抗發炎和抗氧化介入對早期退化性關節炎之療效

Abstract

骨關節炎（OA）是最常見的關節疾病，由於人口老齡化而日益普遍。造成骨關節炎的原因是由於軟骨上出現缺陷、軟骨變薄、表面變得粗糙使關節處上下骨頭的負擔加重，導致關節間隙變窄，形成骨刺。許多研究表明，骨關節炎患者的滑液膜液中氧化壓力上升且抗氧化酶水平下降，因此氧化壓力是骨關節炎進展中的重要因素，故減少關節腔內氧化物質的量並提高抗氧化物質以減緩骨關節炎的進展為本研究之重要目標。已有文獻指出diosmin、hesperidin 和aucubin三種萃取物具有抗氧化及抗發炎之功能，故我們使用diosmin、hesperidin 和aucubin萃取物來處理OA細胞，觀察這些萃取物其抗氧化和抗發炎機制並找出這些萃取物最適濃度及給予時間點。研究結果顯示高劑量(50和100 μM)的hesperidin和diosmin會對軟骨細胞造成死亡，然而 5和10 μM hesperidin可以增加總抗氧化能力並恢復H2O2刺激下之軟骨細胞中的superoxide dismutase和glutathione Peroxidase活性，而5 μM diosmin可改善由H2O2刺激誘導的軟骨發炎反應，故diosmin對H2O2刺激下的軟骨細胞具有保護作用。在mRNA表現量上，因H2O2導致損傷的軟骨細胞中，發現dosmin可降低iNOS，COX-2，IL-1β，COL1A1，MMP-3，MMP-9，提升TIMP-1，SOX9和 COL2A1 mRNA表現量，而hesperidin處理下則降低COX-2，IL-1β，TNF-α，MMP-3，MMP-9，提升IL-10，TIMP-1和SOX9 mRNA表現量，表明hesperidin和diosmin不僅可以穩定表型及降低發炎因子外，而且對軟骨細胞具有合成代謝作用。此外，我們也發現hesperidin和diosmin的抗氧化能力是短暫的 (24小時以內) ，且皆是透過Foxo和Nrf2信號通路調節抗氧化能力。在aucubin萃取物實驗中，結果顯示玻尿酸結合 aucubin有益於軟骨細胞中細胞外基質產生並增強玻尿酸對OA軟骨細胞的抗分解代謝和抗發炎作用。三個萃取物都具有抗發炎效果及有助軟骨細胞外基質維持，而其中hesperidin相較於diosmin具有更好的抗老化及抗氧化能力。由於針對骨關節炎疾病緩解藥物很少，故此研究針對氧化壓力探討將為骨關節炎這種破壞性疾病的潛在治療策略提供有價值的觀點，我們也期許之後可以將研究結果應用於支架或佐劑的開發，以改善並提高早期退化性關節炎之療效。

Osteoarthritis (OA) is the most common joint disorder with increasing prevalence due to aging of the population. In Taiwan, people aged 75 years old and above have a 85% chance to be recognized with degenerative arthritis of the knee, it most commonly affects the knees, hands, feet, the hips, and the spine. Cause of OA is due to the defects appearing on the cartilage, as the cartilage becomes thinner, the surface becomes rough, burdening the upper and lower bone, thus forming the narrowed joint space and bone spurs. These leads to inflammation and swelling of the joints. Many studies have shown an increasing oxidative stress and a decreasing level of antioxidants in synovial fluid in OA patients. Oxidative stress is known as an important factor in the progress of OA. Therefore, reducing the amount of oxidized substances in the joint cavity should slow the progression of OA, and is an important goal to support the cartilage repair. Previous literature has pointed out that the three components of diosmin, hesperidin and aucubin have antioxidant functions. Therefore, we treat diosmin, hesperidin and aucubin components to study their anti-oxidation and anti-inflammatory properties of chondrocytes stimulated with H2O2 or IL-1β. The results showed that high doses (50 and 100 μM) of hesperidin and diosmin caused death in chondrocytes, whereas 5 and 10 μM hesperidin increased total antioxidant capacity and restored superoxide dismutase and glutathione Peroxidase activity in chondrocytes stimulated by H2O2. 5 μM dosmin can improve the cartilage inflammatory response induced by H2O2 stimulation, so diosmin has a protective effect on chondrocytes stimulated by H2O2. Dosmin down-regulated the mRNA levels of iNOS, COX-2, IL-1β, COL1A1, MMP-3, MMP-9, up-regulated TIMP-1, SOX9, COL2A1 and hesperidin down-regulated the mRNA levels of COX-2, IL-1β, TNF-α, MMP-3, MMP-9, up-regulated IL-10, TIMP-1, SOX9 in H2O2-injuried chondrocytes, which revealed that hesperidin and diosmin may not only stabilize the phenotype but also have anabolic effects on chondrocytes. In addition, we also found that hesperidin and diosmin antioxidant capacity is short-lived (within 24 hours) and regulate antioxidant capacity through the Foxo and Nrf2 signaling pathways. In the aucubin experiment, it showed that hyaluronic acid (HA) binding to aucubin is beneficial to the extracellular matrix production in chondrocytes and enhances the anti-catabolic and anti-inflammatory effects of HA on OA. All three extracts (diosmin, hesperidin and aucubin) have anti-inflammatory effects and maintain the extracellular matrix of the cartilage. In addition, hesperidin has better anti-senescence and anti-oxidation ability than diosmin. As disease-modifying drugs for osteoarthritis are rare, targeting the oxidative stress would offer a valuable perspective for exploration of potential therapeutic strategies in the treatment of this devastating disease. We expect this study to be beneficial and applicable to the development of scaffold or adjuvants to improve the efficacy of early OA.