The Investigation Of The Protective Effect And Mechanism Of ?-MSH On Ocular Surface In Rat Dry Eye Model

PurposeDry eye is a multifactorial disease of the tears and ocular surface, which is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface. The symptoms of discomfort, visual disturbance, and tear film instability can cause the patient's discomfort with their eyes and compromise their quanlity of life. The aim of this study was to establish a dry eye model by intraperitoneal injection of scopolamine hydrobromide into rats, and to explore the protective effects and the mechanism of a-MSH on the ocular surface dysfunction in vivo and in vitro.Methods1. Establish a tear-deficient dry eye model in rats by abdominal subcutaneous injection of scopolamine hydrobromide. To investigate the effects of a-MSH on corneal dysfunctions in dry eye rats, the a-MSH at the doses of 2�10-3 mg/ml were topically applied to the corneas on a daily basis; other groups were also included to compare the protective effect with sodium carboxymethyl cellulose. The clinical evaluations,including Schirmer's test, tear breakup time, and corneal fluorescein staining, were conducted every week. The morphology of cornea and conjunctiva goblet cells was examined by histopathological approaches.2. To explore the signaling pathways mediating a-MSH's protective effects on the corneas of dry eye rats, H89 and PD98059, the two widely used pharmacological blockers that block PKA-CREB and MEK-Erk pathways, respectively, were applied with a-MSH every day, then the clinical evaluations were performed on a weekly basis.The cornal morphology, the morphology and number of conjunctiva goblet cells, as well as the apotosis in corneal epithelia were examined by histopathological stainings.The expression of the signaling molecules in PKA and Erk pathways, CREB and Erkl/2,and their phosphorylated forms (p-CREB, p-Erkl/2) were examined by Western blots.3. The QAH-DED-1 mircoarrays were used to detect the protein levels of 42 Dry Eye Disease-associated cytokines in keratoconjunctival tissues. The corneal epithelial cells were stimulated with benzalkonium chloride and treated with a-MSH. Cells viability,apoptosis, and migratory ability were measured to determine whether a-MSH has a good protective effect to the benzalkonium chloride-stimulated corneal epithelial cells.The mechanism of a-MSH's protection on corneal epithelium cells was invstigated by blocking EGF / EGFR signaling pathway with EGFR inhibitor and examining the cell viability, apotosis, and migration.Results1. Abdominal subcutaneous injection of scopolamine hydrobromide caused the decrease of tear secretion and tear film breakup time, impairment of the corneal epithelial integrity, corneal hyperplasia and edema, conjunctival goblet cell depletion;whereas topical applications of a-MSH effectively protected the dry eye rats from ocular surface dysfunctions and lesions. Compared with sodium carboxymethyl cellulose, a-MSH had the advantages in tear film stability and corneal epithelial integrity.2. ?-MSH had the effects of ameliorating ocular surface dysfunctions, anti-inflammation, morphology maintenance, anti-apoptosis, and cytoprotection in the scopolamine-induced dry eye rat model. At the same time, a-MSH activated both PKA-CREB and MEK-Erk pathways in the dry eye corneas and conjunctivas;pharmacological blockade of either pathway abolished a-MSH's protective effects,suggesting that both pathways are necessary for a-MSH's protection under dry eye condition.3. The results of protein arrays revealed that EGFR protein abundance in the dry eye was significantly decreased, and a-MSH increased the EGFR protein levels. At the same time, a-MSH protected HCE cells against benzalkonium chloride in the vital functions, including cell viability, migration, and anti-apoptosis. The protective effects of a-MSH on HCE cells were abolished by blocking the EGFR pathway with a specific EGFR inhibitor, indicating that the protective effects of a-MSH on cornea epithelial cells were exerted through EGF / EGFR system.Conclusionsa-MSH effectively ameliorates ocular surface dysfunctions and lesions in a scopolamine-induced dry eye model via PKA-CREB and MEK-Erk pathways. a-MSH increases tear secretion, prolongs tear break up time, maintains tear film stability and cornea epithelial integrity, reduces corneal edema; prevents conjunctival goblet cells from atrophy and diminishment. Compared with sodium carboxymethyl cellulose, a-MSH has advantages in tear film stability and corneal epithelial integrity. a-MSH activates the PKA-CREB and MEK-Erk signaling pathways in the dry eye model and up-regulates the protein level of EGFR. The activation of the two signaling pathways are required for the protective effects of a-MSH under dry eye condition. At the same time, a-MSH has the protect effects on HCE cell viability, migration, and anti-apoptotic ablity when the cells are subjected to the stimulation of benzalkonium chloride, and these effects are achieved via EGF / EGFR system.