2.1

Glycolysis and DED

The glycolytic pathway, also known as glycolysis, is a crucial metabolic pathway that involves the catalysis of glucose by various enzymes to produce metabolic products and ATP, thereby providing energy to the cell . When transported into the cytoplasm, glucose then undergoes glycolysis, with the resulting pyruvate predominantly utilized for oxidative phosphorylation (OXPHOS) after the tricarboxylic acid (TCA) cycle, thereby facilitating the production of ATP. Glucose-6-phosphate (G-6-P) and 3-phosphoglycerate, conducted as intermediates within glycolysis, are closely related to the pentose phosphate pathway and the biosynthesis of amino acids. Glycolytic intermediates play a crucial role not only in energy provision but also in bolstering the immune cellular response to extrinsic stimuli. Despite its relative efficiency in ATP generation compared to the TCA cycle and OXPHOS, glycolysis stands as a significant metabolic route for the activation of immune cells. In the period of active inflammation, immune cells heavily rely on glycolysis as their principal metabolic pathway to fulfill the exigencies of the inflammatory process, transitioning back to OXPHOS as inflammation subsides. Dysregulation of glycolysis has been identified as a contributing factor to a range of medical conditions, including type 2 diabetes, rheumatoid arthritis, and cancer.

Researcher findings have demonstrated notably elevated concentrations of glucose, lactate, and creatine in tears of DED patients, implying a potential state of heightened energy metabolism. Jiang et al. observed a significant increase in levels of malic acid, L-lactic acid, pyruvate, D-glucose, L-proline, and citric acid, as well as their metabolic pathways, indicating a substantial enhancement in glycolysis/gluconeogenesis. Perumal et al. illustrated, via proteomic analysis of tears from aqueous-deficient and evaporative dry eye patients, an upregulation in the expression of glycolytic enzymes ENO1, PGK1, and ALDOA. Furthermore, Chen et al. elucidated a notable enrichment in amino acid biosynthesis, glycolysis/gluconeogenesis, and glutathione metabolism through proteomic and metabolomic analysis of tears from DED patients, among which six glycolysis-related proteins were upregulated, namely ENO1, PRDX6, PGAM1, PKM, ALDOA, and GAPDH. Moreover, a study conducted by Qingfeng Ni et al. suggested that lactate dehydrogenase A, ENO1, and PKM may serve as potential targets for treating meibomian gland dysfunction.

These findings indicate that the upregulation of glycolysis plays a significant role in the ocular surface metabolic changes of DED patients. As the control of glycolytic flux primarily depends on the activity of three key rate-limiting enzymes, namely hexokinases (HKs), phosphofructokinase-1 (PFK-1), and pyruvate kinases (PKs), we would further elaborate on the glycolytic process in dry eye patients focusing on the alterations in these three enzymes and their effects ( Fig. 1 ).

Changes of glycolysis and key metabolic enzymes in patients with dry eye. HKs, hexokinases; G-6-P, glucose-6-phosphate; F-6-P, fructose-6-phosphate; F-1,6-P, fructose-1,6-biphosphate; PFK-1, phosphofructokinase-1; DHAP, dihydroxyacetone phosphate; G-3-P, glyceraldehyde-3-phosphate; 1,3-DPG, 1,3-diphosphoglycerate; 3-PG, 3-phosphoglycerate; 2-PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; TCA, tricarboxylic acid; OXPHOS, oxidative phosphorylation.

2.2

Lipid metabolism and DED

Lipids in tears constitute a vital component of the tear film, primarily deriving from the meibomian glands. Research has substantiated that wax esters, cholesterol esters, triglycerides, diglycerides, and free fatty acids are the primary lipid constituents found in meibum secretion. These lipids play a crucial role in upholding the surface tension, viscosity, elasticity, and osmotic pressure of the tear film, contributing to maintaining the integrity of the tear film on the ocular surface and reducing tear evaporation. Variations in the lipid profile, both terms of quality and quantity, are indicative of alterations in tear composition.

