珊瑚木苷和藻藍素於壓力誘導之類退化性關節炎軟骨細胞模組之作用探討

A Compression-induced OA-like Chondrocyte Model for Aucubin and C-phycocyanin Evaluation

楊穎奇(In-Chi Young)

指導教授 ： 林峰輝

國立臺灣大學/工學院/醫學工程學研究所/博士(2016年)

https://doi.org/10.6342/NTU201603718

摘要

由於軟骨是關節中負責承重的組織，其自我修復的作用緩慢。在長時間承受外力的作用下往往會對關節軟骨造成難以復原的傷害，進而造成退化關節炎(OA)的發生。外力會影響軟骨細胞對軟骨的代謝作用，並因而改變軟骨的組成。藉由調控軟骨細胞所承受的外力，將有可能找出誘發 OA 的外在條件。本研究藉由對豬軟骨細胞施加一系列不同強度的持續性或間歇性的壓縮力，並觀察軟骨細胞代謝作用的變化，藉此找出誘導豬軟骨細胞退化的外力條件，並建立類退化性關節炎的細胞模組，以作為後續OA 研究或藥物篩選的平台。
本研究結果顯示，軟骨細胞若持續 24 小時承受超過 40 psi 的持續性壓縮力，軟骨細胞外基質(ECM)會因硫酸化糖胺聚醣(sGAG)的合成減少而降解；分解性基因MMP-13的表現量會顯著增加，而為 ECM 主要成分的 aggrecan 與 type II collagen的基因表現也有顯著降低的現象。促炎性基因IL-6的表現量增加以及活性氧(ROS)增加的結果也透露出發炎反應及氧化壓力的徵兆。在另一方面，適度的間歇性壓縮力有助於ECM 成分基因的表現，且不會增加分解性或促發炎性基因的表現。30-60 psi的間歇性壓縮力也會促進合成性蛋白 BMP-7 的產生。結果顯示出豬軟骨細胞會依照外在所施加的持續性或間歇性壓縮力的強度而表現出特定的代謝反應。藉此結果，我們以持續性的壓縮力來製作類退化性關節炎軟骨細胞模組，並隨後應用於藥物篩選上。
在藥物篩選中，中草藥成分中具有抗氧化、抗發炎及抗細胞凋亡作用的珊瑚木苷和藻藍素被選用來進行測試。利用前述的類退化性關節炎細胞作為外力性細胞膜組，搭配過氧化氫誘導的氧化性細胞膜組，共同測試珊瑚木苷和藻藍素對豬軟骨細胞的保護效果。
研究結果顯示珊瑚木苷和藻藍素均具有抑制 ROS 生成、降低 caspase-3 的活性以及抑制軟骨細胞凋亡的功能。珊瑚木苷和藻藍素在經過壓縮力或過氧化氫刺激後的軟骨細胞中仍能維持其 aggrecan 和 type II collagen 的基因表現。此外珊瑚木苷還具有抑制 IL-6 和 MMP-13 基因表現和保護 sGAG 的效果。結果顯示出珊瑚木苷能夠保護軟骨細胞免於外力或氧化壓力所造成的傷害，能抑制OA 初期徵兆的發生。在初期OA 的治療上展現其作為新穎藥物的潛力。

關鍵字

退化性關節炎 ； 外力 ； 氧化壓力 ； 疾病模組 ； 珊瑚木苷 ； 藻藍素

並列摘要

During osteoarthritis (OA) progression, damage from mechanical stress with insufficient self-repair by joints can contribute to the disease. The effects of exerting stress on chondrocyte metabolism can regulate cartilage homeostasis. The specific stress-response condition is therefore a key for OA induction. This study was aimed to produce a cell-based OA system as a disease model by evaluating the metabolic response of porcine chondrocyte with a series of mechanical stimulation via static or cyclic compression.
With static loading over 40 psi, extracellular matrix (ECM) degradation was initiated by a decrease in sulfated-glycosaminoglycans (sGAG) content, up-regulation of catabolic MMP-13 gene, and down-regulation of ECM related aggrecan and type II collagen genes within 24 h. Signs of pro-inflammatory events and oxidative stress were observed with respect to elevated IL-6 expression and reactive oxygen species (ROS) production, respectively. On the other hand, moderate cyclic loading (30-40 psi)-stimulated chondrocytes showed an up-regulation of ECM related genes without significant changes of catabolic and pro-inflammatory genes. BMP-7 concentration is increased after 30 to 60 psi of cyclic loading. These results showed that chondrocytes exhibited specific magnitude-dependent metabolic response to static or cyclic loading. The OA-like chondrocytes was generated accordingly with static loading and was adopted as a disease model for the following compound screening.
In the screening process, herbal compounds aucubin and c-phycocyanin (C-PC) that have been shown to protect cellular components against oxidative stress, along with their anti-inflammation and anti-apoptosis effects, were verified using compression-generated OA-like chondrocyte model. The protective effects of compounds were also tested in oxidative stress-stimulated chondrocytes through H2O2 stimulation. The results showed that both aucubin and C-PC had the ability to inhibit ROS production, reduce caspase-3 activity, and decrease apoptosis cell population. Both compounds can also reverse aggrecan and type II collagen gene expressions after H2O2 or compression stimulation. Besides, aucubin showed a prominent inhibition effect on IL-6 and MMP-13 genes and protection of sGAG after compression and H2O2 stimulation. The results demonstrated that aucubin could prevent early signs of OA caused by compressive and oxidative stresses. Therefore, we suggest that aucubin can be used as a potential drug candidate for early OA treatment.

並列關鍵字

osteoarthritis ； mechanical stress ； oxidative stress ； disease model ； aucubin ； c-phycocyanin

參考文獻

1. Neogi T, Zhang Y. Epidemiology of osteoarthritis. Rheum. Dis Clin North Am 2013; 39:1-19

2. Woolf, AD., Pfleger, B., Burden of major musculoskeletal conditions. Bull World Health Organ 2010; 81: 646–656.

3. Nguyen, UDT., Zhang, Y., Zhu, Y., Niu, J., Zhang, B., Felson, DT., Increasing prevalence of knee pain and symptomatic knee osteoarthritis: survey and cohort data. Ann Intern Med 2011; 155: 725–732.

4. Huang TL, Wu CC, Yue J, Sumi S, Yang KC. l-Lysine regulates tumor necrosis factor-alpha and matrix metalloproteinase-3 expression in human osteoarthritic chondrocytes. Process Biochem 2016;51: 904–11

6. Felson DT. Developments in the clinical understanding of osteoarthritis. Arthritis Res Ther 2009; 11:203.