| Abstract: | 研究目的：蘋果酸被廣泛應用在化妝品中，然而關於蘋果酸的皮膚安全性報告至今仍然很缺乏。本研究將探討蘋果酸對於人類角質細胞株的生物效應及分子機轉。 研究方法及資料：首先以人類表皮角質細胞株(HaCaT)與正常人類表皮角質細胞(NHEKs)添加不同濃度(10 mM、12.5 mM、15 mM、17.5 mM、20 mM)的蘋果酸處理不同時間，分析細胞存活率以及細胞週期。接著再以HaCaT進行後續實驗，使用相位差顯微鏡、流式細胞儀、DAPI染色、DNA斷裂分析、海馬生物能量測定儀及西方墨點法來偵測細胞凋亡及細胞週期停滯分子機轉。 研究結果：實驗結果指出，經由DAPI染色、DNA斷裂分析及偵測到sub-G1期發現蘋果酸誘發人類角質細胞產生細胞凋亡。流式細胞儀觀察到蘋果酸會增加粒線體過氧化物(mito-SOX)並且降低粒線體膜電位。使用海馬生物能量測定儀分析蘋果酸處理的人類角質細胞株，發現粒線體耗氧量下降而細胞外酸化速度增加。蘋果酸促進凋亡相關蛋白FasL、Fas、Bax、Bid、caspases-3、caspases-8、caspases-9、cytochrome c表現量增加而抑制Bcl-2及PARP蛋白表現，蘋果酸並促進內質網壓力路徑相關GRP78、GADD153與ATF6α表現量增加。 結論與建議：本研究證實蘋果酸經由G0/G1細胞週期停滯而抑制人類皮膚角質細胞增生，並經由內質網及粒線體依賴路徑誘發細胞程序性死亡。本研究結果將提醒消費者及醫師留意化妝產品中蘋果酸及其他果酸的潛在風險。
Objective：Malic acid (MA) has been commonly used in cosmetic products, but the safety reports in skin are sparse. In this study, we investigated the biological and molecular effects of MA in human keratinocyte cell lines (HaCaT). Methods and Materials：HaCaT cells and normal human epidermal keratinocytes (NHEKs) were treated with MA at different concentrations (10 mM, 12.5 mM, 15 mM, 17.5 mM and 20 mM) for various time periods. Cell viability and cell cycle were analyzed by using flow cytometry. The molecular mechanisms of anti-proliferation through cell cycle arrest and apoptosis were investigated in HaCaT cells by phase-contrast microscope, flow cytometry, DAPI stain, DNA fragmentation, Seahorse XF 24, and Western blot. Results：The data showed that MA induced DNA damage and apoptosis based on the observations of DAPI staining, DNA fragmentation, and sub-G1 phase in HaCaT cells. Flow cytometric assays also showed that MA increased the production of mitochondrial superoxide (mito-SOX) but decreased the mitochondrial membrane potential. Analysis of bioenergetics function with the XF 24 analyzer Seahorse extracellular flux analyzer demonstrated that oxygen consumption rate (OCR) was significantly decreased whereas extracellular acidification rate (ECAR) was increased in MA-treated keratinocytes. The occurrence of apoptosis was proved by the increased expressions of FasL, Fas, Bax, Bid, caspases-3, -8, -9, cytochrome c, and the declined expressions of Bcl-2, PARP. MA also induced endoplasmic reticulum stress associated protein expression such as GRP78, GADD153, and ATF6α. Conclusion and Suggestion：We demonstrated that MA had anti-proliferative effect in HaCaT cell through the inhibition of cell cycle progression at G0/G1, and the induction of programmed cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways. Our investigation highlights to consumers and physicians the potential risks of malic acid and other alpha-hydroxy acids which may be contained in cosmetic products. |