Previous studies have indicated that levels of triglycerides are heightened in the tears of DED patients and are susceptible to reactive oxygen species (ROS). Biomarkers associated with lipid peroxidation, including HNE and malondialdehyde, are increased in patients diagnosed with meibomian gland dysfunction (MGD). Borchman et al. conducted a principal component analysis of infrared spectra of meibum samples from MGD patients and healthy individuals, and they found that MGD patients had relatively lower levels of cholesterol esters compared to normal individuals. These studies suggest that alterations in tear composition and meibomian gland lipid metabolism occur throughout the progression of DED and could potentially serve as vital factors in its pathophysiology. Delving into lipid research within DED has the potential to unveil novel biomarkers and therapeutic targets essential for the identification and management of DED patients.

Lipid metabolism is closely associated with ocular inflammation in dry eye ( Fig. 2 ). Bioactive lipids generated from the precursors of omega-6 or omega-3 essential polyunsaturated fatty acids (PUFA), including classical eicosanoids, specialized pro-resolving mediators (SPM), sphingolipids, and endocannabinoids, not only play a role in the initiation and amplification of inflammation but also contribute to the resolution and termination of inflammatory responses, exerting synergistic effects at different stages of the inflammatory process. The classic twenty-carbon fatty acids exhibit potent pro-inflammatory properties, while SPMs promote the resolution of inflammation by inhibiting pro-inflammatory cytokines and inducing the production of anti-inflammatory mediators. On the contrary, omega-3 PUFAs undergo enzymatic conversion into SPMs, thereby exerting anti-inflammatory effects.

Changes of lipid metabolism in patients with dry eye. PUFA, polyunsaturated fatty acids; AA, Arachidonic acid; PLA2, phospholipase A2; COX2, cyclooxygenase; SPMs, specialized pro-resolving mediators; Cer, ceramide; S1P, sphingosine 1-phosphate; C1P, ceramide 1-phosphate; SM, sphingomyelin; TCA, tricarboxylic acid.

Ceramide (Cer), sphingosine 1-phosphate (S1P), ceramide 1-phosphate (C1P), and their precursor molecule sphingomyelin (SM) are currently among the most extensively researched sphingolipids, playing pivotal roles in cellular apoptosis and inflammation processes. Ceramide serves as a critical component in SPL metabolism, directly or indirectly associated with inflammation through its downstream metabolites C1P and S1P. Enhanced sphingolipid metabolism is a significantly altered metabolic pathway in the peripheral cornea and aqueous humor of DED. Robciuc et al. reported that sphingomyelin (SM) is one of the most important sphingolipids and is crucial for maintaining homeostasis in the pathological physiological processes of the anterior segment of the eye. Ham et al. studied tear samples from normal and dry eyes of New Zealand female white rabbits and found that the types and concentrations of SM molecules in DED tears were significantly higher than those in the control group. Xiaoniao Chen et al. demonstrated that SM is upregulated in the peripheral cornea and aqueous humor of DED patients, and the hydrolysis of sphingomyelin leads to an accumulation of ceramides in corneal epithelial cells of DED patients. Given the significant role of sphingolipids in regulating inflammatory responses, it is reasonable to speculate that elevated levels of SM and Cer are involved in the occurrence of inflammation in patients with dry eye diseases.

2.3

Amino acid metabolism and DED

Amino acids are essential nutrients in organisms and serve as the building blocks for protein synthesis. Free amino acids in tissues participate in various biological processes and help maintain organ and tissue homeostasis. The tear film contains multiple amino acids, and studies have reported significant differences in amino acid metabolism between DED patients and healthy individuals. Amino acid profiling has the potential to function as a sensitive biomarker for detecting ocular inflammation, offering a novel perspective on comprehending the pathophysiologic mechanism of various ocular surface diseases.

3.1

MAPK signal pathway

The mitogen-activated protein kinase (MAPK) is a crucial cellular signaling mediator involved in the regulation of ocular inflammation and immune responses. MAPK consists of three main subtypes: c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinases (ERK), and p38 mitogen-activated protein kinase (p38 MAPK). Among these, MAPK activated protein kinase 2 (MK2) and functions as a key checkpoint kinase in the cell cycle. Phosphorylation of the p38α MAPK subtype and its formation into a complex with MK2 facilitate transcription, protein synthesis, changes in cell surface receptor expression, and alterations in cell cytoskeletal structure, ultimately impacting cell survival and apoptosis.

