Diabetes mellitus (DM) is one of the major global health challenges today, due to the rapid increase in its incidence. This disease affects individuals of all ages, social conditions and demographic groups, and is spreading virtually everywhere in the world. DM is currently considered a disease with a large number of hidden cases, as those diagnosed represent only a small part of the real prevalence [1]. This chronic metabolic disorder develops when the pancreas fails to produce adequate amounts of insulin or when body tissues cannot effectively respond to the hormone’s action, leading to disturbances in glucose regulation. When diabetes is not kept under control, excessive glucose in the blood (hyperglycemia) occurs, which, over time, can seriously affect several body systems, especially blood vessels and the nervous system [2].

Approximately 50% of people with diabetes develop a form of corneal degeneration known as diabetic keratopathy [3]. The epithelial surface of the cornea is organized into 5–7 strata of non-keratinized squamous cells arranged in a stratified pattern which are essential for maintaining the transparency and structural integrity of the cornea. Due to the avascular nature of the cornea, the supply of nutrients and glucose to the corneal epithelial cells occurs primarily by diffusion from the aqueous humor rather than from the tear film [4]. Because the aqueous humor contains high glucose (HG) concentrations under hyperglycemic conditions, corneal epithelial cells in patients with diabetes mellitus are subject to prolonged contact with glucose, which contributes to a spectrum of pathological epithelial changes [5]. Disturbance of the corneal homeostatic balance has been widely observed in patients with diabetes. This condition is associated with a malfunction of the epithelial barrier, changes in the structure of epithelial cells and reduced corneal sensitivity [6]. Even small changes in the diabetic cornea can impair healing processes, leading to prolonged recovery times or non-healing epithelial lesions. The delayed healing process exposes patients to serious visual complications, including gradual epithelial thinning, the onset of secondary damage, and the triggering of cytokines that facilitate fibrotic scar development [5].

Corneal epithelial cells form the outermost layer of the ocular surface, protecting the eye from microbes, allergens, and chemicals [7]. The prompt renewal of the epithelial layer, along with the restoration of its protective barrier, is crucial for maintaining corneal architecture and vision-related function [8]. It has been demonstrated that exposure to HG levels leads to dysfunction of the corneal epithelial barrier, leading to a notable reduction in transepithelial electrical resistance (TEER). Therefore, gaining insight into the pathophysiological processes affecting the cornea in patients with diabetes is crucial for maintaining its structural and functional integrity [9, 10].

Moreover, increased oxidative stress resulting from elevated reactive oxygen species (ROS) production has been identified as a key factor in the development of diabetic complications [10]. The cornea, due to its location on the external part of the eye, is constantly exposed to high concentrations of oxygen and solar radiation, both sources of oxidative stress. To counteract this problem, the cornea contains various enzymatic antioxidant defenses, including superoxide dismutase (SOD) and catalase (CAT), and non-enzymatic ones, including glutathione (GSH). SOD catalyzes the conversion (dismutation) of the superoxide anion (O₂$\cdot$^–) into the less harmful free radical hydrogen peroxide (H₂O₂). As the only antioxidant capable of directly reacting with O₂$\cdot$^–, SOD is often considered the principal element of the cornea’s antioxidant defense, this primary antioxidant plays a decisive role in neutralizing reactive oxygen species at the earliest stage of oxidative challenge. CAT is a heme-bound enzyme that catalyzes the breakdown of H₂O₂ into water (H₂O) and oxygen (O₂), using Mn as a cofactor. In addition to detoxifying H₂O₂, CAT protects SOD from inactivation and may play a direct role in filtering UVB radiation. GSH is essential for the catalytic activity of the enzyme glutathione peroxidase (GPX), which performs a similar function to CAT by reducing H₂O₂ to H₂O, but it uses reduced glutathione (GSH) as an electron donor [11].

