| Abstract: | 近年來醫學美容和皮膚保養產品是個相當熱門的話題，其中alpha-hydroxyacids (AHAs)成分被相當廣泛的使用。然而，過去對長時間使用AHAs的安全性一直沒有獲得很好的解答。之前的研究已經針對AHAs當中的甘醇酸和乳酸進行細胞毒性的探討，在本篇研究中我們繼續針對AHAs當中的檸檬酸進行研究，探討檸檬酸是否會對人類皮膚角質細胞(HaCaT)產生細胞毒性作用。實驗結果顯示，檸檬酸對人類皮膚角質細胞(HaCaT)會誘導細胞凋亡和造成細胞週期的停滯，進而產生細胞毒性作用。結果顯示檸檬酸不僅是呈現劑量－依賴性的模式來抑制HaCaT細胞增生，同時也會誘導細胞凋亡和引起細胞週期分別停滯於G2/M 期(24小時之前)和S 期(48小時)。西方點墨法的結果指出，檸檬酸於24小時之前會抑制cyclin B、P21蛋白的表現，增加Chk2蛋白表現進一步造成cdc25c磷酸化而使cdc2減低，造成細胞週期停滯在G2/M 期。於48小時會增加cyclin A、cyclin E、CDK2、P21、P27蛋白表現，而造成細胞週期停滯在S 期。我們也觀察到細胞形態上的改變，並利用DAPI染色偵測DNA受損情形。流式細胞儀結果指出，檸檬酸是經由粒線體膜電位下降，進而使粒線體釋放出AIF、Endo G、cytochrome c到細胞質中。此外，檸檬酸也經由增加Bax的表現及抑制Bcl-2及Bcl-xL的表現並活化了caspase 9及caspase 3隨後啟動了caspase-依賴性及caspase-非依賴性的凋亡路徑。檸檬酸促使粒線體膜電位下降及活性氧的增加可能和細胞質鈣離子增加有關。此外，檸檬酸也能經由活化死亡接受器Fas進一步活化下游的caspase-8或使細胞質中的Bid解活化。因此，檸檬酸可經由粒線體路徑及活化死亡接受器路徑來誘發人類皮膚角質細胞的凋亡。對應到臨床的應用，皮膚在接觸檸檬酸之後產生的換膚脫皮現象，依據我們的實驗結果推測可能為檸檬酸引起的角質細胞凋亡所造成。未來我們將持續進行相關實驗，藉以提供更多臨床上應用果酸的學理依據。
Cosmetic and skin care products have been hot topics in dermatology for decades. Alpha-hydroxyacids (AHAs) have been widely used. However, there is concern about its safety for long-term use. We had evaluated the potential cytotoxic effects of glycolic acid and lactic acid in previous studies. In this study, we investigated the cytotoxic effect of citric acid in human keratinocyte cell line (HaCaT). The study results suggested that citric acid was cytotoxic to HaCaT cells via the mechanisms of induction of apoptosis and cell cycle arrest. The results indicated that citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent mode, but also induced apoptosis and cell cycle arrest at G2/M phase (before 24 hours) and S phase (at 48 hours). Western blotting results before 24 hours showed that citric acid decreases the levels of cyclin B, cdc2, cdc25c, P21 and increase the levels of chk2 that causes cell cycle arrest at G2/M phase. Western blotting results at 48 hours showed that citric acid increases the levels of cyclin A, cyclin E, CDK2, P21, P27 that cause cell cycle arrest at S phase. We observed cell morphological changes, and also used DAPI stain to evaluate DNA damage. The results of flow cytometry showed that citric acid induces HaCaT cell apoptosis through mitochondrial membrane potential (MMP) decrease and AIF, Endo G, and cytochrome c release from mitochondria to the cytosol. In addition, citric acid increased the level of Bax and inhibited the levels of Bcl-2, Bcl-xL and activated caspase 9 and caspase 3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathway. We also found that the decrease of MMP and the increase of reactive oxygen species (ROS) may be related to the increase of cytoplasmic calcium level. Citric acid also activated death receptor and increased the levels of caspase 8, or activated Bid protein. Therefore, citric acid could induce apoptosis through mitochondrial pathway and death receptor pathway in human kerationocyte cell line (HaCaT). Our study results suggested that the chemical exfoliation after applying citric acid might be caused by the induction of keratinocyte apoptosis. In the future, we will conduct further experiments to provide more academic basis for clinical application of hydroxyacids. |
