Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, OH, 44272, USA.
Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, OH, 44272, USA.
Biomed Pharmacother. 2017 Dec;96:198-207. doi: 10.1016/j.biopha.2017.09.140. Epub 2017 Oct 6.
BACKGROUND/OBJECTIVE: Osteoarthritis (OA) is a leading cause of joint dysfunction, disability and poor quality of life in the affected population. The underlying mechanism of joint dysfunction involves increased oxidative stress, inflammation, high levels of cartilage extracellular matrix degrading proteases and decline in autophagy-a mechanism of cellular defense. There is no disease modifying therapies currently available for OA. Different parts of the Butea monosperma (Lam.) plant have widely been used in the traditional Indian Ayurvedic medicine system for the treatment of various human diseases including inflammatory conditions. Here we studied the chondroprotective effect of hydromethanolic extract of Butea monosperma (Lam.) flowers (BME) standardized to the concentration of Butein on human OA chondrocytes stimulated with IL-1β.
The hydromethanolic extract of Butea monosperma (Lam.) (BME) was prepared with 70% methanol-water mixer using Soxhlet. Chondrocytes viability after BME treatment was measured by MTT assay. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using TaqMan assays and immunoblotting with specific antibodies. Autophagy activation was determined by measuring the levels of microtubule associated protein 1 light chain 3-II (LC3-II) by immunoblotting and visualization of autophagosomes by transmission electron and confocal microscopy.
BME was non-toxic to the OA chondrocytes at the doses employed and suppressed the IL-1β induced expression of inerleukin-6 (IL-6) and matrix metalloprotease-3 (MMP-3), MMP-9 and MMP-13. BME enhanced autophagy in chondrocytes as determined by measuring the levels of LC3-II by immunoblotting and increased number of autophagosomes in BME treated chondrocytes by transmission electron microscopy and confocal microscopy. BME upregulated the expression of several autophagy related genes and increased the autophagy flux in human OA chondrocytes under pathological conditions. Further analysis revealed that BME activated autophagy in chondrocytes via inhibition of mammalian target of rapamycin (mTOR) pathway. Of importance is our finding that BME-mediated suppression of IL-1β induced expression of IL-6, MMP-3, -9, and -13 was autophagy dependent and was abrogated by inhibition of autophagy.
The above results show that the Butea monosperma (Lam.) extract has strong potential to activate autophagy and suppress IL-1β induced expression of IL-6 and MMP-3, -9 and -13 in human OA chondrocytes. This study shows that BME or compounds derived from BME can be developed as safe and effective chondroprotective agent(s) that function by activating autophagy to suppress the expression of inflammatory and catabolic factors associated with OA pathogenesis.
背景/目的：骨关节炎（OA）是导致受影响人群关节功能障碍、残疾和生活质量下降的主要原因。关节功能障碍的潜在机制涉及氧化应激增加、炎症、软骨细胞外基质降解蛋白酶水平升高和自噬下降——一种细胞防御机制。目前，OA 没有可改变病情的治疗方法。Butea monosperma（Lam.）植物的不同部位在传统的印度阿育吠陀医学系统中被广泛用于治疗各种人类疾病，包括炎症性疾病。在这里，我们研究了 Butea monosperma（Lam.）花（BME）的水-甲醇提取物对 IL-1β刺激的人 OA 软骨细胞的软骨保护作用，该提取物已标准化至 Butein 的浓度。
采用索氏提取法，用 70%甲醇-水混合溶剂制备 Butea monosperma（Lam.）水-甲醇提取物（BME）。通过 MTT 测定法测定 BME 处理后软骨细胞的存活率。使用 TaqMan 测定法和用特异性抗体进行免疫印迹，定量聚合酶链反应（qPCR）测定基因表达水平。通过免疫印迹测定微管相关蛋白 1 轻链 3-II（LC3-II）的水平，并通过透射电子显微镜和共聚焦显微镜观察自噬体，来确定自噬的激活。
BME 在所用剂量下对 OA 软骨细胞无毒，并抑制 IL-1β诱导的白细胞介素 6（IL-6）和基质金属蛋白酶-3（MMP-3）、MMP-9 和 MMP-13 的表达。BME 通过测量免疫印迹法的 LC3-II 水平、透射电子显微镜和共聚焦显微镜观察 BME 处理后的软骨细胞中自噬体的数量，增强了软骨细胞中的自噬。BME 上调了几种自噬相关基因的表达，并增加了人 OA 软骨细胞在病理条件下的自噬流。进一步的分析表明，BME 通过抑制哺乳动物雷帕霉素靶蛋白（mTOR）通路在软骨细胞中激活自噬。重要的是，我们的发现表明，BME 介导的抑制 IL-1β诱导的 IL-6、MMP-3、-9 和 -13 的表达依赖于自噬，并且自噬抑制可阻断其表达。
上述结果表明，Butea monosperma（Lam.）提取物具有很强的潜力，可以激活自噬，并抑制人 OA 软骨细胞中 IL-1β诱导的 IL-6 和 MMP-3、-9 和 -13 的表达。这项研究表明，BME 或源自 BME 的化合物可以被开发为安全有效的软骨保护剂，通过激活自噬来抑制与 OA 发病机制相关的炎症和分解代谢因子的表达。