Studies have linked changes in ROS production to diabetic corneal problems, suggesting that HG and ROS production leads to systemic corneal disease, characterized by peripheral neuropathy, delayed corneal wound healing, and impaired cell migration [12, 13]. Although oxidative stress has been widely studied, the specific mechanisms by which hyperglycemia modulates reactive oxygen species generation and interferes with the repair of corneal epithelial barrier function are not yet fully elucidated.

In previous studies we demonstrated, in HG-treated human retinal microvascular endothelial cells, an activation of cytosolic phospholipases A₂ (cPLA₂) and of mitogen-activated protein kinase (MAPK) ERK1/2 [14] and an increase of cyclooxygenase-2 (COX-2) protein levels and release of inflammatory cytokines [15], highlighting that the activation of the ERK/cPLA2/COX-2 axis represents an early event after exposure to HG, mediating retinal damage. Cytosolic PLA₂ catalyzes the hydrolysis of membrane glycerophospholipids, primarily phosphatidylcholine, at the sn-2 position, releasing arachidonic acid (AA), which is a precursor for eicosanoids such as prostaglandins E2 (PGE2) and leukotrienes (LTs). Cytosolic PLA₂ activation is triggered by various pro-inflammatory signals, initiating a Ca²⁺-dependent translocation from the cytosolic space toward the perinuclear membranes. This process involves phosphorylation of Ser-505 in the catalytic domain by MAPK ERK1/2 and phosphorylation of Ser-727 by other MAPKs [16]. To date, few studies have elucidated the role of the signaling pathway in diabetic corneal diseases.

Cynara cardunculus L. var. altilis (DC.) is a perennial plant native to the Mediterranean region, popularly known as cultivated cardoon and closely related to the more widespread globe artichoke [C. cardunculus L. var. scolymus (L.) Fiori] and the wild cardoon [C. cardunculus L. var. sylvestris (Lamk) Fiori], the progenitor of both cultivated species. Traditionally grown for its edible, fleshy leaf midribs and for the use of its flowers as a source of vegetable rennet [17], the cultivated cardoon has recently attracted interest for its potential in the development of natural extracts for pharmacological applications [18]. The primary reason for this interest is its considerable abundance of bioactive molecules, including phenolic acids, flavonoids, and sesquiterpene lactones [19, 20], which have been shown to possess antioxidant, anti-inflammatory, and hepatoprotective properties [21]. Among the three C. cardunculus taxa, cultivated cardoon is considered the most promising for the extraction of natural bioactive compounds, as it may produce more biomass than wild cardoon and globe artichoke [22].

It has been demonstrated that globe artichoke leaf extract can lower sorbitol levels in rat eye lenses exposed to high glucose conditions. Sorbitol buildup is a hallmark of diabetic retinopathy, and reducing its accumulation could offer protective benefits against this eye condition. Experimental evidence from both ex vivo and in vivo models demonstrates that artichoke leaf extract plays a protective role by decreasing oxidative stress and suppressing inflammatory processes, while simultaneously enhancing the efficiency of the endogenous antioxidant defense system [23, 24, 25]. The cultivated cardoon, a versatile crop in the Mediterranean diet, is particularly rich in bioactive phenolic compounds, recognized for their antioxidant effects, as well as dietary fiber and essential minerals [26]. Polyphenols possess strong antioxidant properties that enable them to neutralize reactive oxygen species, thereby limiting oxidative stress. This protective effect can reduce the risk of oxidative damage to ocular tissues, which is a key factor in the development of age-related eye diseases such as cataracts and macular degeneration [23]. Clearly, these multifaceted actions cannot be performed by a single compound, but rather by a complex mixture of active ingredients, mainly mono- and dicaffeoylquinic acids and flavonoids such as luteolin, which exert additive or synergistic pharmacological effects.

In the present study, we investigated the early cellular and molecular responses of HG-treated human corneal epithelial cells to cultivated cardoon (CC) leaf extract. Our findings indicate that CC extract improved viability, reduced ROS production, increased antioxidant enzyme activities and migration and reduced the inflammatory process after glucose treatment, suggesting its potential as a preventive agent against the development of chronic diabetic complications. The results, obtained after 24- and 48-hour treatments, are preliminary to understanding CC’s potential in diabetic keratopathy.

