Therapeutic Effect And Mechanism Of γ-aminobutyric Acid Type A Receptor On Dry Eye Model

Objective: To study the expression of γ-Aminobutyric acid type A receptor(GABAAR)in mouse conjunctiva tissue and its therapeutic effect and mechanism on the mouse model of dry eye.Methods: 1.A Desiccating stress(DS)environment with constant temperature(24±2℃),constant humidity(30±5%)and constant wind speed(2.5±0.5km/s)was established.Female C57BL/6 mice were given subcutaneous injection of anisodamine hydrobromide solution for 6-8 weeks.After 14 days,phenolic cotton thread and fluorescein sodium staining were used to evaluate the model.The m RNA and protein expression of GABAAR were detected by RT-PCR and Western blot,and the protein expression level and location of GABAAR were detected by immunohistochemistry.2.Mouse conjunctival Goblet cells(CGCs)cultured in vitro were obtained by tissue block culture method,and divided into control group,muscimol group and picrotoxin-imprinted group(After the pretreatment with 300 u M GABAAR antagonist picrotoxin for 1h,100 u M muscimol was given)and picrotoxin group.The m RNA expressions of GABAAR and Muc5 AC in each group were detected by RT-PCR after 12 h treatment with muscimol.The protein expressions of GABAAR and Muc5 AC in each group were detected by Western blot and ELISA after 24 h treatment with muscimol.3.The dry eye mice were injected with 3mg/m L muscimol solution subconjunctivally.The tear secretion of mice was detected by phenol cotton thread method after 1,3,5 and 7 days of treatment.The ocular surface injury was evaluated by fluorescein sodium staining method,and clinical scores were performed.Morphological changes and CGCs were observed by hematoxylin-eosin and Periodic Acid-Schiff stain(PAS).4.The m RNA levels of GABAAR,Muc5 AC,TNF-α and IL-1β were detected by RT-PCR,and the protein levels of Muc5 AC,TNF-α and IL-1β were detected by ELISA.Western Blot and immunohistochemistry were used to detect the difference of GABAAR activation in each group.Results: 1.GABAAR exists in the conjunctiva of mice,mainly in the squamous epithelium.The m RNA and protein levels of GABAAR in conjunctival tissue of the dry eye mice were significantly lower than the normal.2.The m RNA and protein expression levels of GABAAR and Muc5 AC significantly increased after treatment with muscimol,comparing with control group,and the differences were significant.The expression levels of GABAAR and Muc5 AC in the pretreatment group were lower than those in the muscimol group.Comparing with the control group,the expression levels of GABAAR and Muc5 AC in the picrotoxin treatment group significantly decreased,but the difference was not significant.3.After the treatment of muscimol,the tear secretion of the dry eye mice significantly increased,and the corneal injury reduced,clinical scores improved,the number of CGCs and the morphology of corneal conjunctiva recovered.4.The expressions of GABAAR increased with the number of days treated with muscimol.Muc5 AC began to increase after the muscimol treatment,m RNA reached its peak on 3rd day and the protein level on 5th day.The expression levels of TNF-α and IL-1βdecreased gradually with the treatment time.Conclusion: The parallel decline of GABAAR and Muc5 AC in the conjunctiva of the dry eye mice suggests that GABAAR may be involved in regulating the pathogenesis and progression of dry eye.The up-regulation of GABAAR expression in mouse conjunctival tissue can promote the secretion of Muc5 AC by CGCs,inhibit the expression of inflammatory factors of TNF-α and IL-1β,repair the ocular surface damage caused by DS,and has the potential to treat dry eyes.GABAAR is expected to be a new target for clinical diagnosis and treatment of dry eye.