groups: the control group, cultured in normal medium, and erianin concentration gradient treatment groups (5, 10, 20, 40, and 80 nmol/L). Western blotting was used to detect the expression levels of the inflammatory proteins inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Enzyme-linked immunosorbent assay (ELISA) was used to measure the secretion levels of IL-6, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and nitric oxide (NO) in the cell supernatant. Real-time quantitative PCR (qRT-PCR) was used to analyze the mRNA expression of extracellular matrix metabolism-related genes, including Aggrecan, CollagenⅡ, matrix metalloproteinase 13 (MMP13), and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5). Senescence-associated β-galactosidase (SA-β-gal) staining was used to observe chondrocyte senescence. Immunofluorescence was used to detect the nuclear translocation of nuclear factor-κB (NF-κB) signaling and to analyze the effect of erianin on the NF-κB pathway. Results Within the concentration range of 0-40 nmol/L, erianin showed no obvious toxicity to chondrocytes, and there was no statistically significant difference in chondrocyte viability compared with the control group (all P ＞ 0.05). Therefore, 40 nmol/L was selected as the non-toxic concentration. Compared with the control group, IL-1β increased the expression of iNOS and COX-2 in chondrocytes (both P ＜ 0.01), and elevated the levels of PGE2, NO, IL-6, and TNF-α in the supernatant (all P ＜ 0.01). Treatment with 40 nmol/L erianin reversed the expression of the above inflammatory indicators (all P ＜ 0.01). In addition, erianin upregulated the mRNA expression of the extracellular matrix-related genes Aggrecan and CollageⅡ, downregulated the mRNA levels of ADAMTS5 and MMP13, and reduced the proportion of senescent chondrocytes (all P ＜ 0.01). Compared with the IL-1β group, the intensity of NF-κB signaling in the nucleus was significantly weakened after erianin treatment ( P ＜ 0.01). Conclusions Erianin effectively alleviates IL-1β-induced inflammatory responses in chondrocytes, reduces extracellular matrix degradation, and attenuates chondrocyte senescence by inhibiting the NF-κB signaling pathway.

关键词

毛兰素 / 骨关节炎 / 软骨细胞 / 炎症 / 核因子 κB

Key words

Erianin / Osteoarthritis / Chondrocytes / Inflammation / Nuclear factor-κB

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基金资助

浙江省中医药科技计划(2020ZB0008)

浙江省自然科学基金项目(LYY22H280003)