The global rise in diabetes mellitus has led to a worrying increase in complications associated with the disease. Chronic hyperglycemia causes significant biochemical and metabolic changes that result in both macrovascular and microvascular damage. Macrovascular complications primarily affect the heart and brain, while microvascular damage contributes to neuropathy, nephropathy, and ocular tissue injuries [2]. Patients with diabetes often experience a range of ocular surface issues, including epithelial defects, recurrent corneal erosion, ulcers, corneal edema, superficial punctate keratitis, and delayed corneal epithelial wound healing. In particular, diabetic keratopathy is an ocular complication of diabetic peripheral neuropathy, characterized by impaired corneal epithelial wound healing and reduced subbasal corneal nerve plexus density and hypoesthesia [6]. Clinically, this manifests as ocular surface discomfort, often described as burning or dryness, due to impaired corneal innervation and epithelial integrity [36]. Multiple studies have demonstrated that the diabetic corneal epithelium is characterized by increased cellular fragility, decreased epithelial cell density, reduced epithelial thickness, and compromised barrier integrity. These structural and functional abnormalities are indicative of disrupted cellular homeostasis, involving dysregulation of critical processes such as proliferation, differentiation and migration [37].

It has been shown that both oxidative stress and the inflammatory process are responsible for the damage to the ocular tissues of diabetic patients. Recognizing the significant role of oxidative stress in the progression of diabetic complications, extensive research has investigated the protective potential of various antioxidants, particularly those derived from plants. Natural compounds such as $\alpha$- and $\gamma$-tocopherol, retinol, carotenoids and phytochemicals including anthocyanins and polyphenols, have demonstrated beneficial effects. These substances help mitigate complications by inhibiting the synthesis of prostaglandins and pro-inflammatory cytokines and by reducing oxidative stress markers [38].

Polyphenols, a diverse class of secondary metabolites found in plant-based foods such as fruit, vegetables, nuts and tea, contribute to the maintenance of ocular physiological homeostasis and promote tissue repair under pathological conditions [39, 40].

Cynara cardunculus L. botanical complex is recognized as a rich source of health-promoting compounds, including polyphenols, inulin, vitamins, and minerals [26, 27]. Leaf extracts of both globe artichoke and cardoons have traditionally been used extensively in folk medicine, particularly for the treatment of liver disorders. The therapeutic effects of these extracts have often been attributed to key phenolic constituents particularly present in the heads and leaves, such as 1caffeoylquinic acids and flavones [41].

In our study, CC leaf extract alone did not alter cell viability, as reported in Fig. 2A, nor did it affect any of the measured parameters (data not shown). The treatment of HCEpiCs with 25 mM glucose significantly reduced corneal cell viability, confirming results by others that demonstrated a decreased epithelial cell density in the cornea in the presence of elevated glucose levels, both in animal models and in human research studies [42, 43]. To select the CC leaf extract concentration with the best protective effect on HG-treated HCEpiCs, cell viability was determined using MTT assay, which provides information on cellular metabolic activity, thereby discriminating between living and dead cells. The results demonstrated that the pre-treatment of corneal cells with cultivated cardoon extract preserved their viability by protecting them from HG damage.

