Abstract:Objective: This study aimed to investigate the specific molecular mechanism of Yinqiao Powder in affecting macrophage polarization in herpes simplex keratitis (HSK) through the cyclic GMP-AMP synthetase (cGAS)-stimulator of interferon genes (STING)-Interferon regulatory factor 3 (IRF3) molecular pathway. Methods: Human corneal epithelial cells (HCE-T) were divided into control, HSK, and HSK + Yinqiao Powder groups. Cell viability was detected by MTT assay, and apoptosis was detected by TUNEL assay. M0 macrophages were grouped as Ctrl, HSV-1, HSV-1 + oe-cGAS, HSV-1 + Yinqiao Powder, and HSV-1 + oe-cGAS + Yinqiao Powder. ELISA was used to detect the concentrations of Arg-1 and iNOS in cell supernatants, and Western blotting was used to detect the relative protein expressions of cGAS, STING, and IRF3. Conditional medium (CM) from each group of M0 macrophages was collected to intervene in HCE-T cells and divided into Ctrl-CM, HSV-1-CM, HSV-1 + oe-cGAS-CM, and HSV-1 + Yinqiao Powder-CM groups. MTT assay was used to detect cell viability, and TUNEL assay was used to detect apoptosis. Balb/c mice were divided into control, model, and drug groups. The model and drug groups were inoculated with HSV-1 on the cornea of Balb/c mice using the corneal scratch method to construct an HSK mouse model, and the drug group was treated with Yinqiao Powder. The incidence and mortality of the three groups were compared on days 1, 3, 5, 7, and 14 after modeling. ELISA was used to detect the levels of Arg-1 and iNOS in the serum of the three groups, and Western blotting was used to detect the protein expressions of cGAS, STING, and IRF3 in corneal tissues. Results: Compared with the control group, the HCE-T cell viability in the HSK group was decreased but apoptosis was increased, which was reversed by Yinqiao Powder intervention. Compared with the Ctrl group, the Arg-1 concentration in the cell supernatant of the HSV-1 group was decreased, the iNOS concentration was increased, and the protein expressions of cGAS, STING, and IRF3 were decreased. Compared with the HSV-1 group, the Arg-1 concentration was increased, the iNOS concentration was decreased, and the protein expressions of cGAS, STING, and IRF3 were enhanced in the HSV-1 + oe-cGAS group and the HSV-1 + Yinqiao Powder group, and the same results were obtained in the HSV-1 + oe-cGAS + Yinqiao Powder group. Compared with the Ctrl-CM group, the HCE-T cell viability was decreased and apoptosis was increased in the HSV-1-CM group, which was reversed by overexpressing cGAS in macrophages or intervening with Yinqiao Powder. In vivo experiments found that Yinqiao Powder intervention could improve the pathological progression of keratitis. Conclusion: Yinqiao Powder inhibits M1 polarization of macrophages through the cGAS-STING-IRF3 molecular pathway, thereby delaying the progression of HSK.
