The effect of a-Lipoic acid (ALA) on oxidative stress, inflammation, and apoptosis in high glucose–induced human corneal epithelial cells

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.

Genipin relieves diabetic retinopathy by down-regulation of advanced glycation end products via the mitochondrial metabolism related signaling pathway

“…Upon exposure to sugar, proteins, lipids, and nucleic acids are oxidized and rearrange to form stable products, which can further undergo crosslinking, thus yielding AGEs[ 39 ]. AGEs are associated with a series of diseases such as Alzheimer’s disease, skin senescence, face splash capillary ectasia, and pathologies such as AMD, diabetic keratopathy, and DR. AGEs are reactive intermediates of chronic hyperglycemia with proteins, lipids, and nucleic acids in vivo , leading to the accumulation of pro-inflammatory cytokines and ROS.…”

Diabetic Keratopathy: Redox Signaling Pathways and Therapeutic Prospects

“…α-Lipoic acid is a naturally occurring molecule with recognized antioxidative features, which attenuated AGE accumulation, blocking AGE-RAGE-ROS-mediated oxidative stress and cells apoptosis, and suppressed AGE-RAGE-TLR4-NLRP3 cascade-related inflammation in an in vitro study on high glucose-exposed human corneal epithelial cells [147].…”

Cemtirestat, an aldose reductase inhibitor and antioxidant compound, induces ocular defense against oxidative and inflammatory stress in rat models for glycotoxicity

“…13 DM considered as a possible negative factor for corneal endothelial functions as well. 14 Accumulating evidence suggests that metabolism-based regulation of OS and activated polyol pathway are potential targets for developing promising treatments not only for retinopathy, but for cataract and even other ocular disorders in DM. 15,16 In this respect, the beneficial effects of some redox modulatory compounds [16][17][18] and natural 19,20 or synthetic derivatives 21 of AR inhibitors (ARIs) on the diabetic eye has been reported.…”

“…Corneal epithelial cells are also affected by high glucose, OS, CS, and inflammation has proven to play an important role in the progression of diabetic neuropathy 13 . DM considered as a possible negative factor for corneal endothelial functions as well 14 …”