酚酸化合物對 methylglyoxal 誘導 Neuro-2A 神經細胞凋亡之保護效應及其分子作用機轉
Methylglyoxal (MG) 屬於活性雙羰基化合物，為蛋白質糖化過程中間產物，主要經由糖解作用代謝而來。在糖尿病患者血液或組織中皆可發現高濃度之 MG 存在。研究指出，生理上糖化作用與糖尿病所引發之神經病變有密切之關係。越來越多證據顯示，神經細胞凋亡為神經性病變之重要機制。酚酸化合物普遍存在許多蔬果來源中，酚酸化合物所具有的生理活性也被廣泛研究，如抗氧化、抗發炎、降低心血管疾病及抗癌等生理功能，然而對於糖化產物誘導神經細胞凋亡之保護效應卻少有文獻提及。本研究乃探討酚酸化合物對於 MG 誘導 Neuro-2A 神經細胞凋亡之效應及其訊息傳導路徑。結果顯示， MG 與 Neuro-2A 神經細胞共培養後會隨時間 (24-72 h) 與濃度 (100-400 μM) 之增加而顯著降低細胞存活率及增加細胞毒性 (p<0.05)。由細胞週期分佈之結果發現，隨著 MG 處理濃度上升 (100-400 μM)，細胞週期顯著性 (p<0.05) 堆積在 sub-G1 phase。進一步利用 Annexin V-FITC/PI 雙染分析，結果顯示， MG (400 μM) 與細胞培養 72 h，發現凋亡細胞累積達到 27.9 %。由粒線體膜電位變化之結果得知， MG 可能透過破壞粒腺體完整性而誘導神經細胞走向凋亡。進一步探討凋亡相關蛋白質之表現，由 Bax 及 Bcl-2 之分析結果得知，MG 會造成 Bax/ Bcl-2 ratio顯著性提昇 (p<0.05)。此外，在 caspase-3 及 PARP 蛋白質表現方面，caspase-3 活性及蛋白質表現，皆會受到 MG 誘導而提升，進而導致 PARP 裂解。確立了 MG 對神經細胞凋亡之傷害模式後，以共培養方式分別介入十四種酚酸化合物，探討其對於細胞之保護能力。酚酸化合物與 MG 共培養 72 h，發現酚酸化合物能提高細胞存活率與降低 MG造成之毒性效應，其中以 chlorogenic acid、syringic acid 及 vanillic acid 之保護效果最佳。由以上結果得知，酚酸化合物具有減緩 MG 所誘導之粒線體破壞而造成神經細胞凋亡之潛力。進一步探討抗氧化力較佳且較具細胞保護效應之 vanillic acid 於抑制 MG 所誘導之神經細胞凋亡之可能訊息傳導路徑。由實驗結果得知，MG 及 vanillic acid 與 Neuro-2A 神經細胞共培養 3 h，單獨處理 MG 會造成細胞內活性氧之顯著上升 (p<0.05)。介入不同濃度之 vanillic acid (20 及 50 μM) 後，能有效抑制 MG 所引發之氧化壓力 (p<0.05)。由 MAPK 家族之分析結果顯示，MG 之處理會活化細胞中 JNK 及 p38 分子之表現。此外 MG 亦透過活化 PKC 蛋白質而提升 NAD(P)H oxidase 之次單元 p47phox 蛋白質表現，造成大量 ROS 產生。此外，MG 可促使神經細胞內糖化產物 N-ε-carboxymethyllysine (CML) 之大量累積 (p<0.05)。當同時介入 vanillic acid 可明顯減緩 JNK、p38 MAPK 與 PKC 之活化及減少 CML 之累積。綜合以上結果，酚酸化合物具有保護神經細胞因 MG 所誘導凋亡之能力，可能是透過抑制活性氧之生成，進而調控 JNK 及 p38 MAPK 以及 PKC之表現，另外亦可以減少高度糖化終產物 CML 的累積，而達到保護神經細胞之功效，對於預防糖尿病所併發之神經性損傷可能有其生理功效。 In the process of protein glycation, methylglyoxal is a reactive dicarbonyl compound physiologically generated as an unstable intermediate, and is found in high levels in blood or tissue of diabetic subjects. Biological glycation has been commonly implicated in the development of diabetic microvascular complications such as neuropathy. Increasing evidence suggests that disorder of neuronal cell proliferation followed by apoptosis is an important mechanism in the development of diabetic neuropathy complication. Phenolic acids are widely distributed in various vegetables and fruits and possess many physiological functions such as anti-oxidation, anti-inflammation, anti-tumor, and prevention from cardiovascular diseases. However, the protective effect of phenolic acids on glycation product induced apoptosis in neuronal cell remains unclear.In this study, we investigated the inhibitory effects of phenolic acids on methylglyoxal (MG)-induced apoptosis of neuroblastoma cell (Neuro-2A) and the involved molecular mechanisms. The data showed that MG exhibited inhibitory effects on proliferation and viability of Neuro-2A cells in time-and dose dependent manners. Cells treated with MG (400 μM) for 72 h indicated that cell cycle was significantly arrested at sub-G1 phase. Furthermore, the data of Annexin V-FITC/PI double stained assay showed that MG significantly increased the number of apoptotic cells (27.9 % that compared with control). Under MG treatment, the mitochondrial membrane potential was reduced, which was resulted from the modulation of Bax/Bcl-2 ratio. Beside, the expression of caspase-3 was elevated and resulted in the cleavage of PARP. We further investigated the cytoprotective effect of phenolic acids on methylglyoxal-induced damage. Among fourteen phenolic acids, we found that chlorogenic acid, syringic acid, and vanillic acid showed the most inhibitory effect on MG-induced Neuro-2A cells death. The data showed that cells co-incubated with phenolic acid and MG could prevent Neuro-2A cell against apoptosis via maintaining the integrity of mitochondria. Furthermore, vanillic acid, which shows better efficacy in antioxidant capacity and cytoprotection among fourteen phenolic acids which evaluated in this study, was used to investigate its molecularly inhibitory effect on MG-induced apoptosis in Neuro-2A cells . Data showed that vanillic acid (20 and 50 μM) significantly reduced ROS production under the treatment of MG. We found MG could elevate levels of the phosphorylation of JNK and p38 MAPK members. In addition, we also found that MG induced activation of PKC and p47phox. Interestingly, MG also induced accumulation of N-ε-carboxymethyllysine (CML), which is a typical glycotoxin of advanced glycation end products, and Neuro-2A cells co-treated with vanillic acid markedly suppressed JNK, p38, PKC, p47phox activation and CML accumulation.In conclusion, the inhibitory abilities of phenolic acids on MG-induced Neuro-2A cells apoptosis are through decreasing oxidative stress which result in the activation of MAPK pathway, and inhibiting expression of PKC, and preventing the accumulation of CML. Therefore, we suggest that phenolic acids possess cytoprotective ability in the prevention of diabetic neuropathy complication.
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