title: Beneficial Effects of Berberine on Oxidized LDL-Induced Cytotoxicity to Human Retinal Müller Cells.
abstract: OBJECTIVE
  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 Müller cells. We now explore pathogenic effects of modified LDL on Müller cells, and the efficacy of berberine in mitigating this cytotoxicity.
  
  
  METHODS
  Confluent human Müller 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 μM, 1 hour) and/or the adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibitor, Compound C (5 μM, 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-α), and glial cell activation (glial fibrillary acidic protein).
  
  
  RESULTS
  Native-LDL had no effect on cultured human Müller 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 Müller cell injury by activating the AMPK pathway, and merits further study as an agent for preventing and/or treating DR.
authors.affiliation: Centre for Experimental Medicine School of Medicine, Dentistry, and Biomedical Sciences, Queen's University Belfast, Northern Ireland, United Kingdom.
authors.forename: Dongxu
authors.initials: D
authors.lastname: Fu
authors.affiliation: Centre for Experimental Medicine School of Medicine, Dentistry, and Biomedical Sciences, Queen's University Belfast, Northern Ireland, United Kingdom.
authors.forename: Jeremy Y
authors.initials: JY
authors.lastname: Yu
authors.affiliation: Centre for Experimental Medicine School of Medicine, Dentistry, and Biomedical Sciences, Queen's University Belfast, Northern Ireland, United Kingdom.
authors.forename: Anna R
authors.initials: AR
authors.lastname: Connell
authors.affiliation: Section of Endocrinology and Diabetes, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States.
authors.forename: Shihe
authors.initials: S
authors.lastname: Yang
authors.affiliation: Centre for Experimental Medicine School of Medicine, Dentistry, and Biomedical Sciences, Queen's University Belfast, Northern Ireland, United Kingdom.
authors.forename: Michelle B
authors.initials: MB
authors.lastname: Hookham
authors.affiliation: Centre for Experimental Medicine School of Medicine, Dentistry, and Biomedical Sciences, Queen's University Belfast, Northern Ireland, United Kingdom.
authors.forename: Rebecca
authors.initials: R
authors.lastname: McLeese
authors.affiliation: Centre for Experimental Medicine School of Medicine, Dentistry, and Biomedical Sciences, Queen's University Belfast, Northern Ireland, United Kingdom.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Lyons
mesh_terms: D000704:Analysis of Variance; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D001599:Berberine / Q000494:pharmacology*; D015415:Biomarkers / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003930:Diabetic Retinopathy / Q000378:metabolism; D063928:Ependymoglial Cells / Q000187:drug effects* / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000494:pharmacology; D009414:Nerve Growth Factors / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015843:Serpins / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
chemical_list: D000975:Antioxidants; D001599:Berberine; D015415:Biomarkers; D005136:Eye Proteins; D015850:Interleukin-6; D016209:Interleukin-8; D008077:Lipoproteins, LDL; D009414:Nerve Growth Factors; D052247:Nitric Oxide Synthase Type II; D017382:Reactive Oxygen Species; D015843:Serpins; D014409:Tumor Necrosis Factor-alpha; D000099174:Pigment Epithelium-Derived Factor; C099252:oxidized low density lipoprotein; C496319:NOS2 protein, human