MicroRNA146a Expression In Ocular Surface Tissue Of Dry Eye Mouse Model And Its Correlation With Inflammation

Purpose: Dry eye is a common eye disease in clinical work,which can result in bad influence on patients' living quality.Due to the complicated pathogenisis of dry eye disease,there is no effective treatment in clinical work.In recent years,many researches proved that inflammation plays a critical role in the pathogenesis of dry eye disease,besides,more and more evidence suggested that micro RNA146 a has a close replationship with some important regulatory pathwaies in inflammation.Because there was no discovery about the expression and role of micro RNA146 a in dry eye disease,so in our experiment,we established moderate or severe dry eye disease mouse model,and used dry eye related clinical tests,real time PCR,histopathologic examination,and immunofluorescent staining to explore the expression of micro RNA146 a in dry eye disease and its regulatory effect on targets in ocular inflammation.Method: 1.The establishment of dry eye mouse model and assessment The left eyes of forty five-week-old Balb/c mice were categorized into normal control group(40 eyes);the right eyes of these mice were categorized into dry eye group(40 eyes).Right eyes of these mice were topically treated with 0.2% benzalkonium chloride for fourteen days to establish moderate or severe dry eye mouse model.At the same time,in order to estimate the situation of ocular surface,we need to examine BUT,FLS score,inflammation index of two groups termly.2.Histopathologic examination and immunoflurescence After the establishment of dry eye mouse model,20 mice were chosen from these 40 mice randomly,HE(hematoxylin eosin staining)and PAS(periodic acid –Schiff staining)were performed to observe the differences of cornea and conjunctiva between the normal control group and the dry eye group.The number of goblet cells in the conjunctiva were counted after PAS(periodic acid-Schiff staining),and the difference of them in two groups were compared.Besides,immunofluorescent staining was implemented in two groups to compare the specific fluorescence staining and fluorescence intensity of NF-?B p65 and TNF-?.3.Real-time fluorescence quantitative PCR As for the remaining 20 mice,total RNA of the cornea and conjunctiva were extracted,and the expression of micro RNA146 a,interleukin-1(IL-1),interleukin-6(IL-6),interleukin-8(IL-8),interleukin-1 receptor associated kinase 1(IRAK1)and tumor necrosis factor receptor associated factor 6(TRAF6)were detected through real-time fluorescent quantitative PCR.Results: 1.After establishment,BUT in the dey eye goup was(3.640±0.493)s,which was significantly declined in comparison with in the normal control froup(10.645±0.583)s,(P<0.05).FLS scores in dry eye group was(14.650±0.860),which was significantly higher than that in the normal control group(1.233±0.927),(P<0.05).Inflammation index in dry eye group was(0.667±0.032),which was significantly higher than that(0.236±0.068)in the normal control group,(P<0.05).2.Compared to the normal control group,HE of the dry eye group showed that the corneal epithelial cell layer was uneven,and the number of cells increased.Collagenous fibers in the corneal stromal layer were irregular and accompanied with fibroblast activation.The number of conjunctival epithelial cells was also increased and ineven and accompanied with the deect.PAS showed that the number of goblet ells in dry eye group(9.500±4.506)/view was significantly lower than that in the normal control group(29.667±8,756)/view,(P<0.05).Immunofluorescent staining showed that compared to the normal control group,in dry eye group,the specific fluorescence staining of NF-?B p65 and TNF-? were existed significantly,and fluorescence intensity was also increased.3.Real-time fluorescent quantitative PCR showed that the expression levels of micro RNA146 a,IL-1,IL-6,IL-8were increased in the cornea and conjunctiva in dry eye group incomparison with the normal control group,(P<0.05).Compared to the cornea in the normal control group,the expression of IRAK1 was down-regulated,and TRAF6 was up-regulated in the cornea of the dry eye group,(P<0.05).However,compared to the conjunctiva in the normal control group,the expression of IRAK1 was up-reguated,and TRAF6 was down-regulated in the conjunctiva of the dry eye group(P<0.05).4.Literature arrangement and Targetscan: In recent decades,there are 53 scientific papers about the relationship between micro RNA146 a and IRAK1,and there are 61 scientific papers about the relationship between micro RNA146 a and TRAF6.The result of Targetscan shows that the seed region of micro RNA146 a in IRAK 3'UTR and TRAF6 3'UTR of human.The werighted context scores are 0.29 and 0.15 respecttively and the context score percentile are 97 and 95 respectively.In addition,it also shows the seed region of micro RNA146 a in IRAK 3'UTR and TRAF6 3'UTR of mouse.The werighted context scores are 0.31 and 0.10 respecttively and the context score percentile are 96 and 92 respectively.These result shows that there is high percentage that IRAK1 and TRAF6 are targets of micro RNA146 a,and also provide evidence for the research data.Conclusions: The use of 0.2% benzalkonium chloride can induce appropriate dry eye disease mouse model,which means benzalkonium chloride can become a valuable method to investigate dry eye disease related ocular inflammation.The change of corneal and conjunctival structure in dry eye disease through pathological staining,and the result of immunofluorescent staining can prove the important role of inflammation in dry eye disease.The elevated expression of micor RNA146 a and inflammatory factors in dry eye disease,and the selectively inhibiting effect of micro RNA146 a on its inflammatory targets can prove that micro RNA146 a has inhibiting regulatory function in infalmmaiton related to dry eye disease,and it has tissue specificity in the regulation of its inflammatory targets.