目的：本研究旨在探讨银翘散通过环状GMP-AMP合酶（cyclic GMP-AMP synthetase，cGAS）-干扰素基因刺激因子（stimulator of interferon genes，STING）-干扰素调节因子3（Interferon regulatory factor 3，IRF3）分子通路影响巨噬细胞极化在单纯疱疹病毒性角膜炎（Herpes Simplex Keratitis, HSK）中的具体分子机制。方法：将人角膜上皮细胞HCE-T分为如下组：对照组、HSK组、HSK+银翘散组。MTT法检测细胞活力、TUNEL法检测细胞凋亡。将M0巨噬细胞分组：Ctrl组、HSV-1组、HSV-1+oe-cGAS组、HSV-1+银翘散组、HSV-1 +oe-cGAS+银翘散组。ELISA检测细胞上清液中Arg-1、iNOS的浓度，蛋白质印迹法检测cGAS、STING、IRF3的相对蛋白表达。收集各组M0巨噬细胞条件培养基（Conditional medium，CM）干预HCE-T细胞并且分为Ctrl-CM组、HSV-1-CM组、HSV-1+oe-cGAS-CM组、HSV-1+银翘散-CM组。MTT法检测细胞活力、TUNEL法检测细胞凋亡。取Balb/c小鼠并将其分为：对照组、模型组、药物组。模型组和药物组采用角膜划痕法将HSV-1接种于Balb/c小鼠角膜以构建HSK小鼠模型，药物组使用银翘散干预治疗。对比三组建模后第1、3、5、7、14天三组的发病情况和死亡率。ELISA法检测三组血清中Arg-1、iNOS水平，蛋白质印迹法检测角膜组织cGAS、STING与IRF3蛋白表达。结果：与对照组相比，HSK组HCE-T细胞活力降低、凋亡增加，而银翘散干预则可逆转此结果。与Ctrl组相比，HSV-1组细胞上清液中Arg-1浓度降低、iNOS浓度增加，细胞cGAS、STING、IRF3蛋白表达下降；与HSV-1组相比，HSV-1+oe-cGAS组、HSV-1+银翘散组Arg-1浓度增加、iNOS浓度降低，cGAS、STING、IRF3蛋白表达增强，HSV-1 +oe-cGAS+银翘散组得到相同结果。与Ctrl-CM组相比，HSV-1-CM组HCE-T细胞活力降低、凋亡增加，通过过表达巨噬细胞中的cGAS或者使用银翘散干预逆转了这一结果。体内实验发现银翘散干预能够改善角膜炎病理进展。结论：银翘散可能通过cGAS-STING-IRF3分子通路，抑制巨噬细胞M1型极化，从而延缓HSK进展。

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.

兰州市科技计划项目（23-B07）