Fu Dongxu, Yu Jeremy Y, Connell Anna R, Yang Shihe, Hookham Michelle B, McLeese Rebecca, Lyons Timothy J
Centre for Experimental Medicine School of Medicine, Dentistry, and Biomedical Sciences, Queen's University Belfast, Northern Ireland, United Kingdom.
Section of Endocrinology and Diabetes, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States.
Invest Ophthalmol Vis Sci. 2016 Jun 1;57(7):3369-79. doi: 10.1167/iovs.16-19291.
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.
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).
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).
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.
对糖尿病视网膜病变（DR）的机制理解有限阻碍了治疗进展。小檗碱是一种异喹啉生物碱，在动物和人体研究中已显示出对葡萄糖和脂质代谢有有益作用，但对DR的影响尚不清楚。我们之前证明了在人类糖尿病中视网膜内低密度脂蛋白（LDL）的外渗和修饰，以及修饰后的LDL对人视网膜Müller细胞的毒性。我们现在探讨修饰后的LDL对Müller细胞的致病作用，以及小檗碱减轻这种细胞毒性的功效。
将汇合的人Müller细胞暴露于体外修饰的“高度氧化、糖化（HOG-）LDL与天然LDL（N-LDL；200mg蛋白/L）中6或24小时，有或没有用小檗碱（5μM，1小时）和/或单磷酸腺苷（AMP）激活的蛋白激酶（AMPK）抑制剂Compound C（5μM，1小时）进行预处理。使用包括蛋白质印迹、活性氧（ROS）检测测定和定量实时PCR在内的技术，评估以下结果：细胞活力（CCK-8测定）、自噬（LC3、Beclin-1、ATG-5）、凋亡（裂解的半胱天冬酶3、裂解的聚ADP核糖聚合酶）、氧化应激（ROS、核因子红细胞2相关因子2、谷胱甘肽过氧化物酶1、NADPH氧化酶4）、血管生成（血管内皮生长因子、色素上皮衍生因子）、炎症（诱导型一氧化氮合酶、细胞间粘附分子1、IL-6、IL-8、肿瘤坏死因子-α）和神经胶质细胞活化（胶质纤维酸性蛋白）。
天然LDL对培养的人Müller细胞没有影响，但HOG-LDL表现出明显的毒性，显著降低细胞活力并诱导自噬、凋亡、氧化应激、血管生成因子表达、炎症和神经胶质细胞活化。小檗碱减弱了HOG-LDL的所有作用（所有P<0.05），并且其作用通过AMPK抑制而减弱（P<0.05）。
小檗碱通过激活AMPK途径抑制修饰后的LDL诱导的Müller细胞损伤，作为预防和/或治疗DR的药物值得进一步研究。
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