Novellasdemunt et al. demonstrated that stress stimuli, such as NaCl, can phosphorylate MK2 and promote the transcription and conformational activation of PFKFB3 via the p38 MAPK/MK2 pathway, leading to enhanced activation of PFK1 and subsequent increase in glycolytic rate. In an experimental DED mouse model, the activation of the MAPK signaling pathway led to a notable increase in phosphorylated forms of ERK, JNK, and p38 within corneal and conjunctival epithelial cells. Additionally, there was a marked upregulation of pigment epithelium-derived factor (PEDF) expression in tears of DED patients. Furthermore, the administration of recombinant PEDF showed promising therapeutic effects by suppressing the production of inflammatory cytokines such as IL-β, IL-6, TNF-α, and IL-17a, through the inhibition of the MAPK p38 and JNK signaling pathways, thereby effectively treating DED mice. Su Li et al. have developed a highly efficient treatment for DED by developing an eye drop formulation of Losmapimod, a p38 MAPK inhibitor with dual anti-inflammatory and antioxidant effects, coupled with ROS scavenger Tempo in cationic peptide micelles to break the vicious cycle of dry eye inflammation. Inhibiting the MAPK signaling pathway to alleviate ocular inflammation in dry eyes is an effective strategy.

3.2

NF-κB signaling pathway

Nuclear factor kappa B (NF-κB) is a group of widely distributed gene pleiotropic transcription factors that exist in eukaryotic cells. It is composed of NF-κB1 (p50, its precursor p105), NF-κB2 (p52, its precursor p100), RelA (p65), RelB, and C-Rel, forming homologous or heterologous dimeric protein complexes. Different combinations of NF-κB and Rel proteins form different NF-κB complexes, with p50/p65 being the most widely distributed and active, also known as NF-κB. Typically, NF-κB remains in the cytoplasm by binding to its inhibitory factor (IκB) protein family members or by associating with its precursors, p100 or p105, and therefore lacks transcriptional activity. Among them, IκBα is the most important regulatory protein that plays a significant role in masking the nuclear localization signal of NF-κB, thereby impeding its translocation into the nucleus for the regulation of gene transcription.

Upon cellular stimulation, IκBα undergoes phosphorylation and subsequent degradation by IκB kinase (IKK), resulting in the dissociation of NF-κB, exposure of the nuclear localization signal, and subsequent translocation into the nucleus to regulate the transcription of target genes. Furthermore, NF-κB can bind to κB binding sites present in gene promoters responsible for encoding cytokines such as TNF-α, IL-1β, and MMP-9, thereby regulating the expression of these genes. The classical NF-κB pathway is predominantly triggered by receptors for pro-inflammatory cytokines and pattern recognition receptors. This pathway involves the activation of the IKK complex, which comprises NF-κB essential modulator, leading to the degradation of IκBα and the nuclear translocation of dimers containing p50, p65, and c-Rel. Subsequently, these dimers initiate the transcription of pro-inflammatory genes.

The NF-κB signaling pathway is a vital target in the innate and adaptive immune response in DED. Hyperosmolarity of tear film, as the primary pathogenic factor in DED, triggers the phosphorylation and translocation of NF-κB p65 to the nucleus, resulting in the regulation of various pro-inflammatory genes such as IL-6, TNF-α, IL-1β. Research by Joo Youn Oh et al. has shown that targeting the HSPB4/TLR2/NF-κB axis in macrophages holds promise for therapeutic interventions in DED. Chang He et al. discovered that in cases of DED related to chronic graft-versus-host disease, there was an activation of the TLR2-mediated NF-κB signaling pathway, which led to an inflammatory state. Donghui Yu et al. reported that PM2.5, as a risk factor of DED, caused the activation of p65 and downstream molecules, leading to DED-related inflammatory response. It is worth mentioning that Tisochrysis lutea , which is rich in PUFA, has been proven to improve DED symptoms as a functional food supplement by inhibiting the degradation of IκB-α and activation of NF-κB. These indicated that the NF-κB signaling pathway is a pivotal pathway and viable therapeutic target in DED inflammation.

