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Baorui Ma,* Linnuo Pang,* Pingqing Huang, Jie Bai, Zhiqin Zhang, Huimin Wu, Mengru Cai, Jin Yang, Yuchen Xu, Xingbin Yin, Changhai Qu, Jian Ni School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Changhai Qu; Jian NiSchool of Chinese Materia Medica, Beijing University of Chinese Medicine, Higher Education Zone, Fangshan District, Beijing, 102488, People’s Republic of ChinaEmail quchanghai@bucm.edu.cn; njtcm@263.netPurpose: To prepare the levocarnitine thermosensitive in situ gel (LCTG) and evaluate its effect on dry eye disease (DED).Methods: Draize eye irritation test and other examinations were used to evaluate the eye irritation after multiple administration of LCTG. The Schirmer test, fluorescein sodium staining, HE staining and TUNEL staining were used to detect the tear secretion, corneal injury, histopathological changes of the cornea and lacrimal gland, and the apoptosis rate of cornea epithelial cells after 3 days of the administration. The conjunctival goblet cell density was detected by PAS staining, and the expression levels of matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-9 (MMP-9) of corneal epithelial cells were detected by immunofluorescence staining after 7 days of the administration.Results: LCTG is non-irritating to rabbit eyes and has good biocompatibility. LCTG administration for 3 days can significantly increase the amount of tear secretion in mice with DED, promote corneal epithelial integrity and central corneal epithelium thickness recovery, and improve the pathological morphology and structure of corneal and lacrimal gland tissues, and reduce the apoptosis rate of the corneal epithelial cells. After 7 days of the administration, the preparation can promote the proliferation of conjunctival goblet cells and down-regulate the cornea expression levels of MMP-3 and MMP-9 in epithelial cells