Physiologically, to fulfill its protective barrier function, the cornea maintains a high TEER, guaranteed by the presence of tight junctions between epithelial cells, as well as a marked regenerative capacity which, in response to an insult, activates mechanisms of cell proliferation and migration [4]. Ultrastructural analysis via scanning electron microscopy revealed disrupted intercellular architecture in diabetic rats, associated with compromised integrity among corneal cells, responsible of the epithelial barrier [44] and in vitro studies also demonstrated significantly lower TEER values in corneal epithelial cell monolayers under diabetic conditions [12]. Our findings confirmed an HG-induced significative reduction in TEER, indicating a compromise in the integrity of the corneal epithelial barrier, and demonstrated that CC leaf extract maintained high TEER values in the presence of 25 mM glucose. Furthermore, we confirmed that HG treatment slows down the wound healing of HCEpiC [13]. In the present study, the treatment with HG in the presence of CC leaf extract effectively accelerated migration and wound closure to normal levels. Rapid re-epithelialization is essential to ensure the physiological function of the cornea; CC leaf extract, by ensuring a re-epithelialization process, potentially prevents corneal damage. In diabetic patients, delayed epithelial healing increases the risk of ocular complication and microbial infections, particularly in the context of an already compromised immune system [45, 46]. Consequently, considerable efforts have been directed toward identifying agents capable of enhancing epithelial repair in the diabetic cornea such as cytokines [47], silk-derived proteins [48] and natural substances [49]. Our findings showed the effect of CC extract in promoting and accelerating cell migration, counteracting the damage of HG, and represent a promising outcome in the context of therapies based on the use of natural substances.

A close correlation has been also shown between diabetic corneal dysfunction and increased ROS production. Prolonged hyperglycemia can trigger glucose-induced protein auto-oxidation and promote ROS generation [35].

Oxidative stress is the consequence of a disrupted balance in which ROS are generated at a rate that surpasses the cell’s antioxidant capacity to neutralize or repair the resulting oxidative damage, particularly present in the conjunctiva and cornea, highly exposed to UV radiation and particularly vulnerable to oxidative stress [50]. In diabetic mice, corneal injury leads to heightened oxidative stress, which significantly hinder corneal epithelial healing and elevate the risk of persistent epithelial defects [51].

The endogenous antioxidant defense system includes both enzymatic and nonenzymatic antioxidants. Enzymatic antioxidants consist of SOD, CAT, glutathione peroxidase and glutathione oxidase, and, among the non-enzymatic antioxidants, a significant role is played by GSH, synthesized by metabolic processes [52]. In patients with type 2 diabetes mellitus, insulin treatment has been shown to only partially reduce oxidative stress, as evidenced by persistently low GSH levels [10].

We therefore examined the effect of CC leaf extract on ROS concentrations and on several antioxidant enzyme activities and we found that CC leaf extract significantly decreased the production of ROS, increased SOD and CAT antioxidant activities as well as GSH content in HCEpiCs in a high glucose environment.

In eukaryotic cells, the bulk of oxidative metabolism is carried out in the mitochondria and endoplasmic reticulum. Within these organelles, standard redox reactions inevitably lead to the generation of free radicals, which, if not efficiently neutralized, can contribute to oxidative stress. The mitochondrial electron transport chain is responsible for producing approximately 90% of cellular ROS [53]. Moreover, metabolic pathways involving enzymes such as COX-2 and cPLA₂ play a key role in ROS generation. A study on an in vivo/ex vivo hypoxia model showed that the activation of cPLA₂ is involved in ventilator-induced diaphragm dysfunction by promoting mitochondrial ROS production, highlighting a positive interaction between cPLA₂ activity and oxidative stress at the mitochondrial level [54]. It also demonstrated in microglial cells, stimulated with lipopolysaccharide (LPS), an increase in the cPLA₂ activation alongside ERK1/2 activation and the ROS production induced by LPS was significantly reduced by cPLA₂ inhibitors such as arachidonyl trifluoromethyl ketone (AACOCF3) but not by the iPLA₂ inhibitor BEL, indicating that cPLA₂ is specifically involved in mediating LPS-induced oxidative stress in microglial cells [55]. Moreover, in lens epithelial cells, cPLA₂ plays a crucial role in PDGF-stimulated cell growth, with its activation tightly regulated by calcium-dependent membrane translocation. This membrane targeting is essential for cPLA₂ to activate NADPH oxidase, leading to increased ROS production. These findings highlight that cPLA₂ plays a key role in linking calcium signaling to oxidative stress in corneal epithelial cells [56].

