TNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA). Here, we examined HA synthase and inflammatory gene expression; synovial fluid HA, TNF-α, and viscosity; and TSG-6-mediated HC-HA complex formation in an equine OA model. The objectives of this study were to (1) evaluate the TNF-α-TSG-6-HC-HA signaling pathway across multiple joint tissues, including synovial membrane, cartilage, and synovial fluid, and (2) determine the impact of OA on synovial fluid composition and biophysical properties. HA and inflammatory cytokine concentrations (TNF-α, IL-1β, CCL2, 3, 5, and 11) were analyzed in synovial fluid from 63 OA and 25 control joints, and HA synthase (HAS1-3), TSG-6, and hyaluronan-degrading enzyme (HYAL2, HEXA) gene expression was measured in synovial membrane and cartilage. HA molecular weight (MW) distributions were determined using agarose gel electrophoresis and solid-state nanopore measurements, and HC-HA complex formation was detected via immunoblotting and immunofluorescence. SEC-MALS was used to evaluate TSG-6-mediated HA crosslinking, and synovial fluid and HA solution viscosities were analyzed using multiple particle-tracking microrheology and microfluidic measurements, respectively. TNF-α concentrations were greater in OA synovial fluid, and TSG6 expression was upregulated in OA synovial membrane and cartilage. TSG-6-mediated HC-HA complex formation was greater in OA synovial fluid and tissues than controls, and HC-HA was localized to both synovial membrane and superficial zone chondrocytes in OA joints. SEC-MALS demonstrated macromolecular aggregation of low MW HA in the presence of TSG-6 and inter-α-inhibitor with concurrent increases in viscosity. Synovial fluid TNF-α concentrations, synovial membrane and cartilage TSG6 gene expression, and HC-HA complex formation were increased in equine OA. Despite the ability of TSG-6 to induce macromolecular aggregation of low MW HA with resultant increases in the viscosity of low MW HA solutions in vitro, HA concentration was the primary determinant of synovial fluid viscosity rather than HA MW or HC-HA crosslinking. The TNF-α-TSG-6-HC-HA pathway may represent a potential therapeutic target in OA.

中文翻译：

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马骨关节炎中的透明质酸合成、降解和交联：TNF-α-TSG-6 介导的 HC-HA 形成


TNF-α刺激基因 6 (TSG-6) 蛋白是一种 TNF-α 响应性透明质酸，具有酶活性，可以催化多糖透明质酸 (HA) 与另一种蛋白质的共价交联，形成重链透明质酸 (HC) -HA）在骨关节炎（OA）等病理状况下形成复合物。在这里，我们检查了 HA 合酶和炎症基因的表达；滑液HA、TNF-α和粘度；以及马 OA 模型中 TSG-6 介导的 HC-HA 复合物形成。本研究的目的是 (1) 评估跨多个关节组织（包括滑膜、软骨和滑液）的 TNF-α-TSG-6-HC-HA 信号通路，以及 (2) 确定 OA 对关节功能的影响滑液成分和生物物理特性。对 63 个 OA 和 25 个对照关节的滑液中的 HA 和炎性细胞因子浓度（TNF-α、IL-1β、CCL2、3、5 和 11）以及 HA 合酶 (HAS1-3)、TSG-6 和测量滑膜和软骨中的透明质酸降解酶（HYAL2、HEXA）基因表达。使用琼脂糖凝胶电泳和固态纳米孔测量确定 HA 分子量 (MW) 分布，并通过免疫印迹和免疫荧光检测 HC-HA 复合物的形成。使用 SEC-MALS 评估 TSG-6 介导的 HA 交联，并分别使用多粒子跟踪微流变学和微流体测量来分析滑液和 HA 溶液粘度。 OA 滑液中 TNF-α 浓度较高，并且 OA 滑膜和软骨中 TSG6 表达上调。 OA 滑液和组织中 TSG-6 介导的 HC-HA 复合物形成量高于对照组，并且 HC-HA 定位于 OA 关节的滑膜和浅层软骨细胞。 SEC-MALS 证明，在 TSG-6 和 inter-α-抑制剂存在下，低 MW HA 发生大分子聚集，同时粘度增加。马 OA 中滑液 TNF-α 浓度、滑膜和软骨 TSG6 基因表达以及 HC-HA 复合物形成均增加。尽管 TSG-6 能够诱导低分子量 HA 大分子聚集，从而导致体外低分子量 HA 溶液粘度增加，但 HA 浓度是滑液粘度的主要决定因素，而不是 HA MW 或 HC-HA 交联。 TNF-α-TSG-6-HC-HA 途径可能代表 OA 的潜在治疗靶点。