Dry Eye Disease Symptoms And NHE8Gene Regulation Under Ocular Surface Inflammatory Environment

ObjectiveDry eye is a multi-factorial disease, inflammation as a defense reaction is the mostcommon pathology. Studies have shown the levels of inflammatory cytokines TNF-αhas elevated in tears and conjunctival epithelial cells under dry eye condition. Wecollected the age, gender and symptoms of dry eye disease, as well as monitoring theexpression of TNF-α in conjunctival epithelial cells of dry eye patients to explore therelationship between ocular inflammation and clinical symptoms.Sodium/hydrogen exchanger8(NHE8) is a novel protein, the physiological role isto be proportional to the cell membrane by proton exchange of extracellular sodium ions,thereby adjusting the pHi (intracellular pH)stability, the cell volume and collaboratingHCO3-, NH4+, Cl-transport and other electrolytes. NHE8protein mainly located in theintestinal brush border, the expression of NHE8was inhibited by TNF-α, IFN-γ andother inflammatory factors in intestinal inflammation diarrhea. We compared theexpression level of NHE8in conjunctiva and intestine, surprisingly we found NHE8was highly expressed in conjunctival cells. This study investigated the NHE8in vitroand the regulation of NHE8by TNF-α.Methods1. The study was a cross-sectional descriptive hospital-based survey conducted atthe Department of Eye clinic of the Northern Hospital between October,2012andJanuary,2013.The participants comprised adult ophthalmic outpatients aged25years or older.2. Dry eye disease was assessed objectively with Schirmer’s test and Tear-filmBreak-up Time (BUT). Schirmer s test reading of <10mm with a TBUT of <10s wasconsidered diagnostic ofDED. Separately,16people were randomly selected as thecontrol group and the case group.3. Collect conjunctiva epithelial cells from2groups by impression cytology, thenextract RNA and reverse transcription immediately. Design the plate for target geneTNF-α and run Real-time PCR.4. Immunohistochemical analysis of the distribution of NHE8protein on humanconjunctiva.5. Cell culture.6. Fluorescence immunocytochemistry staining.7. RNA extraction and protein extraction.8. Real-time PCR and Western Blot9. Transient transfection and analysis of promoter activity.10. Using Stata12.0software, measurement data were compared using t-test, twodisease incidence comparison was measured by χ2test, correlation between indicatorsusing linear correlation analysis. P <0.01indicates significant differences.ResultsIn dry eye outpatients:1.The incidence of dry eye is49%.2.The incidence offemale patients is higher than of male patients significantly, χ2=29.78, P <0.01.3.Higher expression of TNF-α in conjunctiva epithelial cells appears in the casegroup, t=7.31, P <0.01.4.TNF-α expressed in conjunctiva epithelial cells in patients with dry eye disease isinto a negative correlation with the Schirmer s Test, R2=0.2583, p <0.01; withBUT is negative correlated by R2=0.1435, p=<0.01.5.In human conjunctivatissue, NHE8protein located on the cell membrane 6.On human conjunctival epithelial cells CCL-20.2cells, NHE8protein localized inthe plasma membrane; lower concentration (25ng/ml) TNF-α significantlyinhibited NHE8protein expression; cells treated by actinomycin D were foundthe inhibition of NHE8gene transcription compared the treatment of TNF-α andthe control; transient transfection data showed that person NHE8recombinantplasmid transfected cells inhibition of NHE8promoter sequences was between-671bp and-89bp.ConclusionThe common symptoms of dry eye disease include dryness, irritation, sensation,light sensitivity, and itching. There is higher incidence of dry eye in outpatients, and theincidence has great relevance with age and gender. In conjunctiva epithelial cells of thepatients with dry eye the expression of TNF-α is increased to approximately45%. Andthere is a negative correlation between TNF-α and subjective&objective index of dryeye disease. In human conjunctiva Na+-H+exchanger NHE8has higher expressionlevel, and the distribution is in the conjunctival plasma membrane.On conjunctiva epithelial cells, TNF-α inhibit NHE8protein expression by hinderNHE8gene transcription. Inflammatory cytokines TNF-α can induce inflammation,inhibit NHE8gene promoter activity.