Targeting translocator protein (TSPO) ameliorates inflammation and maintains mitochondrial homeostasis to protect against dry eye.

To investigate the expression and role of translocator protein (TSPO) in dry eye disease (DED). In vitro experiments were performed using immortalized human corneal epithelial cells (HCECs) cultured under hyperosmolar conditions (500 mOsM). Cell viability was analyzed by the Cell Counting Kit-8 assay. Intracellular reactive oxygen species (ROS) generation was detected by 2',7'-dichlorodihydrofluorescein diacetate staining. Mitochondrial membrane potential was assessed using the JC-1 assay. Intracellular calcium (Ca2+) levels were measured by Fluo-4 AM staining. Apoptosis was analyzed by flow cytometry. Mitochondrial morphology was observed using transmission electron microscopy. mRNA expression was quantified by real-time PCR. Protein expression was assessed by western blotting, enzyme-linked immunosorbent assay, and immunofluorescence. In vivo experiments were conducted using a scopolamine-induced DED model in female Wistar rats. Clinical parameters of tear secretion, corneal epithelium defects, and conjunctival goblet cells density were measured. mRNA and protein expression in the cornea and conjunctiva were assessed by real-time PCR, western blotting, and immunofluorescence. Apoptosis in the cornea and conjunctiva was assessed by TUNEL staining. In vitro, TSPO was overexpressed in HCECs exposed to hyperosmotic stress, accompanied by elevated levels of pro-inflammatory cytokines, ROS overproduction, intracellular Ca2+ overload, and mitochondrial dysfunction, ultimately leading to apoptosis. TSPO knockdown significantly enhanced cell survival by attenuating these cellular injuries and inhibiting apoptosis via a mitochondria-dependent pathway. In vivo, increased TSPO expression was observed in both the corneal and conjunctival tissues of DED rats, along with elevated inflammation. Treatment with the TSPO-specific ligand PK11195 significantly antagonized TSPO expression, alleviated inflammation, preserved corneal epithelial integrity, and suppressed apoptosis without reversing tear deficiency. TSPO overexpression participates in the pathogenesis of DED. Inhibiting TSPO alleviates inflammation and oxidative stress and promotes cell survival through a mitochondrial-dependent pathway.