Recombinant Human Lysozyme Attenuates Hyperosmolarity-Induced Dry Eye Disease by Suppressing Oxidative Stress and Modulating NLRP3 Inflammasome Signaling

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Abstract
Given the escalating global prevalence of dry eye disease (DED) and the inadequacy of current palliative management, this study was to evaluate thetherapeutic potential of recombinant lysozyme (rlysozyme) and investigate underlying mechanisms. Human corneal epithelial cells (HCEC) were subjected to hyperosmotic stress and treated with graded rlysozyme. Cell viability, wound healing, barrier integrity, oxidative stress, cytokines, and mitochondrial function were evaluated by MTS and scratch assays, immunofluorescent staining, ELISA, qRT-PCR, and flow cytometry. Results: No cytotoxicity was observed at any of the tested concentrations (2.76 × 10−9–2.76 × 10−4 M) in HCEC. In hyperosmolarity-induced DED cells, rlysozyme at the highest concentrations (2.76 × 10−4 M) significantly increased cell viability by 12.30% (p < 0.001) and promoted corneal repair by 45.04% (p < 0.05), which were comparable to those of 0.4% hyaluronic acid (the positive control). It also improved via restoring ZO-1 organization. Mechanistically, qRT-PCR results revealed that rlysozyme reduced mRNA expression of IL-1β (by 28.7%), TNF (by 39.2%) and key NLRP3 inflammasome components, including NLRP3 (by 62.0%) and PYD and CARD domain-containing protein gene(PYCARD, 21.4%), while caspase-1 (CASP1) and IL-6 expression remained unchanged. Moreover, oxidative stress was attenuated, as evidenced by significant decreases in reactive oxygen species (ROS, by 59.1%), 8-hydroxy-2-deoxyguanosine levels (8-OHdG, by 23.6%) and total matrix metalloproteinase-9 (MMP-9, by 64.3%) protein levels (p < 0.001), while mitochondrial membrane potential was assessed qualitatively. In conclusion, this study demonstrates thatrlysozyme effectively enhanced corneal epithelial cell viability, promoted wound healing, and restored ZO-1 structural organization without inducing cytotoxicity. These protective effects were associated with downregulation of NLRP3 inflammasome-related genes and attenuation of oxidative stress, supporting further investigation of its therapeutic potential in DED.
Funding Information
  • Shaanxi Huikang Biotech Co., Ltd.
  • The Hong Kong Polytechnic University (H-ZDGP)

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