PFKFB3-Mediated Glycolytic Metabolic Reprogramming Regulates Inflammatory Response in Dry Eye Disease

To investigate glycolytic and inflammatory changes on the ocular surface caused by dry eye disease (DED) and the regulatory effect of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3)-dependent glycolysis on the nuclear factor kappa B (NF-κB) pathway.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and a lactate assay were used to evaluate the expression of glycolytic genes, lactate secretion, and inflammatory factors in human corneal epithelial cells (HCECs) under hyperosmotic conditions, which served as an in vitro DED model. Transcriptome sequencing identified key regulatory genes in HCECs under hyperosmotic stimulation. PFKFB3 overexpression plasmids and the small molecule inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one or small interfering RNA (siRNA) were used to validate the role of PFKFB3 in glycolytic reprogramming and NF-κB pathway activation.

Hyperosmotic stress significantly upregulated glycolytic metabolic enzymes, increased lactate production, and induced inflammatory cytokine secretion in HCECs. Transcriptomics revealed a marked upregulation of the glycolytic regulator PFKFB3 and NF-κB-related genes. Overexpression of PFKFB3 further enhanced NF-κB pathway activation. Inhibition of PFKFB3 reversed hyperosmotic-induced glycolytic activation, suppressed NF-κB phosphorylation, and reduced tumor necrosis factor alpha (TNF-α) secretion.

Hyperosmotic stress activated the NF-κB pathway through PFKFB3-dependent glycolytic reprogramming, forming a vicious metabolic-inflammatory cycle. Targeting PFKFB3 may block this interaction and provide a novel therapeutic strategy for DED.