Heilongjiang Key Laboratory Animals and Comparative Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China.
Int J Mol Sci. 2024 Jun 4;25(11):6202. doi: 10.3390/ijms25116202.
Cartilage, a flexible and smooth connective tissue that envelops the surfaces of synovial joints, relies on chondrocytes for extracellular matrix (ECM) production and the maintenance of its structural and functional integrity. Melatonin (MT), renowned for its anti-inflammatory and antioxidant properties, holds the potential to modulate cartilage regeneration and degradation. Therefore, the present study was devoted to elucidating the mechanism of MT on chondrocytes. The in vivo experiment consisted of three groups: Sham (only the skin tissue was incised), Model (using the anterior cruciate ligament transection (ACLT) method), and MT (30 mg/kg), with sample extraction following 12 weeks of administration. Pathological alterations in articular cartilage, synovium, and subchondral bone were evaluated using Safranin O-fast green staining. Immunohistochemistry (ICH) analysis was employed to assess the expression of matrix degradation-related markers. The levels of serum cytokines were quantified via Enzyme-linked immunosorbent assay (ELISA) assays. In in vitro experiments, primary chondrocytes were divided into Control, Model, MT, negative control, and inhibitor groups. Western blotting (WB) and Quantitative RT-PCR (q-PCR) were used to detect Silent information regulator transcript-1 (SIRT1)/Nuclear factor kappa-B (NF-κB)/Nuclear factor erythroid-2-related factor 2 (Nrf2)/Transforming growth factor-beta (TGF-β)/Bone morphogenetic proteins (BMPs)-related indicators. Immunofluorescence (IF) analysis was employed to examine the status of type II collagen (COL2A1), SIRT1, phosphorylated NF-κB p65 (p-p65), and phosphorylated mothers against decapentaplegic homolog 2 (p-Smad2). In vivo results revealed that the MT group exhibited a relatively smooth cartilage surface, modest chondrocyte loss, mild synovial hyperplasia, and increased subchondral bone thickness. ICH results showed that MT downregulated the expression of components related to matrix degradation. ELISA results showed that MT reduced serum inflammatory cytokine levels. In vitro experiments confirmed that MT upregulated the expression of SIRT1/Nrf2/TGF-β/BMPs while inhibiting the NF-κB pathway and matrix degradation-related components. The introduction of the SIRT1 inhibitor Selisistat (EX527) reversed the effects of MT. Together, these findings suggest that MT has the potential to ameliorate inflammation, inhibit the release of matrix-degrading enzymes, and improve the cartilage condition. This study provides a new theoretical basis for understanding the role of MT in decelerating cartilage degradation and promoting chondrocyte repair in in vivo and in vitro cultured chondrocytes.
软骨是一种包裹滑膜关节表面的柔韧而光滑的结缔组织，依赖于软骨细胞来产生细胞外基质（ECM）并维持其结构和功能的完整性。褪黑素（MT）以其抗炎和抗氧化特性而闻名，具有调节软骨再生和降解的潜力。因此，本研究致力于阐明 MT 对软骨细胞的作用机制。体内实验包括三组：假手术组（仅切开皮肤组织）、模型组（采用前交叉韧带切断术（ACLT）方法）和 MT 组（30mg/kg），给药 12 周后提取样本。采用番红 O-快绿染色评估关节软骨、滑膜和软骨下骨的病理改变。免疫组织化学（ICH）分析评估基质降解相关标志物的表达。采用酶联免疫吸附试验（ELISA）检测血清细胞因子水平。在体外实验中，将原代软骨细胞分为对照组、模型组、MT 组、阴性对照组和抑制剂组。采用 Western blot（WB）和定量 RT-PCR（q-PCR）检测沉默信息调节转录因子 1（SIRT1）/核因子 kappa-B（NF-κB）/核因子红细胞 2 相关因子 2（Nrf2）/转化生长因子-β（TGF-β）/骨形态发生蛋白（BMPs）相关指标。采用免疫荧光（IF）分析检测 II 型胶原（COL2A1）、SIRT1、磷酸化 NF-κB p65（p-p65）和磷酸化 mothers against decapentaplegic homolog 2（p-Smad2）的状态。体内结果显示，MT 组软骨表面相对光滑，软骨细胞丢失较少，滑膜轻度增生，软骨下骨厚度增加。ICH 结果显示，MT 下调了基质降解相关成分的表达。ELISA 结果显示，MT 降低了血清炎症细胞因子水平。体外实验证实，MT 上调了 SIRT1/Nrf2/TGF-β/BMPs 的表达，同时抑制了 NF-κB 通路和基质降解相关成分。加入 SIRT1 抑制剂 Selisistat（EX527）后，MT 的作用被逆转。综上所述，这些发现表明 MT 具有改善炎症、抑制基质降解酶释放和改善软骨状况的潜力。本研究为理解 MT 在体内和体外培养的软骨细胞中减缓软骨降解和促进软骨细胞修复中的作用提供了新的理论依据。