3.3

PI3K/AKT/mTOR signaling pathway

Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT (also known as protein kinase B, PKB) signaling pathway is essential for the regulation of various cellular processes within corneal epithelial homeostasis, including cell proliferation, apoptosis, glucose metabolism, and protein synthesis. Growth factors such as epidermal growth factor, insulin-like growth factor 1, substance P/neurokinin 1, and insulin trigger the phosphorylation of PI3K by binding to their respective membrane receptors, and activating the PI3K/AKT signaling pathway, resulting in promoting corneal epithelial cell proliferation, migration, anti-apoptosis, anti-inflammatory response, and wound healing functions. The mammalian target of rapamycin (mTOR) is an atypical serine/threonine protein kinase and acts as a sensor for ATP, amino acids, and hormones within the cellular environment, which regulates cell growth, autophagy, and metabolism. AKT primarily regulates cell growth by stimulating synthetic routes and inhibiting degradation processes like apoptosis and autophagy via the mTOR1 pathway. PTEN activates the PI3K/AKT signaling pathway to suppress glycolysis by diminishing the levels of PFKFB3 and PKM2.

In DED, the hyperosmolar conditions induced oxidative stress and an excessive accumulation of ROS, thereby inhibiting the PI3K/AKT pathway, and culminating in corneal inflammation, and tissue damage. Consequently, reinvigorating the PI3K/AKT pathway may be a promising therapeutic strategy for DED. Astaxanthin, a carotenoid, has been shown to restore the activity of the PI3K/AKT pathway and notably reduced the expression of high mobility group box 1 (HMGB1) as well as inflammatory cytokines TNF-α and IL-1β in a dose-dependent manner, potentially serving as a treatment for DED. Moreover, the immunosuppressant cyclosporine A (CsA) impedes the expression of TNF-α, p-NF-κB p65, and Bax, consequently mitigating cell apoptosis and inflammation by reviving the PI3K/AKT pathway in corneal epithelial cells in vitro.

3.4

AMPK signaling pathway

Adenosine 5′-monophosphate-activated protein kinase (AMPK) serves as a crucial cellular energy sensor responding to signals of energy consumption, particularly stimulated by higher levels of the AMP/ATP ratio. Its regulatory functions extend to diverse metabolic processes encompassing mTORC1, fatty acid oxidation, glycolysis, and maintenance of mitochondrial homeostasis. AMPK suppresses synthetic metabolism to reduce ATP consumption, while simultaneously enhancing catabolic metabolism to generate ATP. This pivotal function of AMPK significantly impacts cellular functions including cell growth, proliferation, and autophagy. In addition to its role in metabolic processes, AMPK is recognized as a key anti-inflammatory target. Acting as a crucial inducer of macrophage polarization, the activation of AMPK suppresses M1 polarization of macrophage by upregulating the expression of SIRT1 and CREB to inhibit the NF-κB pathway. Conversely, AMPK activation facilitates M2 polarization of macrophage and exerts anti-inflammatory effects by upregulating the expression of STAT6 and PPARγ.

Numerous studies have reported the significant involvement of AMPK in the regulation of inflammation process and immune reactions. Sung et al. discovered that phosphorylated AMPK is decreased in conjunctival tissues, and the activation of AMPK significantly improves clinical symptoms and reduces ocular inflammation in DED mice. Besides various pharmacological activators of AMPK, research has demonstrated that omega-3 polyunsaturated fatty acids (ω3-PUFA) can enhance AMPK activity in skeletal muscle and intestinal epithelial cells. Moreover, as an endogenous activator of AMPK, betaine could promote the improvement of corneal damage and down-regulation of inflammatory factors TNF-α, IL-1β, IL-6, and IL-8. This finding holds promising implications for the improvement of dry eye conditions utilizing regulating the AMPK signaling pathway.

3.5

JAK/STAT signaling pathway

The JAK/STAT signaling pathway is composed of three key components, namely, the tyrosine kinase-associated receptors, the transcription factor STAT, and the tyrosine kinase JAK. Upon the binding of ligands to cytokine receptors, the JAK/STAT pathway can regulate the expression of target genes. Research has highlighted the role of the JAK-STAT pathway in Sjögren’s syndrome. Patients with primary Sjögren’s syndrome (pSS) exhibited reduced autophagy levels in salivary glands, resulting in elevated levels of pro-inflammatory mediators and activation of the JAK-STAT pathway. Furthermore, JAK inhibitor tofacitinib can reverse the overexpression of IL-6 caused by autophagy defects and exert anti-inflammatory effects.

