Rehearsal For Thesis Presentation

Dry eye disease (DED) is a common eye disorder characterized by reduced tear production or increased tear evaporation, leading to tear film instability and damage to the ocular surface. Benzalkonium chloride (BAC), a preservative commonly found in eye drops, is one of the factors known to induce DED. Raw milk serves as a favorable medium for the growth of environmental probiotics, and kefir, a traditionally alcoholic fermented milk, is rich in probiotics and served as a good source for their isolation. In this study, human conjunctival epithelial cell line Clone 1-5c-4 were used in vitro to screen potential probiotic strains that may reduce BAC-induced damage. The selected strains were further tested in a mouse model of DED to evaluate their therapeutic effects in vivo. Four strains were tested: Lactobacillus kefiranofaciens K6, K49, and K50, isolated from kefir, and Lacticaseibacillus paracasei RM081 from raw milk. Each strain’s cell-free fermentation supernatant (CFS) and heat-killed (HK) preparations were used to pretreat the cells before exposure to 10 ppm BAC. Cell viability, cytokine levels, and reactive oxygen species (ROS) production were measured. Results showed that all CFS and HK treatments, except RM081 HK, significantly improved cell viability (P < 0.05). All CFS groups significantly increased the anti-inflammatory cytokine IL-10 (P < 0.05) and decreased the inflammatory factor MMP-9 (P < 0.05), while IL-1β, TNF-α, and IL-6 levels showed a downward trend. In terms of ROS, K6 and K49 CFS significantly reduced ROS activity (P < 0.05). Based on the in vitro results, strains K6 and K49 were selected for in vivo testing. Six-week-old BALB/c mice were used. Each eye was treated daily with 5 μL of low (10%) or high (20%) concentrations of K6 or K49 CFS, followed by 5 μL of 0.2% BAC to induce DED twice per day for 14 days. Clinical symptoms of DED, serum cytokines, superoxide dismutase (SOD) activity in extraorbital lacrimal glands (ELG), and histological changes in the eye tissue were evaluated. The high-dose K49 group (K49-H) showed a significant increase in tear volume (P < 0.05), and both K6-H and K49-H showed significantly smaller areas of corneal damage (P < 0.05). All CFS-treated groups exhibited significantly fewer eye blinks (P < 0.05). Serum analysis revealed significantly lower levels of inflammatory cytokines TNF-α, IL-6, IFN-γ, and IL-17A in all CFS groups (P < 0.05). IL-1β levels were significantly reduced in K6-H, K49-L, and K49-H groups (P < 0.05). MMP-9 levels were significantly reduced in K6-L, K6-H, and K49-H groups (P < 0.05). IL-10 levels showed no significant change. No significant differences were found in ELG’s SOD activity among groups. Histological analysis showed that the K49-H group had improvements in corneal epithelial thickness and stromal density. In summary, this study identified potential probiotic strains from kefir that alleviate BAC-induced DED, with L. kefiranofaciens K49 showing the most promising effects. Future research will focus on clarifying the underlying mechanisms and exploring its application in human and animal health.
Keywords: dry eye disease, benzalkonium chloride, probiotics, raw milk, kefir

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