Effects Of Lipopolysaccharide Pretreatment On Ocular Inflammation And The Expression Of MicroRNA146a In EIU Rats

Objective: The aim of this study was to explore the expression changes of micro RNA146 a,its target proteins IRAK1,TRAF6 and NF-κB P65 in LPS pretreatment alleviating the ocular inflammation of EIU rats.Methods: Wistar rats in SPF grade were randomly assigned to three groups: normal control group（NC）,endotoxin tolerance group（ET）,endotoxin-induced uveitis group（EIU）,with fifteen rats in each group.Endotoxin tolerance was induced by injecting0.1 mg/Kg LPS to rats for five consecutive days while rats in NC and EIU groups received an equal amount of sterile saline.On the sixth day,rats in the ET group and EIU group received 300 ug LPS to initiate ocular inflammation while rats in NC group received an equal amount of sterile saline.All of the drugs were injected into the right hind footpad.Signs of ocular inflammation in all rats were observed under a slit lamp after 24 hours and evaluated by clinical and histopathological analysis.ELISA was used to determine the concentration of TNF-а and IL-6 in aqueous humor,and RT-PCR and Western Blot were used to quantify the relative expression levels of miR-146 a,IRAK1and TRAF6.Meanwhile,immunofluorescence staining was performed to assess the activation of NF-κB P65 in the iris-ciliary body.Results: In EIU rats,significant dilation of iris blood vessels,fibrin exudation in the anterior chamber,and posterior adhesion were observed under a slit lamp.The infiltration of inflammatory cells and the edema of the iris-ciliary body were confirmed in histopathological slides.The clinical signs and histopathological manifestations of rats in ET group were alleviated and no obvious manifestations of uveitis were found in NC group.The average concentrations of TNF-a and IL-6 in the aqueous humor of NC rats were 64.09 pg/ml and 40.42 pg/ml.The mean levels of TNF-a and IL-6 were428.60 pg/ml and 107.90 pg/ml in EIU rats,and the mean levels of TNF-a and IL-6 in ET rats were 289.70 pg/ml and 65.33 pg/ml.RT-PCR results showed that the relative expressions of miR-146 a,IRAK1 and TRAF6 m RNA in the iris-ciliary body of EIU rats were 2.33 ± 0.06,1.68 ± 0.23,1.57 ± 0.28,respectively.The relative expression levels of miR-146 a,IRAK1 and TRAF6 m RNA in ET rats were: 3.50 ± 0.37,0.90 ±0.04,0.78 ± 0.03,respectively.The relative protein expressions of IRAK1 and TRAF6 detected by Western Blot in the iris-ciliary body of EIU rats were: 0.57 ± 0.05 and 0.56± 0.07,respectively,while the relative protein expressions of IRAK1 and TRAF6 in the ET rats were: 0.47 ± 0.02 and 0.25 ± 0.06,respectively.In short,compared with the rats in EIU group,reduced expression of TNF-a and IL-6 in aqueous humor,decreased expression of IRAK1 and TRAF6 while increased expression of miR-146 a in the irisciliary body of ET rats was observed,and all of the differences were statistically significant（P<0.05）.Meanwhile,immunofluorescence staining showed that the fluorescence intensity of NF-κB P65 in the iris-ciliary body of ET group was weakened.Conclusions: LPS pretreatment reduced the ocular inflammatory response in EIU rats,which may be related to inhibiting the expression of IRAK1,TRAF6 and activation of NF-κB P65 after upregulating the expression of miR-146 a.