Abstract
Purpose:
Fenofibrate 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 contributes to 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 Akita mice and ARPE-19 cells were used in this study. Akita mice were randomly given normal rodent chow or rodent chow with 0.15% fenofibrate. ARPE19 cells were treated with different doses of fenofibrate (0.5-25mM) in normal glucose (5 mM) or high glucose (HG, 25 mM). Expression of NADPH oxidases (Nox) and superoxide dismutases (SOD), canonical Wnt pathway proteins (phosphorylated low-density lipoprotein receptor-related protein 6 (pLRP6) and nuclear β-catenin) were examined by immnunofluorescence, western blot analysis or real-time RT-PCR in mouse retinas and cultured cells. Generation of reactive oxygen species (ROS) was measured using DCF assay. Overexpression of Nox4 in ARPE19 cells were achieved by infection with adenovirus expressing wild type Nox4 or Nox4 siRNA.
Results:
Administration with fenofibrate significantly decreased Nox2 and Nox4 upregulation and reversed SOD1 and SOD2 downregulation in Akita mouse retinas. Meanwhile, increased nuclear β-catenin levels in retinas of Akita mice were reduced by fenofibrate treatment. In cultured ARPE19 cells, fenofibrate significantly inhibited HG-induced Nox2 and Nox4 upregluation and attenuated HG-induced SOD1 and SOD2 decreases. Consistently, fenofibrate dose-dependently suppressed HG-induced ROS generation. Moreover, HG-induced LRP6 phosphorylation was reduced in fenofibrate-treated ARPE19 cells. Further mechanism study demonstrated that in ARPE19 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.
Keywords: 499 diabetic retinopathy •
634 oxidation/oxidative or free radical damage