Zhen Li1,2, Yu Han2, Yan Ji3, Kexin Sun1,4, Yanyi Chen1,4, Ke Hu5,6. 1. Department of Ophthalmology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology and Chongqing Eye Institute, Chongqing, China. 2. Department of Ophthalmology, The People's Hospital of Leshan, Leshan, Sichuan Province, China. 3. Department of Ophthalmology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology and Chongqing Eye Institute, Chongqing, China. j_ycw@163.com. 4. Chongqing Medical University, Chongqing, China. 5. Department of Ophthalmology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology and Chongqing Eye Institute, Chongqing, China. 42222@qq.com. 6. Chongqing Medical University, Chongqing, China. 42222@qq.com.
Abstract
PURPOSE: Oxidative 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. METHODS: HCECs 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. RESULTS: When 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. CONCLUSION: Our 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.
PURPOSE: Oxidative 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. METHODS: HCECs 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. RESULTS: When 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. CONCLUSION: Our 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.
Authors: Y Kaji; T Usui; T Oshika; M Matsubara; H Yamashita; M Araie; T Murata; T Ishibashi; R Nagai; S Horiuchi; S Amano Journal: Invest Ophthalmol Vis Sci Date: 2000-02 Impact factor: 4.799