The Roles Of Chrysin In The Treatment Of Experimental Autoimmune Uveoretinitis And Its Mechanism

Objective:To establish the animal model of experimental autoimmune uveoretinitis in C57BL/6J mouse.And to observe the therapeutic effect of chrysin on EAU,also to explore the possible mechanisms.Methods:(1)Fouty-eight 6-8 week-old female C57BL/6J mice,were randomly divided into a control group and a model group,which were immunized with emulsified IRBP_1-20/CFA subcutaneously to induce EAU,inhanced with intraperitoneal injection of pertussis toxin.The ocular fundus ware observed daily since day 12 until euthanasia at day 21.Fundus photographs were taken,clinical and histopathological scoring were performed at day 21.The integrity of the blood-retinal barrier and the expression of tight junction proteins were measured by Evans Blue,western blot and q RT-PCR respectively.(2)Six to eight week-old female C57BL/6J mice,randomly divided into a control group and a chrysin group.All immunized with IRBP_1-20/CFA to induce EAU.Mice in chrysin group were administrated chrysin 25mg/kg/day since 3 days before immunization(day-3)to day 21 everyday orally.Eye-ground photographs were taken,clinical and histopathological scores were given at day 21.The integrity of the blood-retinal barrier and the expression of tight junction proteins were measured by Evans Blue,western blot and q RT-PCR respectively.The activities of macrophages infiltrated into the retina were evaluated through immunohistochemistry and immunofluorescence.The apoptosis of the retinal cells was detected by TUNEL.The proportion of Th1,Th17,Treg and Th0 cells in the spleen were examined by flow cytometry.Inflammatory molecules and STAT1,p-STAT1,STAT3,p-STAT3 and NF-?Bp65 were detected by q RT-PCR and western blot.Results:(1)The fundus appearance of mice in model group was retinal infiltrations,vasculitis and papilledema,the histopathologic manifestation was the irregularity of the arrangement of retinal layers,vasculitis and vitreous infiltrations,which agreed with the typical manifestation of EAU.Compared with the mice in control group,significantly higher clinical scores(t=-3.953,P<0.05)and histopathologic scores(t=-7.906,P<0.01)were found in model group mice.Poorer integrity of the blood-retinal barrier and lower expression of tight junction proteins were observed in immunized mice.Part II:Significantly lower clinical and histopathological scores,better integrity of the blood-retinal barrier and higher expression of tight junction proteins were observed in chrysin-treated mice.Both macrophage infiltration and the expression of i NOS in the retina were inhibited efficiently by chrysin,activated macrophages infiltrated into the retina were decresed.The apoptosis of the retinal cells was inhibited.The proportion of Th1,Th17,and Th0 cells were decreased,while the proportion of regulatory T cells was increased.(t=3.963,P<0.01;t=9.997,P<0.01;t=6.093,P<0.01;t=-3.162,P<0.05)IFN-?,IL-17A,IL-6,IL-1?and TNF-?were reduced in retina,while higher levels of TGF-?were detected in chrysin-treated mice.Further,STAT1,p-STAT1,STAT3,p-STAT3 and NF-?Bp65 was downregulated with chrysin treatment.Conclusions:I.The animal model of EAU can be successfully established by subcutaneously injection of emulsified IRBP_1-20/CFA.II.Chrysin ameliorates both clinical and histopathological manifestions of EAU.III.Chrysin exerts its immunological regulating effect on EAU by down-regulating the proportion of Th1,Th17,and Th0 cells and up-regulating the proportion of Treg cells.It also maintains the integrity of blood-retinal barrier,suppresse the infiltration of macrophage in the retina,inhibit the intraocular inflammation.The anti-inflammatory effect of chrysin may be achieved by inhibiting the STAT1,STAT3 and NF-?B signal transduction pathways.