香豆素對高葡萄糖培養周邊神經膠細胞之影響

Abstract

根據2016-2019年國民營養健康狀況變遷調查顯示，國人18歲以上罹患糖尿病的盛行率高達9.82%，其中更有約50%的患者會進展為糖尿病周邊神經病變 (diabetic peripheral neuropathies)，並增加日後截肢、死亡的風險。許旺細胞 (Schwann's cell)是周邊神經系統中的神經膠細胞，在神經修復、營養支持上扮演重要角色。但在糖尿病高糖環境中，會因為細胞內氧化壓力增加導致細胞凋亡而造成周邊神經受損，並出現麻木、疼痛等症狀。香豆素 (coumarin)廣泛存在於蔬菜、水果及中草藥之中，具有抗氧化、抗癌、抗發炎等功效。本研究探討三種化學結構相似的天然香豆素化合物對體外高葡萄糖培養周邊神經膠細胞之影響。實驗使用RSC96 (rat Schwann cells)許旺細胞株，並在高糖濃度 (150 mM glucose)培養基中分別加入結構相似的香豆素化合物，包括繖型酮 (umbelliferone)、七葉樹苷 (esculetin)及瑞香素 (daphnetin)。首先，以MTT試驗分析細胞的存活率，結果顯示，與高糖組相比，以繖型酮、七葉樹苷處理時，許旺細胞之存活率並無顯著差異。然而，25 μM的瑞香素卻能顯著提升細胞存活率7%，並且可顯著增加Nrf2 (nuclear factor erythroid 2-related factor 2)在細胞核中含量、GCLC (glutamate cysteine ligase catalytic subunit)和GSH (glutathione)的生成以及降低細胞中H2O2濃度。並進一步發現，瑞香素可藉由調節Bax (BCL2-Associated X)、Bcl-2 (B-cell lymphoma-2)以及降低caspase-3蛋白表現量抑制許旺細胞的凋亡。然而，瑞香素對Nrf2路徑中的抗氧化相關蛋白HO-1 (heme oxygenase-1)、NQO1 (Quinone Oxidoreductase 1)並非有顯著增加的效果。由上述結果推測，150 mM葡萄糖會使許旺細胞中的氧化壓力及細胞凋亡增加，而瑞香素可藉由調節Nrf2入核量，降低高糖對許旺細胞造成的傷害。但瑞香素處理高糖誘導許旺細胞第24小時，可能並非Nrf2作用最高之時間點，又或是尚有其他抗氧化調節路徑，導致HO-1、NQO1表現量並無顯著提升，因此未來此方面可再深入探討。

According to the 2016-2019 Nutrition and Health Survey in Taiwan, the prevalence rate of people over the age of 18 suffering from diabetes is as high as 9.82%, of which around 50% of patients will develop into diabetic peripheral neuropathies (DPN). Schwann cells are the glial cells that exist in the peripheral nerve system, which play an important role in nerve repair and nutritional support. Coumarin derivatives, which is abundant in vegetables, fruits and Chinese herbal medicine, exhibited antioxidation, anticancer and anti-inflammatory effects. This study investigates the effect of coumarin derivatives on peripheral glial cell incubated in high glucose medium. The RSC96 (rat Schwann cells) cell line incubated in medium contains 150 mM glucose, and then treated with umbelliferone, esculetin or daphnetin. The results from MTT assay showed that the high glucose group compared to that treated with umbelliferone and esculetin in the cell viability rate was no significant difference. However, 25 μM daphnetin can significantly increase the cell viability by 7%. Additionally, daphnetin can significantly increase the content of Nrf2 (nuclear factor erythroid 2-related factor 2) in the nucleus, the production of GCLC (glutamate cysteine ligase catalytic subunit) and GSH (glutathione) and reduce the concentration of H2O2 in cells. It was further found that daphnetin can inhibit the apoptosis of Schwann cells by regulating Bax (BCL2-Associated X), Bcl-2 (B-cell lymphoma-2) and reducing the expression of caspase-3. Nevertheless, daphnetin did not significantly increase the antioxidant-related proteins, including HO-1 (heme oxygenase-1) and NQO1 (Quinone Oxidoreductase 1) in the Nrf2 pathway. From the above results, it is speculated that 150mM glucose can lead to increase the oxidative stress and apoptosis in Schwann cells, but daphnetin can reduce the damage by regulating the Nrf2 pathway. However, RSC96 cells were incubated with 150 mM glucose with daphnetin for 24 h may not be the best time for Nrf2 effect, or there are other antioxidant regulatory pathways, resulting in no significant increase in the expression of HO-1 and NQO1. Therefore, this aspect can be further explored in the future.