Melatonin Delays Arthritis Inflammation and Reduces Cartilage Matrix Degradation through the SIRT1-Mediated NF-κB/Nrf2/TGF-β/BMPs Pathway

Mingchao Zhao, Di Qiu, Xue Miao, Wenyue Yang, Siyao Li, Xin Cheng, Jilang Tang, Hong Chen, Hongri Ruan, Ying Liu, Chengwei Wei, Jianhua Xiao
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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.
褪黑激素通过 SIRT1 介导的 NF-κB/Nrf2/TGF-β/BMPs 通路延缓关节炎炎症并减少软骨基质降解
软骨是一种包裹在滑膜关节表面的柔韧光滑的结缔组织,依靠软骨细胞产生细胞外基质(ECM)并维持其结构和功能的完整性。褪黑激素(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:Sham组(仅切开皮肤组织)、Model组(采用前十字韧带横断法(ACLT))和MT组(30 毫克/千克)。使用 Safranin O-快绿染色法评估关节软骨、滑膜和软骨下骨的病理变化。免疫组化(ICH)分析用于评估基质降解相关标记物的表达。血清细胞因子水平通过酶联免疫吸附试验(ELISA)进行量化。在体外实验中,原代软骨细胞被分为对照组、模型组、MT 组、阴性对照组和抑制剂组。采用 Western 印迹(WB)和定量 RT-PCR (q-PCR)检测沉默信息调节转录物-1(SIRT1)/核因子卡巴-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。免疫荧光(IF)分析用于检测Ⅱ型胶原蛋白(COL2A1)、SIRT1、磷酸化NF-κB p65(p-p65)和磷酸化抗十瘫同源物2母体(p-Smad2)的状态。体内研究结果显示,MT 组软骨表面相对光滑,软骨细胞适度丢失,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。酶联免疫吸附试验(ELISA)结果显示,MT 降低了血清中炎症细胞因子的水平。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的表达。引入SIRT1抑制剂Selisistat(EX527)可逆转MT的影响。这些研究结果表明,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。这项研究为理解 MT 在体内和体外培养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。
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