Inhibiting the JAK-STAT pathway has been proven to have therapeutic effects in DED models. Studies have shown that local treatment with tofacitinib improves corneal fluorescein staining, reduces inflammatory cell infiltration, and decreases pro-inflammatory cytokine levels in an experimental DED model. Furthermore, elevated levels of phosphorylated STAT3 have been observed in three DED models induced by benzalkonium chloride exposure, lacrimal gland ablation as well as meibomian gland dysfunction. Local administration of the STAT3 inhibitor S3I-201 has downregulated the expression of MMP-3/9 and inflammatory cytokine IL-1β and IL-6. It also prevented apoptosis in corneal and conjunctival epithelial cells, enhanced the corneal epithelial barrier function, and promoted increased tear production and conjunctival goblet cell density. Particularly, previous research has provided evidence of the interaction between JAK-STAT signaling transduction and metabolic pathways. Tofacitinib has been discovered to inhibit the expression of key enzymes PFKFB and HK2 in glycolysis and suppress the expression of pro-inflammatory mediators IL-6, IL-8, and IL-1β in Rheumatoid Arthritis.

3.6

cGAS-STING signaling pathway

The cGAS-STING pathway represents a recently discovered inflammatory pathway instigated by the recognition of cytoplasmic double-stranded DNA (dsDNA), which is closely related to metabolic disorders including obesity, nonalcoholic fatty liver disease, insulin resistance, and cardiovascular diseases. Under diverse stress conditions, the release of DNA from the cell nucleus and mitochondria leads to the activation of the STING pathway, with cytoplasmic dsDNA recognized by cGAS. Consequently, the activation of TBK1 and IRF3 is initiated, facilitating the secretion of downstream inflammatory mediators like IFN-α/β and CXCL10.

Studies have demonstrated the accumulation of dsDNA in the tears of DED patients, accompanied by a decrease in DNAse levels. Additionally, increased expression of cGAS and STING proteins have been noted in experimental dry eye animal models and hyperosmotic stress-induced HCECs, ^, revealing a correlation between dry eye and activation of the cGAS-STING pathway. Researchers have also found that the damage to the ocular surface caused by BAC could be suppressed in mice through genetic or pharmacological inhibition of STING, indicating the potential therapeutic value of targeting the cGAS-STING pathway in dry eyes. It is noteworthy that lactate-induced mitochondrial DNA (mtDNA) damage in salivary gland epithelial cells of patients with pSS, leading to its leakage and subsequent activation of the cGAS-STING pathway. Sodium dichloroacetate (DCA), a lactate scavenger, has displayed effectiveness in inhibiting inflammation both in vivo and in vitro. This mechanism significantly contributes to understanding the involvement of the cGAS-STING pathway in dry eye.

4.1

Pyroptosis

Pyroptosis is characterized as a proinflammatory form of programmed cell death mediated by gastrin. The initiation of inflammasome activation triggers the caspase-mediated cleavage of gasdermin D (GSDMD), the pore-forming protein, which consequently facilitates the maturation and secretion of inflammatory cytokines via GSDMD membrane pores. The current studies have demonstrated that inflammasome-induced pyroptosis, dependent on GSDMD, is heightened in DED patients. This is manifested by increased expression levels of caspase-1, the active cleavage of GSDMD (N-terminal domain, N-GSDMD), as well as IL-18, IL-1β, and LDH.

Researchers have demonstrated that the expression of myocardial infarction-associated transcript (MIAT) is observably involved in the induction of pyroptosis and apoptosis under conditions of hyperosmotic stress. Notably, the silence of MIAT has been found to enhance pyroptosis and apoptosis, while impeding the migration and proliferation of HCECs. Moreover, the administration of Calcitriol effectively alleviated hyperosmotic stress-stimulated pyroptosis in HCECs via NLRP3-ASC-caspase-1-GSDMD pathway, with pyroptosis cells remarkably decreasing by 41.6% after calcitriol treatment. The activation of NLRP12 and NLRC4 inflammasomes-induced GSDMD-mediated pyroptosis, along with IL-33 release, has also been identified in DED. Our research also confirmed the increased levels of NLRP3, cleaved-caspase-1, cleaved-GSDMD, and cleaved-IL-1β proteins upon the indirect co-culturing model of THP-1 macrophages and HCECs. This upregulation can be modulated by the glycolysis inhibitor 2-DG, linking the NLRP3-driven pyroptosis of macrophages to the glycolysis of corneal epithelial cells in DED. The Pyroptosis pathways involved in DED are summarized in Fig. 3 .

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