Journal
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
Volume 57, Issue 7, Pages 3369-3379Publisher
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.16-19291
Keywords
angiogenesis; apoptosis; autophagy; berberine; diabetic retinopathy; GFAP; inflammation; Muller cell; oxidative stress; oxidized LDL
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Funding
- Oklahoma Center for the Advancement of Science and Technology (Oklahoma City, OK, USA) [HR08-067]
- Centers of Biomedical Research Excellence Program of the National Center for Research Resources (Bethesda, MD, USA) [P20 RR 024215]
- LINJO fund (Oklahoma City, OK, USA)
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PURPOSE. Limited mechanistic understanding of diabetic retinopathy (DR) has hindered therapeutic advances. Berberine, an isoquinolone alkaloid, has shown favorable effects on glucose and lipid metabolism in animal and human studies, but effects on DR are unknown. We previously demonstrated intraretinal extravasation and modification of LDL in human diabetes, and toxicity of modified LDL to human retinal Muller cells. We now explore pathogenic effects of modified LDL on Muller cells, and the efficacy of berberine in mitigating this cytotoxicity. METHODS. Confluent human Miller cells were exposed to in vitro-modified highly oxidized, glycated (HOG-) LDL versus native-LDL (N-LDL; 200 mg protein/L) for 6 or 24 hours, with/without pretreatment with berberine (5 mu M, 1 hour) and/or the adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibitor, Compound C (5 mu M, 1 hour). Using techniques including Western blots, reactive oxygen species (ROS) detection assay, and quantitative real-time PCR, the following outcomes were assessed: cell viability (CCK-8 assay), autophagy (LC3, Beclin-1, ATG-5), apoptosis (cleaved caspase 3, cleaved poly-ADP ribose polymerase), oxidative stress (ROS, nuclear factor erythroid 2-related factor 2, glutathione peroxidase 1, NADPH oxidase 4), angiogenesis (VEGF, pigment epithelium-derived factor), inflammation (inducible nitric oxide synthase, intercellular adhesion molecule 1, IL-6, IL-8, TNF-alpha), and glial cell activation (glial fibrillary acidic protein). RESULTS. Native-LDL had no effect on cultured human Miller cells, but HOG-LDL exhibited marked toxicity, significantly decreasing viability and inducing autophagy, apoptosis, oxidative stress, expression of angiogenic factors, inflammation, and glial cell activation. Berberine attenuated all the effects of HOG-LDL (all P < 0.05), and its effects were mitigated by AMPK inhibition (P < 0.05). CONCLUSIONS. Berberine inhibits modified LDL-induced Miller cell injury by activating the AMPK pathway, and merits further study as an agent for preventing and/or treating DR.
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