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Abstract：
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.
摘要：
软骨,包裹滑膜关节表面的柔韧光滑的结缔组织，依赖于软骨细胞的细胞外基质（ECM）的生产和其结构和功能的完整性的维持。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。体内实验分为三组：假手术（仅切开皮肤组织），模型（使用前交叉韧带横断（ACLT）方法），和MT（30毫克/千克），在给药12周后进行样品提取。关节软骨的病理改变，滑膜,使用SafraninO-fast绿色染色评估软骨下骨。采用免疫组织化学(ICH)分析来评估基质降解相关标志物的表达。通过酶联免疫吸附测定(ELISA)测定定量血清细胞因子的水平。在体外实验中,将原代软骨细胞分为对照，型号,MT,阴性对照,和抑制剂组。采用Westernblotting(WB)和定量RT-PCR(q-PCR)检测沉默信息调节因子转录因子-1(SIRT1)/核因子-κB(NF-κB)/核因子-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。免疫荧光（IF）分析用于检查II型胶原蛋白（COL2A1）的状态，SIRT1，磷酸化NF-κBp65（p-p65），和磷酸化的母亲对十骨截瘫同系物2（p-Smad2）。体内结果显示，MT组表现出相对光滑的软骨表面，适度的软骨细胞损失，轻度滑膜增生,软骨下骨厚度增加。ICH结果表明，MT下调了与基质降解相关的成分的表达。ELISA结果显示MT降低血清炎性细胞因子水平。体外实验证实，MT上调SIRT1/Nrf2/TGF-β/BMPs的表达，同时抑制NF-κB通路和基质降解相关成分。SIRT1抑制剂Selissistat（EX527）的引入逆转了MT的作用。一起,这些研究结果表明，MT具有改善炎症的潜力，抑制基质降解酶的释放，改善软骨状况.本研究为了解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的作用提供了新的理论基础。
