Dan Luo,1,* Hui-jie Ji,1,* Xue-qing Yan,1 Zi-meng Wang,1 Liu-jiao Li,2 Li Shi,1 Wei-ping Gao,1 Kai Li1 

1Department of ophthalmology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, People’s Republic of China; 2Liuzhou Traditional Chinese Medicine Hospital, Liuzhou, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Kai Li; Weiping Gao, Department of Ophthalmology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, People’s Republic of China, Email likai8922@163.com; 260790@njucm.edu.cn

Purpose: Run-Mu-Ling granules (RMLG), a traditional Chinese medicinal formula, are used to treat dry eye (DE); however, the underlying mechanism remains poorly understood. This study aimed to elucidate the potential molecular mechanisms by which RMLG alleviates ocular surface inflammation in DE.
Methods: We established an in-vivo DE rat model and in-vitro human corneal epithelial cell line (HCEC) injury models. Corneal damage severity was evaluated using various tests, including corneal fluorescein staining, tear break-up time, and phenol red tear test. Hematoxylin and eosin staining was used to examine histopathological changes in corneal tissues. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling detected corneal cell damage in rats. Transmission electron microscopy was used to observe the microstructures of corneal tissue. Immunofluorescence and Western blotting analyses were used to assess NLRP3, GSDMD, ASC, caspase-1, IL-18, IL-1β, and TNF-α expression levels in corneal tissues and HCEC. Cell viability was determined using CCK-8 and colony formation assays, and pyroptosis was examined using Annexin V-PI staining.
Results: RMLG significantly improved tear film stability, promoted tear secretion, attenuated corneal tissue damage, enhanced HCEC activity, and suppressed pyroptosis. It also inhibited the activation of the NLRP3/GSDMD signaling pathway in corneal tissues and HCEC, reducing the release of downstream pro-inflammatory cytokines.
Conclusion: This study suggests that NLRP3/GSDMD-mediated pyroptosis plays a crucial role in the pathogenesis of DE and that inhibition of this pathway is a key mechanism by which RMLG alleviates ocular surface inflammation in DE. These findings suggest that RMLG could be a promising therapeutic option for DE, offering new insights into its molecular action and potential clinical application.

Keywords: RMLG, dry eye, corneal damage, inflammation, pyroptosis, traditional Chinese medicine