- 學位論文
兒茶素（-）-epigallocatechin-3-gallate (EGCG)對骨關節疾病的影響
The Effect of (-)-epigallocatechin-3-gallate (EGCG) on Bone and Joint Disease
摘要
茶是亞洲地區最受歡迎的飲料。（-）-epigallocatechin-3-gallate（EGCG）是綠茶中萃取物中最豐富而且也是最有效的兒茶素，而且因為具有強效的抗氧化作用，所以也是目前被研究最多的兒茶素。因為EGCG具有促進骨組織生成和抗發炎作用，因此對骨骼和關節健康多有助益。在促進骨髂健康方面，過去的流行病學、臨床和動物研究中，報導了綠茶或其生物活性成分對骨骼健康的保護作用，綠茶的攝取可以改善骨質流失並降低骨質疏鬆性骨折的風險。但是EGCG與受傷後骨頭修復及再生能力之間的關係仍不清楚。在本研究中，我們分別使用兩種動物模式，股骨骨缺損模式及單獨脛骨骨折模式，分別來研究EGCG對於骨組織缺損中新骨再生的影響，及探討EGCG其對骨折癒合促進的作用。 此外EGCG能夠調節發炎反應，因此可能是骨關節炎的潛在治療選擇。先前的研究顯示，EGCG會顯著降低基質金屬肽酶(MMPs)(MMP-1和MMP-13)和聚集蛋白聚醣酶-1和-2的產生，因此可能是治療關節炎的新方向。除此之外EGCG還表現出抗氧化和抗發炎的特性，研究顯示EGCG能夠抑制人類軟骨細胞和滑膜細胞發炎性物質的產生，並可以抑制介白素-1β誘導的軟骨糖胺聚醣的釋放。創傷性骨關節炎是因為關節損傷而繼發性骨關節炎。關節創傷後發生的即時反應包括細胞壞死和細胞凋亡及各種組織分解反應。由於EGCG具有抗氧化和抗發炎的特性，對於創傷性骨關節炎發病機轉的抑制是有益的，我們認為EGCG可應用於創傷性骨關節炎的治療。在本研究中，我們將使用創傷關節炎動物模式，來研究EGCG對於創傷性關節炎的治療及其可能的機轉。 此研究中將分成兩部份。第一部分，我們將專注於EGCG對骨組織的作用。我們將利用股骨缺損模式及單獨脛骨骨折模式來研究局部使用EGCG對骨組織增生及骨折修復的影響。我們將使用組織形態分析，骨形成蛋白-2(BMP-2)的免疫組織化學分析，放射學評估（X射線和顯微電腦斷層）以及生物力學測試來證明，EGCG對骨組織櫎生及骨折修復的促進作用。在第二部份骨關節炎的研究中，我們將使用前十字韌帶截斷誘導創傷性骨關節炎模型，驗證EGCG對於關節炎的作用。我們將進行關節功能分析，組織形態分析，第Ⅱ型、第Ｘ型膠原蛋白、自噬作用相關蛋白的免疫組織化學分析，軟骨細胞凋亡的染色，來評估EGCG對軟骨保護的效果。 此研究已經成功證明，EGCG在骨缺損處的局部應用，可以增加新生的骨組織量，並且改善機械性能（包括最大負荷，斷裂點，剛度，最大負荷曲線下的面積，斷點曲線下的面積和極限應力）。此外局部施用EGCG也可通過增加骨痂組織來促進骨折癒合，幫助恢復骨骼的機械強度，包括最大負荷量，楊氏系數，剛度和斷裂負荷。而免疫組織化學分析也顯示，EGCG對骨組織新生和骨折癒合的促進作用，與骨形成蛋白-2的增加表現有關。這些結果證實EGCG對骨缺損和骨折修復具有明確的骨促進作用。 而在創傷性關節炎的研究中，我們也發現關節注射EGCG可以減輕前十字韌帶切除誘導的骨關節炎症狀，且提高老鼠關節炎後患肢的承重能力及跑步耐力。從組織學上來看，EGCG能降低軟骨的破壞，有較低的OARSI組織評分，且會增加糖胺聚醣和第II型膠原蛋白的含量，並降低第X型膠原蛋白。除此之外，我們也發現EGCG的治療可以減少軟骨創傷後的細胞凋亡，並且增加細胞自噬作用。因此，此項研究的結果顯示EGCG對於創傷性關節炎的軟骨具有明確保護作用，能夠減少軟骨糖胺聚醣流失且降低第II型膠原蛋白破壞，而此項的保護作用可能是藉由減少細胞凋亡及增加細胞自噬作用的機轉。 整體而言，我們的研究顯示，局部注射EGCG可以促進骨組織的新生及骨折處癒合。而關節內注射EGCG可以減少軟骨細胞凋亡，促進細胞自噬作用，減少創傷性關節炎的進展。EGCG能提供促進骨創傷修復及減少骨關節炎惡化新的治療方向。
並列摘要
Tea is the most popular beverage in the Asian area. (-)-epigallocatechin-3-gallate (EGCG), the most plentiful catechin extracted from green tea, was the most studied bioactive compound because of its potent antioxidant effects. EGCG was also reported to provide a beneficial impact on bone and joint health due to its osteogenesis promotion and anti-inflammatory effect. Numerous epidemiological, clinical, and animal studies have reported the health benefits of green tea or its bioactive extracts and indicated that green drinking can reduce bone loss and decrease the risk of osteoporotic fractures, however, the relationship between tea consumption and bone repair or regeneration after injury remained unclear. In this study, we used a femoral bone defect model to investigate the effect of EGCG on bone regeneration in the femoral bone defect and also used an isolated tibial bone fracture model to investigate its effects on enhancing bone fracture healing. EGCG can modulate inflammation and can be a potential treatment option for arthritis. EGCG significantly decreases the production of matrix metallopeptidases (MMPs) (MMP-1 and MMP-13) and aggrecanase-1 and -2 and is considered a promising agent for inhibiting the pathogenesis of arthritis. EGCG also exhibits anti-oxidant and anti-inflammatory activities and has been reported to be able to inhibit the production of inflammatory mediators in human chondrocytes and synovial fibroblasts and to suppress the