Efferocytosis Participates In Ocular Surface Inflammation By Regulating Macrophage Polarization

Objective: To explore whether efferocytosis participates in ocular surface inflammation by regulating macrophage polarization.Methods: A total of 50 healthy C57BL/6 male mice aged 6-8 weeks were randomly divided into normal control group,iron group,inhibitor group,enhancer group and solvent control group,with 10 mice(20 eyes)in each group.The normal control group was injected intraperitoneally with 0.2 m L of normal saline,and the other groups were injected intraperitoneally with 0.2 m L of iron dextran(50mg/m L),once every 3 days.From the 14 th day,the inhibitor group,the enhancer group and the solvent control group were injected intraperitoneally with the same volume(0.2m L)of 50mg/kg XMD8-92,10mg/kg simvastatin and50%DMSO solvent once a day,respectively.The anterior segment of the eyes was observed under slit lamp microscope on the 0th,7th,14 th and 28 th day after intraperitoneal injection,and the ocular surface inflammation index,breaking up time(BUT)and corneal fluorescein staining score were evaluated.The mice were killed 28 days later,and the cornea,conjunctiva and lacrimal gland tissues were taken for the following detection: HE staining;immunofluorescence staining and RT-PCR were used to detect the expression of macrophage polarization related indexes(CD86,CD206,iNOS,Arg-1);RT-PCR and Western blot were used to detect the expression of efferocytosis related signal factors(Gas6,Mer TK);ELISA was used to detect the expression of inflammatory factors(IL-1β,TNF-α,MMP-9).Results: 1.In the normal control group,there was no obvious conjunctival congestion,transparent cornea,good stability of tear film and no obvious staining spots.HE staining showed that corneal epithelium was intact,conjunctival epithelium was scattered with goblet cells,lacrimal gland structure was intact and cells were closely arranged.2.Compared with the normal control group,the ocular surface inflammatory index and corneal fluorescein staining score were increased and BUT was shortened in the iron 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-9were up-regulated(P < 0.05).3.There was no significant difference in the score of ocular surface inflammation index,BUT,corneal fluorescein staining,HE staining and related indexes between the solvent control group and the iron group.4.Compared with the solvent control group,the ocular surface inflammation index and corneal fluorescein staining score of the inhibitor group were further increased,and the BUT was further shortened.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 Mer TK was inhibited(P < 0.05),the expression of polarization-related indexes of M1 macrophages such as CD86 and iNOS was further up-regulated,and the expression of inflammatory factors such as IL-1β,TNF-α and MMP-9 was further up-regulated(P < 0.05).5.Compared with the solvent control group,the ocular surface inflammation index and corneal fluorescein staining score decreased and BUT was relatively prolonged in the enhancer group.HE staining showed the repair of corneal epithelial integrity,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 Mer TK was increased(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 inhibited(P < 0.05).Conclusion: 1.High-iron environment induces macrophages to polarize to M1,which aggravates ocular surface inflammation and tissue damage.2.XMD8-92 and simvastatin inhibit and enhance the efferocytosis of macrophages respectively,induce the phenotypic and functional changes of macrophages,and thus inhibit or promote the resolution of ocular surface inflammation and damage repair.3.Efferocytosis may participates in ocular surface inflammation by regulating the polarization of macrophages.