The effect of a-Lipoic acid (ALA) on oxidative stress, inflammation, and apoptosis in high glucose-induced human corneal epithelial cells.
The effect of a-Lipoic acid (ALA) on oxidative stress, inflammation, and apoptosis in high glucose-induced human corneal epithelial cells.
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α-硫辛酸 (ALA) 对高糖诱导的人角膜上皮细胞氧化应激、炎症和细胞凋亡的影响
DOI：
10.1007/s00417-022-05784-6
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发表时间：
2023-03
影响因子：
2.7
通讯作者：
Hu, Ke
中科院分区：
文献类型：
--
作者：
Li, Zhen;Han, Yu;Ji, Yan;Sun, Kexin;Chen, Yanyi;Hu, Ke
关键词：
目的氧化应激和炎症反应在糖尿病性角膜病变（diabetic keratopathy，DK）的发生、发展中起重要作用。AGEs的过度积累及其与角膜中AGE受体（RAGE）的结合导致氧自由基的形成和炎症细胞因子的释放，诱导细胞凋亡。本研究旨在探讨ALA对高糖诱导的人脐静脉内皮细胞（HCECs）中AGEs积累的影响，并探讨ALA抗高糖诱导的HCECs中AGEs轴介导的氧化应激、凋亡和炎症的分子机制，为治疗DK提供细胞学依据（5.5、10、25、30、40和50 mM）孵育48 h。CCK-8法检测细胞增殖;应用Annexin V-异硫氰酸荧光素（V-FITC）/PI试剂盒检测细胞凋亡，流式细胞仪和TUNEL细胞凋亡试剂盒检测凋亡细胞。根据细胞增殖和细胞凋亡的结果，在以下HG实验中使用25 mM葡萄糖培养基。评估ALA对HG诱导的HCEC的作用。正常葡萄糖组（NG组）、渗透压对照组（OP组）、高糖组（HG组）、高糖+ALA组（HG + ALA组）分别用5.5mM葡萄糖（NG组）、5.5mM葡萄糖+22.5mM甘露醇（OP组）、25 mM葡萄糖（HG组）和25 mM葡萄糖+ ALA（HG + ALA组）处理HCECs 24和48 h。ELISA试剂盒检测细胞内AGEs的积累。采用RT-PCR、Western blotting和免疫荧光法分别定量检测炎性小体、过氧化氢酶（CAT）、超氧化物歧化酶2（SOD 2）、切割半胱氨酸-天冬氨酸蛋白酶-3（Cleaved caspase-3）、Toll样受体4（TLR 4）、Nod样受体蛋白3（NLRP 3）、白细胞介素1 β（IL-1 β）和白细胞介素18（IL-18）。结果当葡萄糖浓度高于25 mM时，细胞增殖明显受到抑制，凋亡率明显增加（P< 0.001）;在HG环境中，ALA处理可缓解HCECs的抑制作用，且呈剂量依赖性，25 μM ALA为最低有效剂量。ALA可显著降低细胞内AGEs的积累（P< 0.001），激活CAT和SOD 2蛋白和基因的表达（P< 0.001），从而抑制ROS诱导的氧化应激和细胞凋亡。此外，ALA还能有效下调TNF α、TLR 4、NLRP 3、IL-1B、IL-18的蛋白和基因水平（P< 0.05），结论本研究表明ALA可能是治疗DK的理想药物，因为它具有减少晚期糖基化终末产物（AGEs）积累和下调AGEs表达的潜在能力。高糖诱导的人角膜上皮细胞（HCECs）中，地塞米松介导的氧化应激、细胞凋亡和炎症，这可能为最终具有临床益处的治疗靶点提供细胞学基础。
PurposeOxidative stress and inflammation had been proved to play important role in the progression of diabetic keratopathy (DK). The excessive accumulation of AGEs and their bond to AGE receptor (RAGE) in corneas that cause the formation of oxygen radicals and the release of inflammatory cytokines, induce cell apoptosis. Our current study was aimed to evaluate the effect of ALA on AGEs accumulation as well as to study the molecular mechanism of ALA against AGE-RAGE axis mediated oxidative stress, apoptosis, and inflammation in HG-induced HCECs, so as to provide cytological basis for the treatment of DK.MethodsHCECs were cultured in a variety concentration of glucose medium (5.5, 10, 25, 30, 40, and 50 mM) for 48 h. The cell proliferation was evaluated by CCK-8 assay. Apoptosis was investigated with the Annexin V- fluorescein isothiocyanate (V-FITC)/PI kit, while, the apoptotic cells were determined by flow cytometer and TUNEL cells apoptosis Kit. According to the results of cell proliferation and cell apoptosis, 