作者：林宝琴,邱春亿
作者单位：美国德克萨斯农工大学医学院眼科药理研究所
【摘要】目的: 研究柚皮素对人视网膜色素上皮细胞(ARPE19)和人脐静脉内皮细胞(HUVEC)的抗氧化作用。
方法：采用MTT的方法检测ARPE19 和 HUVEC细胞的生存率及增殖率。
结果：3，10mg/L柚皮素能显著增加ARPE19细胞的增殖率达10.8% 和11.4%。10mg/L柚皮素能提高ARPE19细胞在缺氧，0.3mmol/L NaN3及200μmol/L H2O2条件下的生存率分别为55.2%, 69.2%及 50.3%。1mg/L柚皮素能提高ARPE19细胞在50μmol/L tBHP和30mg/L NaIO3条件下的生存率达20.2% 和30.4%。30mg/L柚皮素能够促进ARPE19细胞在50 μmol/L tBHP条件下的增殖率达32.2%,而1mg/L柚皮素可以提高30, 100,300mg/L NaIO3处理的 ARPE19 细胞的增殖率达 30.3%, 10.3%及18.5%。3, 10及 30mg/L柚皮素抑制HUVEC的增殖率分别为23.9%, 70.4%及77.9%。1, 3mg/L柚皮素能提高HUVEC细胞在缺氧条件下的生存率达10.7%和 13.1%，以及提高在300mg/L NaIO3条件下的生存率达41.2%和 37.7%。3mg/L柚皮素能提高HUVEC细胞在200，400μmol/L H2O2条件下的生存率达20.1%和 21.5%。
结论：柚皮素能够促进ARPE19细胞的增殖率，抑制HUVEC生长，同时对这两种细胞均有抗氧化作用。因此，柚皮素是治疗老年黄斑变性的很有前景的候选药物。
【关键词】 柚皮素 人视网膜色素上皮细胞 人脐静脉内皮细胞 老年黄斑变性
INTRODUCTION
Agerelated macular degeneration(AMD) is the leading cause of blindness in older persons in developed countries. It initially occurs in a "dry" form (pathological changes in the RPE and drusen formation), and can progress to geographic atrophy or "wet" form with choroidal neovascularization (CNV). RPE forms a monolayer between the neurosensory retina and choroid. Its main functions are to supply nutrients to the adjacent photoreceptors and to dispose of shed photoreceptor outer segments by phagocytosis [1]. One hypothesis places RPE dysfunction as the major etiology of AMD pathogenesis. When RPE is unable to remove the metabolic waste, it results in accumulation of drusen. RPE dysfunction causes the breakdown of the bloodretinal barrier and the leakage of plasma and proteins that leads to exudative retinal detachment. The break down of Bruchs memebrane under the detached RPE serves as an entrance for new and immature choroidal vessels to grow into the subretinal space that leads to the formation of CNV. Furthermore, loss of RPE may cause loss of choriocapillaris [24]. Therefore, RPE is a target for therapeutic approaches aimed at enhancement of photoreceptor survival in such ocular disease [5]. The mechanisms of the dysfunction or cell death of RPE may involve various factors, such as oxidative injury, degenerative changes in Bruchs membrane and damage to the choroidal vasculature [67]. Different types of oxidative stress results in different patterns of oxidative damage to proteins in RPE cells and different patterns of loss of viability [8].
CNV, the hallmark of wet AMD, is responsible for approximately 90% of cases of severe vision loss due to AMD. CNV is the result of angiogenesis, which include endothelial cell proliferation, migration and adhesion [9].
Naringenin is a flavonoid that is considered to have bioactivity in vivo[1012] and in vitro[13] as an antioxidant.
The aim of this research was to study whether naringenin has the antioxidant activity on various oxidants induced injuries in RPE cells and HUVEC. If so, naringenin could become a candidate for the treatment of AMD.
MATERIALS AND METHODS
Materials Naringenin was purchased from Pfaltz & Bauer, Inc. (Waterbury, CT, USA). Sodium iodate (NaIO3, purity ≥99.5%), thiazolyl blue tetrazolium bromide (MTT, purity ≥97.5 %), hydrogen peroxide (H2O2, 500g/L solution in water), tertburyl hydroperoxide (tBHP, 700g/L in water), dimethyl sulfoxide (DMSO, purity ≥99.9%), sodium azide (NaN3, purity ≥99.5%) , and Dulbeccos modified Eagles medium/Hams F12 (DMEM/F12, 1∶1) were purchased from SigmaAldrich Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from GIBCO (Grand Island, NY, USA). HUVEC and EGM2 Bulletkit that contains EBM2 basal medium and EGM2 singlequot Kit were bought from Lonza Walkersville, Inc. (Walkersville, MD, USA). ARPE19 cells were purchased from ATCC (Manassas, VA, USA).
Cell Culture ARPE19 cells were grown in DMEM/F12 medium supplemented with 100mL/L FBS, 1×105/L penicillin G, and 100mg/L streptomycin sulfate. HUVEC were grown in EBM2 basal medium supplemented with EGM2 singlequot Kit. Cells were incubated in a humidified incubator at 37℃ under 50mL/L CO2 and 950mL/L air.
Naringenin on RPE Cells and HUVEC MTT assay was used to measure the viability and proliferation of ARPE19 cells and HUVEC. 8×104 cells were seeded in 96well plates (100L/well) and allowed to grow overnight. Blanks were prepared by adding 100L medium (Ablank). The cells were then treated with fresh medium with naringenin and/or oxidizing agents at the same time for 12, 24, or 72 hours (200L/well, Acompound). The vehicle control group (Acontrol) was treated with 7.6mL/L alcohol and/or PBS. 20L MTT (5g/L) was added to wells, and incubated for another 4 hours. After incubation, the medium was discarded and 150 L DMSO was added to solubilize formazan produced from MTT by the viable cells. Absorbance was measured at 570nm using a microplate reader (Packard BioScience Co. Meriden, CT, USA). Cell viability was calculated according to the following formula: Viability of cells (%) = (AcompoundAblank)/ (AcontrolAblank)×100%.
Hypoxia Treatment Cells were allowed to attach overnight, and then exposed to naringenin and solvent under hypoxic condition for 72 hours. Hypoxic conditions (10mL/L O2, 50mL/L CO2 and 940mL/L N2) were maintained by using a temperature and humiditycontrolled environmental Cchamber by O2 and CO2 controllers (Proox Model 110 and Pro CO2 Model 120, Bio Spherix Ltd., Redfield, NY) with N2 and CO2 gas sources.
Statistical Analysis Results were presented as means±SEM. Multiple comparisons between groups were made with Students ttests (twotail). Different values at P<0.05 were considered significant.
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