方法：选取6～8周龄健康C57BL/6雄性小鼠50只随机分为正常对照组、单纯铁剂组、抑制剂组、增强剂组及溶剂对照组，每组10只。正常对照组腹腔注射生理盐水0.2mL，其它组均腹腔注射50mg/mL右旋糖酐铁0.2mL，每3d注药1次； 第14d起抑制剂组、增强剂组及溶剂对照组每天分别增加1次腹腔注射同体积(0.2mL)50mg/kg XMD8-92、10mg/kg辛伐他汀和50% DMSO溶剂。于注药后第7、14、28d裂隙灯下观察各组小鼠眼前节情况，进行眼表炎症指数评估及角膜荧光素染色评分； 28d后取角膜、结膜及泪腺组织进行HE染色、免疫荧光染色，并采用RT-PCR检测巨噬细胞极化相关指标(CD86、CD206、iNOS、Arg-1)； Western blot检测胞葬作用相关信号因子(Gas6、MerTK)的表达； ELISA检测炎症因子(IL-1β、TNF-α、MMP-9)的表达。

结果：注药28d，与正常对照组比较，单纯铁剂组及溶剂对照组眼表炎症指数及角膜荧光素染色评分增加； HE染色显示角膜上皮欠完整，结膜杯状细胞减少，泪腺结构欠清晰且细胞排列相对杂乱； 各组织中CD86、iNOS等M1型巨噬细胞标志物表达上调，CD206、Arg-1等M2型巨噬细胞标志物表达下调，IL-1β、TNF-α、MMP-9等炎症因子表达上调(均P<0.05)。与单纯铁剂组和溶剂对照组比较，抑制剂组小鼠眼表炎症指数及角膜荧光素染色评分进一步增长； HE染色显示角膜上皮脱落明显，结膜杯状细胞进一步减少甚至消失，泪腺结构紊乱且细胞排列杂乱不规则； 各组织中Gas6、MerTK等胞葬作用相关信号因子表达下调(均P<0.05)，CD86、iNOS等M1型巨噬细胞标志物及IL-1β、TNF-α、MMP-9等炎症因子表达进一步上调(均P<0.05)； 而增强剂组小鼠眼表炎症指数及角膜荧光素染色评分下降，HE染色显示角膜上皮完整，结膜杯状细胞增多，泪腺结构形态改善； 各组织中Gas6、MerTK等胞葬作用相关信号因子表达上调(均P<0.05)，且CD206、Arg-1等M2型巨噬细胞标志物表达上调，IL-1β、TNF-α、MMP-9等炎症因子表达下调(均P<0.05)。

结论：高铁环境诱导巨噬细胞向M1型定向极化，加重眼表炎症反应及组织损伤，胞葬作用可调控巨噬细胞极化影响高铁环境下眼表炎症反应的发生。

METHODS: A total of 50 healthy C57BL/6 male mice aged 6-8wk were randomly divided into normal control group, iron group, inhibitor group, enhancer group and solvent control group, with 10 mice in each group. The normal control group was injected intraperitoneally with 0.2mL of normal saline, and the other groups were injected intraperitoneally with 50mg/mL iron dextran of 0.2mL, once every 3d. From the 14d, the inhibitor group, the enhancer group and the solvent control group were injected intraperitoneally with the same volume(0.2mL)50mg/kg XMD8-92, 10mg/kg simvastatin and 50% DMSO solvent once a day, respectively. The anterior segment of the eyes was observed under slit lamp microscope on the 7, 14, 28d after intraperitoneal injection, and the ocular surface inflammation index and corneal fluorescein staining score were evaluated. The cornea, conjunctiva and lacrimal gland tissues were taken at 28d for the HE staining and immunofluorescence staining, and RT-PCR were used to detect the expression of macrophage polarization related indexes(CD86, CD206, iNOS, Arg-1); Western blot were used to detect the expression of efferocytosis related signal factors(Gas6, MerTK); ELISA was used to detect the expression of inflammatory factors(IL-1β, TNF-α, MMP-9).

RESULTS: After injection for 28d, compared with the normal control group, the ocular surface inflammatory index and corneal fluorescein staining score were increased in the iron group and the solvent control group. HE staining showed incomplete corneal epithelium, reduced conjunctival goblet cells, unclear lacrimal gland structure and relatively disordered arrangement of cells. In all tissues, the expressions of polarization related indexes of M1 macrophages such as CD86 and iNOS were up-regulated, while those of M2 macrophages such as CD206 and Arg-1 were down-regulated, and the expressions of inflammatory factors such as IL-1β, TNF-α and MMP-9 were up-regulated(all P<0.05). Compared with the iron group and the solvent control group, the ocular surface inflammation index and corneal fluorescein staining score of the inhibitor group were further increased. HE staining showed obvious exfoliation of corneal epithelium, further decrease or even disappearance of conjunctival goblet cells, disorder of lacrimal gland structure and irregular arrangement of cells. In all tissues, the expression of signal factors related to efferocytosis such as Gas6 and MerTK was down-regulated(all P<0.05), the expression of polarization related indexes of M1 macrophages such as CD86 and iNOS and the expression of inflammatory factors such as IL-1β, TNF-α and MMP-9 were further up-regulated(all P<0.05). But the ocular surface inflammation index and corneal fluorescein staining score decreased in the enhancer group. HE staining showed the integrity of corneal epithelial, the increase of conjunctival goblet cells and the improvement of lacrimal gland structure and morphology. In all tissues, the expression of signal factors related to efferocytosis such as Gas6 and MerTK was up-regulated(all P<0.05), and the expression of polarization related indexes of M2 macrophages such as CD206 and Arg-1 was up-regulated, while the expression of inflammatory factors such as IL-1β, TNF-α and MMP-9 was down-regulated(all P<0.05).

CONCLUSION: High-iron environment induces macrophages polarize to M1, which aggravates ocular surface inflammation and tissue damage. Efferocytosis by regulating the polarization of macrophages impact the occurrence of ocular surface inflammation in high-iron environment.