Corneal Autophagy And Its Regulation On Ocular Surface Inflammation In Dry Eye Mice

BackgroundDry Eye Disease(DED)is a multifactorial ocular surface disease affecting millions of people around the world.Its incidence rate has increased in recent years.It is characterized by loss of steady state of the tear film,accompanied by ocular surface symptoms,and its pathogenesis includes tear film instability,tear high osmotic pressure,ocular surface inflammation and damage,and neurosensory abnormalities.Inflammation plays an important role in the pathogenesis of dry eye syndrome.Autophagy is a process in which cells self-phagocytose,fulfilling the metabolic needs of cells and the renewal of certain organelles.Autophagy affects the survival,stability and development of inflammatory cells and participates in the development of inflammatory diseases.Studies have shown that autophagy can prevent lacrimal gland damage in DED mice and also play a role in the pathogenesis of DED patients.The level of autophagy in the DED cornea and its relationship with inflammation are still unclear.Thus,the aim of out study is to investigate the autophagy level in DED mice cornea and the therapeutic effect of autophagy regulators on the inflammation of DED.MethodDry eye mice models were prepared by subcutaneous injection of scopolamine in a dry environment.According to the intervention time,40 normal mice were divided into 4 groups:DE-Od,DE-5d,DE-10d,and DE-20d.The tear secretion test and the corneal fluorescein sodium staining score were used to evaluate the modeling results.Autophagosomes were observed on cornea by transmission electron microscopy.The expression of LC3 protein in each group was detected by immunofluorescence.The expreession of autophagy related proteins LC3,ATG5,p62 and Inflammatory response mediator proteins TNF-?,MMP-3,MMP-9 were detected by Western Blot;According to the medicine treatment,40 dry eye mice were divided into Control group(blank control group),DE-PBS group(PBS solution),DE-AA group(autophagy inducer LYN-1604)and DE-AI group(autophagy inhibitor 3-MA),the groups treated with eye drops.Electron microscopy,Western Blot and immunofluorescence were used to detect corneal autophagy levels.Tear secretion detection,corneal fluorescein sodium staining tested tear secretions and ocular surface damages,HE staining observed corneal histopathology.Western Blot,immunofluorescence detected the expression of TNF-a,MMP-3 and MMP-9.ResultSuccessfully established mice mixed dry eye models;The level of autophagy in the dry eye cornea increased,the number of autophagosomes increased in the DE-5d,DE-10d,DE-20d groups,the expression of autophagy marker protein increased(P<0.05)and there was no significant difference among the DED groups(P>0.05).The expression of inflammatory mediator increased(P<0.05).Electron microscopy,Western Blot,immunofluorescence showed that the autophagy level in corneas of DE-AA group and DE-AI group increased and decreased respectively(P<0.05);The secretion of tears in the DE-AA group increased,the corneal damages was alleviated but the corneal injury in the DE-AI group was severe(P<0.05).HE staining showed the damages of corneal cells in the DE-AA group was alleviated;Immunofluorescence and Western Blot results showed that the expression of TNF-a,MMP-3 and MMP-9 in DE-AA group decreased significantly and increased in the DE-AI group(P<0.05).ConclusionsAutophagy participates in the pathogenesis of DED.The level of autophagy in the dry eye cornea increases but does not continue to increase with the progression of DED.The autophagy activator LYN-1604 can increase the level of autophagy in the cornea,reduce the damage of corneal tissue and the expression of inflammatory mediators,which can be used to treat DED.The autophagy inhibitor 3-MA aggravates dry eye symptoms and corneal damage.Therefore,the activation of autophagy regulates the level of ocular surface inflammation and provides a new direction for dry eye treatment.