| ObjectiveDry Eye Syndrome（DES）is a common clinical ocular surface disease caused by increased evaporation of the tear film or decreased tear secretion,and its prevalence increases significantly by year.The aim of this study was to establish a mice model of dry eye which induced by AQP5 knockout,and explore its pathogenesis,to clarify the role of AQP5 in lacrimal gland homeostasis.To provide new targets and theoretical support for the treatment of dry eye syndrome patients.MethodsAQP5 knockout(AQP5-/-)mice were constructed by C57BL/6N mice through CRISPR/Cas9 technology.C57BL/6N mice of the same age were selected as Wild-type control mice.Mice were routinely reared,and the endoplasmic reticulum stress-related factors（CHOP,GRP78,Casp12,Bax,Bcl-2）and inflammation-related factors（CXCL1,CXCL2,CCL5,IL-1β,IL-6,TNF-α,MCP-1）were detected by PCR and Western blot.Expression of AQP5 andα-SMA in lacrimal glands and infiltration of inflammatory cells（F4/80,Ly6g,CD4）were detected by immunofluorescence.Hematoxylin and Eosin（H&E）staining and transmission electron microscopy（TEM）were used to observe the structure of lacrimal gland epithelium.Lipid aggregation in lacrimal gland cells was observed by Oil red O staining.Exogenous intraperitoneal injection of quercetin was performed on AQP5-/-mice,observed the ease of endoplasmic reticulum stress and relief inflammatory in lacrimal glands.Results（1）AQP5 is widely expressed in cells of lacrimal gland epithelial cells of Wild-type mice,but hardly in AQP5-/-mice.And compared with Wild-type mice,the weight of lacrimal gland in AQP5-/-mice had increased;decreased in tear secretion,and corneal epithelium had punctate defects also in AQP5-/-mice.（2）Compared with Wild-type mice,AQP5-/-mice had aggravated endoplasmic reticulum stress response in lacrimal glands;and ultrastructure also showed severe damage to endoplasmic reticulum structure.（3）The inflammatory cells in the lacrimal gland of AQP5-/-mice was increased,and the inflammatory related factors were significantly up-regulated;the distribution of myoepithelial cells around the acini was disordered（4）Exogenous quercetin for AQP5-/-mice intervention treatment,and it was found that the endoplasmic reticulum stress in the lacrimal gland epithelial cells of the mice in quercetin treatment group was alleviated;the destroyed in ultrastructure of the endoplasmic reticulum was improved;inflammatory cell was reduced,and inflammatory related factors were down-regulated;tear secretion increased,and corneal epithelial punctate defects were decreased.ConclusionThis study illustrated that AQP5-/-mice is a stable mice model of dry eye.The mechanism of dry eye is through the inflammatory induced by endoplasmic reticulum stress in lacrimal epithelial cells.Quercetin significantly relieved endoplasmic reticulum stress after induced inflammatory in lacrimal gland cells of AQP5-/-mice through its anti-inflammatory effect.These results provide the evidence that aquaporin is involved in maintaining the homeostasis of lacrimal epithelial cells,and suggest that AQP5 is a possible therapeutic target for the treatment of dry eye. |
