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Objective Fenofibrate,a peroxisome proliferator-activated receptor alpha(PPAR alpha)agonist,has been shown to be beneficial in the treatment of diabetic retinopathy(DR).However,its underlying mechanisms remain largely unclear.Our previous studies demonstrated that oxidative stress and nitrosative stress-mediated Wnt pathway activation contribute to the retinal inflammation and retinal vascular leakage in DR.The purpose of this study is to evaluate the effect fenofibrate on regulating oxidative stress-mediated Wnt pathway activation and explore the possible mechanisms.Methods Male C57BL/6-Ins2Akita/J mice and immortalized human retinal pigment epithelial cell line(ARPE19 cells)were used in this study.At the age of 8 weeks,C57BL/6-Ins2Akita/J mice were randomly given with normal rodent chow or rodent chow with 0.15％fenofibrate.At the 8th week of treatment,the littermate control mice and C57BL/6-Ins2Akita/J mice were euthanized.ARPE19 cells were treated with different doses of fenofibrate(0.5-25mM)in normal glucose(5 mM)or high glucose(25 mM).Expression of oxidative/anti-oxidative enzymes(NADPH oxidases(Nox)and superoxide dismutases(SOD)),canonical Wnt pathway proteins(phosphorylated low-density lipoprotein receptor-related protein 6(pLRP6)and nuclear beta-catenin)were examined by immnunofluorescence,western blot analysis or real-time RT-PCR in mouse retinas and culture ARPE19 cells.Generation of intracellular reactive oxygen species(ROS)was measured using carboxy-H2DCFDA probe.Overexpression of NADPH oxidase4(Nox4)in ARPE19 were achieved by infection with adenovirus expressing wild type Nox4 or Nox4 siRNA.Results Expression of Nox4 and Nox2 were up-regulated and expression of SOD1 and SOD2 were down-regulated in the retinas of C57BL/6-Ins2Akita/J mice when compared to the age-matched littermate controls.In addition,increased phosphorylation of LRP6 and nuclear beta-catenin level were observed in the retinas of C57BL/6-Ins2Akita/J mice.Treatment with fenofibrate significantly decreased Nox2 and Nox4 expression and increased SOD1 and SOD2 expression in C57BL/6-Ins2Akita/J mice.Meanwhile,retinal nuclear beta-catenin levels in C57BL/6-Ins2Akita/J mice were reduced by fenofibrate treatment.In cultured ARPE19 cells,fenofibrate significantly inhibited high glucose-induced Nox2 and Nox4 upregluation and attenuated high glucose-induced SOD1 and SOD2 decreases.Consistently,fenofibrate dose-dependently suppressed high glucose-induced intracellular ROS generation.Moreover,high glucose-induced LRP6 phosphorylation was reduced in fenofibrate treated ARPE19 cells.Further mechanism study demonstrated that in cultured ARPE 19 cells,overexpression of Nox4 could induce LPR6 phosphorylation; however,knockdown of Nox4 inhibited high glucose-induced LPR6 phosphorylation.Conclusions Nox4-mediated oxidative stress contributes to Wnt pathway activation in diabetic conditions.Attenuation of oxidative stress by fenofibrate is a potential mechanism responsible for its beneficial effect on Wnt pathway activation in DR.