IL-1β-induced glycosaminoglycan release from cartilage. Post-traumatic osteoarthritis (PTOA) is a secondary OA that occurred after a significant joint injury. The immediate responses that occur after joint trauma involve cell death by necrosis and apoptosis combining with the activation of various catabolic events. According to the above possible beneficial mechanisms of EGCG on PTOA pathogenesis, we proposed that EGCG is effective in the treatment of PTOA. This research will be divided into two parts, bone study and joint study. In the first part, we will focus on the effect of EGCG on bone promotion. In this section, we will use a femoral bone defect model and an isolated tibia fracture model to determine the effect of local application of EGCG on bone tissue regeneration and fracture repairment. We will use histomorphological analysis, immunohistochemical analysis of bone morphogenetic protein-2 (BMP-2), radiological evaluation (X-ray and micro-CT), and biomechanical testing to investigate its effects on bone tissue. In the second part of the study, we will use the anterior cruciate ligament transection (OA) induced PTOA model to verify the effect of EGCG on joint arthritis. We will perform joint function analysis, tissue morphology analysis, immunohistochemical analysis of type II and type X collagen, autophagy-related proteins, and staining of chondrocyte apoptosis to evaluate the effect of EGCG on cartilage protection. We have successfully proved that the local application of EGCG at bone defect can de novo bone formation by increasing bone volume and subsequently improve mechanical properties including max load, breakpoint, stiffness, area under the max load curve, area under the breakpoint curve, and ultimate stress. Moreover, locally administered of EGCG can promote fracture healing by improving the callus size and then aid in recovering the mechanical strength of the bone, including the max load, modulus, stiffness, and break load. The promotion effect of EGCG on bone regeneration and fracture healing is related to the upregulation of BMP-2 by EGCG. These results demonstrated the positive bone promotion effect of EGCG on bone defect and fracture. In PTOA study, we found that intra-articular injection of EGCG can alleviate OA-induced OA and improves the functional performance in rats in terms of the weight-bearing ability of OA limb and running endurance. Histologically, EGCG decreases cartilage degradation and results in less OARSI scores. It not only preserves the contents of collagen type II and glycosaminoglycan but also decreases the degradation marker protein (collagen type X). Eventually, EGCG reduces the chondrocyte apoptosis by increase the function of autophagy. In conclusion, our results indicated that EGCG may be useful for the prevention of cartilage degradation in PTOA. Overall, our research shows that local injection of EGCG can promote bone tissue regeneration and fracture healing. Intra-articular injection of EGCG can reduce chondrocyte apoptosis, promote autophagy, and reduce the progression of PTOA. Accordingly, EGCG can provide a new treatment direction to promote bone wound repair and reduce the deterioration of PTOA.