Therefore, elevated intracellular ROS not only damage macromolecules but also function as signaling molecules, triggering a cascade of biochemical and molecular responses that disrupt normal cellular signaling pathways [57, 58]. Our results showed the significant inhibitory effect of CC extract on HG-induced cPLA₂ and ERK1/2 phosphorylated forms and COX-2 and on PGE2 and IL-6 production. These findings suggest that CC extract may exert wide protective effects on glucose stressed HCEpiCs. One could hypothesize that, during ocular pathologies due to diabetes complications, free radicals could activate the ERK1/2/cPLA₂/COX-2 signaling, triggering the inflammatory process and, at the same time, the activation of this pathway could lead to the production of other ROS, creating a positive feedback loop (Fig. 9). We have not directly tested whether ROS act exclusively as upstream activators or are generated as a downstream product of this cascade. Further investigation will be necessary to validate the regulatory role of ROS in this context.

Fig. 9.

Potential signaling pathways modulated by Cynara cardunculus L. cultivated cardoon (CC) leaf extract in high glucose exposed human corneal cells. Cynara cardunculus L. extract enhances the antioxidant defense enzymes including SOD, CAT activities and GSH content and inhibits ROS production and pro-inflammatory pathways, especially ERK 1/2/cPLA₂/COX-2 signaling in high glucose exposed human corneal cells, highlighting the possibility that it could perform a protective function in the management of diabetic keratopathy. BioRender. Scuto, M. (2025) https://BioRender.com/an2rc7d.

In this scenario, identifying therapeutics that could alleviate these conditions is crucial for advancing new treatment strategies for diabetic keratopathy. Our results indicate that CC leaf extract could have strong potential as a therapeutic agent, controlling both the inflammatory process and the production of ROS, thus maintaining intact the protective and regenerative functions of the cornea.

This study reports preliminary results on the effect of CC extract after in vitro treatment of corneal cells with HG. It is hoped that the results obtained will lay the foundation for future in vivo and clinical studies. Clearly, CC extract alone will not be able to replace the traditional therapy that diabetic patients undergo, but it could be used as a dietary supplement to reduce the damage in diabetic keratopathy.

Despite decades of research into oxidative stress as a key pathogenic factor in the development of diabetes mellitus and its complications, this area remains highly relevant. Contemporary approaches to diabetes management are increasingly centered on personalized antioxidant therapies, incorporating targeted delivery systems and strategies that focus on specific sources of ROS.

Further investigation should be carried out to identify reliable biochemical markers of free radical activity and to explore the interplay between oxidative stress and inflammation, aiming to better understand their combined roles in ocular disease progression.

We investigated the effects of CC extract on cell migration and viability using an in vitro model of hyperglycemia-induced corneal cells. Our results demonstrated that cultivated cardoon leaf extract (i) reduced oxidative stress by lowering ROS production and enhancing SOD, CAT activities and GSH content in high glucose exposed human corneal cells; (ii) maintained a high TEER and promoted corneal epithelial wound healing; (iii) induced down-regulation of the ERK 1/2/cPLA₂/COX-2 signaling pathway involved in the inflammatory process and ROS production.

Collectively, these findings indicate that cultivated cardoon extract has the potential to enhance human corneal epithelial cell function. This provides a scientific basis for considering its development as a therapeutic agent in the prevention or treatment of diabetic keratopathy.

All data reported in this paper will also be shared by the lead contact upon request.

GL, ATS, and CDA designed the research study. AC, AA, CP, SL, GP and MS performed the research. GL, ATS, CDA and CL analyzed the data. GL, ATS, and CDA wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

The authors thank Antony Bridgewood for language assistance. For some experimental procedures the authors used instruments available in the Service Center B.R.I.T. BIO-Nanotech Research and Innovation Tower. The authors thank AUDI project proposal.

This research was funded by “University of Catania under Grant Scheme PIACERI”, Linea di Intervento 1, University of Catania, Italy.

The authors declare no conflict of interest. Given her role as the Editorial Board member, Gabriella Lupo had no involvement in the peer-review of this article and has no access to information regarding its peer review. Given her role as the Guest Editor, Maria Scuto had no involvement in the peer-review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Adrian Gericke.