25 mM glucose medium was used in the following HG experiment. The effect of ALA on HG-induced HCECs was evaluated. The HCECs were treated with 5.5 mM glucose (normal glucose group, NG group), 5.5 mM glucose + 22.5 mM mannitol (osmotic pressure control group, OP group), 25 mM glucose (high glucose group, HG group) and 25 mM glucose + ALA (HG + ALA group) for 24 and 48 h. The accumulation of intracellular AGEs was detected by ELISA kit. The RAGE, catalase (CAT), superoxide dismutase 2 (SOD2), cleaved cysteine-aspartic acid protease-3 (Cleaved caspase-3), Toll-like receptors 4 (TLR4), Nod-like receptor protein 3 (NLRP3) inflammasome, interleukin 1 beta (IL-1 ß), and interleukin 18 (IL-18) were quantified by RT-PCR, Western blotting, and Immunofluorescence, respectively. Reactive oxygen species (ROS) production was evaluated by fluorescence microscope and fluorescence microplate reader.ResultsWhen the glucose medium was higher than 25 mM, cell proliferation was significantly inhibited and apoptosis ratio was increased (P< 0.001). In HG environment, ALA treatment alleviated the inhibition of HCECs in a dose-dependent manner, 25 μM ALA was the minimum effective dose. ALA could significantly reduce the intracellular accumulation of AGEs (P< 0.001), activate protein and genes expression of CAT and SOD2 (P< 0.001), and therefore inhibited ROS-induced oxidative stress and cells apoptosis. Besides, ALA could effectively down-regulate the protein and gene level of RAGE, TLR4, NLRP3, IL-1B, IL-18 (P< 0.05), and therefore alleviated AGEs-RAGE-TLR4-NLRP3 pathway–induced inflammation in HG-induced HCECs.ConclusionOur study indicated that ALA could be a desired treatment for DK due to its potential capacity of reducing accumulation of advanced glycation end products (AGEs) and down-regulating AGE-RAGE axis–mediated oxidative stress, cell apoptosis, and inflammation in high glucose (HG)–induced human corneal epithelial cells (HCECs), which may provide cytological basis for therapeutic targets that are ultimately of clinical benefit.
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影响因子：
3.2
作者：
EL Midaoui, A;Elimadi, A;de Champlain, J
通讯作者：
de Champlain, J
DOI：
10.3760/cma.j.issn.0412-4081.2018.06.018
发表时间：
2018-06-11
期刊：
[Zhonghua yan ke za zhi] Chinese journal of ophthalmology
影响因子：
--
作者：
Dan, J;Zhou, Q J;Xie, L X
通讯作者：
Xie, L X
抑制TLR4减轻高糖条件下视网膜神经节细胞的炎症和凋亡
DOI：
10.1007/s00417-017-3772-0
发表时间：
2017-11-01
影响因子：
2.7
作者：
Hu, Lili;Yang, Hongxia;Jiang, Shuanghong
通讯作者：
Jiang, Shuanghong
影响因子：
3.7
作者：
AnandBabu, Kannadasan;Sen, Parveen;Angayarkanni, Narayanasamy
通讯作者：
Angayarkanni, Narayanasamy
影响因子：
3.4
作者：
Bejarano E;Taylor A
通讯作者：
Taylor A