α-硫辛酸对黑素细胞氧化应激下的保护作用及抑制自噬机制的研究
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摘要
目的:探讨α-硫辛酸(α-LA)对体外培养的人黑素细胞氧化损伤的保护作用。方法:以不同浓度的α-LA(100、200及400μmol/L)预处理正常人黑素细胞12 h,再加入500μmol/L H_2O_2处理6 h。采用CCK-8试剂盒检测黑素细胞活力,流式细胞仪分析细胞内活性氧(ROS)含量、钙离子水平及细胞周期,线粒体膜电位检测试剂盒(JC-1)分析线粒体膜电位,图像分析软件测量树突回缩速率,透射电镜观察超微结构以及western blot检测LC3和p62蛋白表达情况。结果:正常情况下α-LA能显著提高黑素细胞的活力,同时还能明显降低氧化应激时黑素细胞内ROS水平,减轻细胞内超微结构的损伤,阻止线粒体去极化及细胞内钙离子的累积,延缓树突的回缩,促进增殖及降低过度激活的自噬水平(P<0.05)。结论:α-LA不仅能在正常情况下显著提高黑素细胞的活力,而且可明显降低氧化应激下黑素细胞过度激活的自噬水平,对黑素细胞起保护作用。
Objective: To investigate the protective effect of α-lipoic acid on H_2O_2 induced oxidative stress in human melanocytes in vitro. Methods: Human melanocytes were pretreated with different concentrations of α-lipoic acid(100, 200 and 400 μmol/L)for 12 h, followed by incubation with 500 μmol/L H_2O_2 for 6 h. Melanocyte proliferation was assessed using CCK-8 assay, while flow cytometry was used to measure intracellular ROS content, intracellular calcium and cell cycle. JC-1 assay was used to measure mitochondrial membrane potential. Dendritic retraction rate was evaluated by image analysis software, and cell ultrastructure was observed under a transmission electron microscopy. The expression levels of LC3Ⅱ/LC3Ⅰ and p62 protein were assessed by western blot. Results: Under normal conditions, α-lipoic acid significantly increased the viability of melanocytes,while under H_2O_2-induced oxidative stress, α-lipoic acid prevented melanocytes from oxidative stress, including reductions in intracellular ROS levels, reduced ultrastructural damage, prevention of mitochondrial depolarization and accumulation of calcium, delayed dendritic retraction, stimulation of proliferation, and inhibition of hyperactive autophagy. Conclusions: α-lipoic acid not only increases the viability of melanocytes under normal condition, but also protects human melanocytes against oxidative stress by inhibiting hyperactive autophagy.
Objective: To investigate the protective effect of α-lipoic acid on H_2O_2 induced oxidative stress in human melanocytes in vitro. Methods: Human melanocytes were pretreated with different concentrations of α-lipoic acid(100, 200 and 400 μmol/L)for 12 h, followed by incubation with 500 μmol/L H_2O_2 for 6 h. Melanocyte proliferation was assessed using CCK-8 assay, while flow cytometry was used to measure intracellular ROS content, intracellular calcium and cell cycle. JC-1 assay was used to measure mitochondrial membrane potential. Dendritic retraction rate was evaluated by image analysis software, and cell ultrastructure was observed under a transmission electron microscopy. The expression levels of LC3Ⅱ/LC3Ⅰ and p62 protein were assessed by western blot. Results: Under normal conditions, α-lipoic acid significantly increased the viability of melanocytes,while under H_2O_2-induced oxidative stress, α-lipoic acid prevented melanocytes from oxidative stress, including reductions in intracellular ROS levels, reduced ultrastructural damage, prevention of mitochondrial depolarization and accumulation of calcium, delayed dendritic retraction, stimulation of proliferation, and inhibition of hyperactive autophagy. Conclusions: α-lipoic acid not only increases the viability of melanocytes under normal condition, but also protects human melanocytes against oxidative stress by inhibiting hyperactive autophagy.
引文
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[2]Jian Z,Tang L,Yi X,et al.Aspirin induces Nrf2-mediated transcriptional activation of haem oxygenase-1 in protection of human melanocytes from H2O2-induced oxidative stress[J].J Cell Mol Med,2016,20(7):1307-1318.
[3]Chang Y,Li S,Guo W,et al.Simvastatin protects human melanocytes from H2O2-induced oxidative stress by activating Nrf2[J].JInvest Dermatol,2017,137(6):1286-1296.
[4]Dell’Anna ML,Picardo M.A review and a new hypothesis for non-immunological pathogenetic mechanisms in vitiligo[J].Pigment Cell Res,2006,19(5):406-411.
[5]Dell’Anna M L,Mastrofrancesco A,Sala R,et al.Antioxidants and narrow band-UVB in the treatment of vitiligo:a double-blind placebo controlled trial[J].Clin Exp Dermatol,2007,32(6):631-636.
[6]Goder A,Nagel G,Kraus A,et al.Lipoic acid inhibits the DNArepair protein O 6-methylguanin-DNA methyltransferase(MGMT)and triggers its depletion in colorectal cancer cells with concomitant autophagy induction[J].Carcinogenesis,2015,36(8):817-831.
[7]Wang Y,Li S,Li C.Perspectives of new advances in the pathogenesis of vitiligo:from oxidative stress to autoimmunity[J].Med Sci Monit,2019,25:1017-1023.
[8]Kimura S,Fujita N,Noda T,et al.Monitoring autophagy in mammalian cultured cells through the dynamics of LC3[J].Methods Enzymol,2009,452:1-12.
[9]Namazi MR.Neurogenic dysregulation,oxidative stress,autoimmunity,and melanocytorrhagy in vitiligo:can they be interconnected?[J].Pigment Cell Res,2007,20(5):360-363.
[10]Gauthier Y,Cario-Andre M,Lepreux S,et al.Melanocyte detachment after skin friction in non lesional skin of patients with generalized vitiligo[J].Br J Dermatol,2003,148(1):95-101.
[11]Kumar R,Parsad D,Kanwar AJ.Role of apoptosis and melanocytorrhagy:a comparative study of melanocyte adhesion in stable and unstable vitiligo[J].Br J Dermatol,2011,164(1):187-191.
[12]Mizushima N,Komatsu M.Autophagy:renovation of cells and tissues[J].Cell,2011,147(4):728-741.
[13]Chen Y,McMillan-Ward E,Kong J,et al.Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells[J].Cell Death Differ,2008,15(1):171-182.
[14]Cao X,Chen A,Yang P,et al.Alpha-lipoic acid protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy[J].Biochem Biophys Res Commun,2013,441(4):935-940.
[15]Zhang J,Deng H,Liu L,et al.α-Lipoic acid protects against hypoxia/reoxygenation-induced injury in human umbilical vein endothelial cells through suppression of apoptosis and autophagy[J].Mol Med Rep,2015,12(1):180-186.