Effects of Runmu Xiaoyao Powder(润目逍遥散)for Dry Eyes Mice with Liver-Meridian Constraint-Heat Syndrome on miR-146a-5p and IRAK 1/TRAF6/NF-κB Signalling Pathway in Cornea and Lacrimal Gland Tissue
Objective To explore the possible mechanism of the treatment of dry eye with liver-meridian constraint-heat syndrome by Runmu Xiaoyao Powder(润目逍遥散)by miR-146a-5pand interleukin-1 receptor-associated kinase 1/tumour necrosis factor receptor associated factor 6/nuclear factor-κB(IRAK1/TRAF6/NF-κB)signalling pathway.Methods Eighty C57BL/6J mice were randomly divided into normal group,model group,agonist group,inhibitor group,sodium hyaluronate group,and Runmu Xiaoyao Powder high-,medium-,and low-dose groups,with 10 mice in each group.Except for the normal group,the mice of dry eye with liver-meridian constraint-heat syndrome were modeled by using benzalkonium chloride solution eye drops combined with chronic pain stimulation.Beginning on the 30th day of modelling,mice in Runmu Xiaoyao Powder high-,medium-,and low-dose groups were given 29,14.5,and 7.25 g/kg of Runmu Xiaoyao Powder respectively twice daily by gavage;mice in sodium hyaluronate group were given 5 pl of sodium hyaluronate drops twice daily;mice in the agonist group were given 2 nmol of agomir-146a-5p drops in each eye at a time,and those in the inhibitor group were given 5 nmol of antagomir-146a-5p drops in each eye,with every other day,3 times per week;mice in the normal and model groups were gavaged with deionised water at 1 ml/(100 g·d).The intervention was continued for 14 days in each group,and mice in each group were examined for tear secretion,tear film rupture time,corneal fluorescein staining,and irritability scores on the day following the last intervention;HE staining was used to observe the pathological conditions of the cornea and lacrimal glands in each group;corneal and lacrimal gland inflammatory factors,such as interleukin 1 β(IL-1β),tumour necrosis factorα(TNF-α),miR-146a-5p expression,were examined;matrix metalloproteinase 3(MMP-3)and matrix metallopro-teinase 9(MMP-9)expression in cornea,IRAK1,TRAF6,nuclear factor κB p65(NF-κB p65)protein and mRNA expression in cornea and lacrimal gland,and phosphorylated nuclear factor κB p65(p-NF-κB p65)protein expres-sion were detected.Results Compared with the normal group,mice in the model group showed reduced tear secre-tion,shorter tear film rupture time,higher irritability score(P＜0.05),and pathological examination showed stain-ing in the centre of the cornea,obvious corneal damage,increased volume of lacrimal gland follicular cells,disor-dered arrangement,a large number of inflammatory cell infiltration,and increased neovascularisation;corneal and lacrimal gland tissues showed elevated expression of IL-1 β and TNF-α,decreased expression of miR-146a-5p,ele-vated expression of IRAK1,TRAF6,NF-κB p65,p-NF-κB p65 protein and IRAK1,TRAF6,NF-κB p65 mRNA,and elevated expression of MMP-3,MMP-9 protein in the cornea(P＜0.05).Compared with the model group,all of the above indexes were significantly improved in high-dose group of Runmu Xiaoyao Powder,while some indexes were improved in the sodium hyaluronate group and the middle-and low-dose Runmu Xiaoyao Powder groups(P＜0.05).Compared with the model group,corneal and lacrimal IRAK1 and TRAF6 mRNA and IRAK1,TRAF6 and p-NF-κB p65 protein expression decreased in the agonist group;compared with the inhibitor group,IRAK1,TRAF6,NF-κB p65 mRNA and protein expression in the cornea and lacrimal gland in the Runmu Xiaoyao Powder groups decreased(P＜0.05).Conclusion Runmu Xiaoyao Powder can negatively regulate the IRAK1/TRAF6/NF-κB signalling pathway in the cornea and lacrimal gland of mice with dry eye of liver-meridian constraint-heat syndrome by up-regulation of miR-146a-5p,so as to inhibit inflammatory response and reduce the damage of the ocular surface tissues,and the high doses group showed the best effect.
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