【摘要】 目的: 观察黄酮对氧化剂所诱导的视网膜色素上皮细胞(RPE)的保护作用。方法：在体研究中，预先给予5g/L黄酮滴眼液(3次/d)，1wk后舌下静脉注射NaIO3诱导大鼠RPE变性，在2和4wk末，采用视网膜电图(ERG)测量C波。离体研究中，采用缺氧、H2O2、NaN3和t-BHP诱导RPE细胞损伤，并用MTT法检测细胞的存活率。结果：ERG的C波结果表明，第4wk末，黄酮抑制了由NaIO3诱导的大鼠RPE变性。离体研究结果表明，黄酮对多种氧化剂所诱导的RPE细胞损伤具有保护作用。结论：黄酮对氧化诱导的在体和离体视网膜色素上皮细胞均具有保护作用。
【关键词】 黄酮;老年黄斑变性;视网膜色素上皮;氧化作用
INTRODUCTION
Agerelated macular degeneration (AMD) is one of the most common irreversible causes of severe loss of vision, in the elderly population[13]. In 2002, the World Health Organization estimated that 14 million persons worldwide are blind or severely visually impaired because of AMD. As the population in the United States and Europe is growing older, and higher expectations of better quality of life, including the ability of reading and driving, are being demanded by patients, the morbidity resulting from AMD is becoming increasingly significant[4]. Given the enormous impact of AMD on an aging population, much public interest and research has been focused on this condition in the past decade.
AMD initially occurs in a "dry" form with pathological changes in the retinal pigment epithelium (RPE) and drusen formation[5]. RPE plays a key role in maintaining retinal function by assuming a strategic position as the metabolic gatekeeper between photoreceptors(PRs) and the choriocapillaries[6]. The aging RPE monolayer incurs annual cell losses at a rate of approximately 0.3%[7] and may as a result impose an increased metabolic demand for each cell[8]. When RPE disabled to remove the metabolic wastes, it will result in the 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.
Flavonoids are a class of more than 4000 phenylbenzopyrones that occur in many edible plants, like fruits and vegetables[9]. These polyphenolic compounds display a remarkable spectrum of biochemical activities including antioxidant activities[10]. This study is to observe the effects of flavone on NaIO3 induced RPE degeneration and various oxidants induced injury in human retinal pigment epithelium (ARPE19) cells.
MATERIALS AND METHODS
Materials Eightweekold male BrownNorway rats were purchased through LARR (Texas A&M University, USA). Animal care and treatment were followed by the institutional guidelines.
Flavone, thiazolyl blue tetrazolium bromide (MTT, purity≥97.5%), Dulbeccos phosphate buffered saline (DPBS), hydrogen peroxide (H2O2, 50wt% solution in water), tertbutyl hydroperoxide (tBHP, 70wt% in water), sodium iodate (NaIO3, purity≥99.5%), sodium azide (NaN3, purity≥99.5%) and Dulbeccos modified Eagles medium/Hams F12 (DMEM/F12, 1∶1) were all purchased from SigmaAldrich Chemical Co. (St. Louis, MO, USA). Human retinal pigment epithelium (ARPE19) cells and fetal bovine serum (FBS) were purchased from ATCC (Manassas, VA, USA).
Effect of Flavone on NaIO3induced RPE Degeneration in Rat Eyes The rats were randomly divided into 3 groups. Control group was instilled with vehicle (30% HPβCD); NaIO3 group was instilled with vehicle plus 35mg/kg NaIO3 injection; flavone+NaIO3 group was instilled with 5g/L flavone eye drops plus 35mg/kg NaIO3 injection. All eye drops were instilled 3 times a day for 1 week before and 4 weeks after NaIO3 injection. At the end of 2 and 4 weeks, all rats were measured cwave of ERG.
The rats were dark adapted for 2 hours, and then anesthetized with ketamine 35mg/kg plus xylazine 5mg/kg intramuscular injection. Half of the initial dose was given every one hour thereafter. The rat pupils were dilated with one drop of 10g/L atropine and 25g/L phenylephrine respectively. One drop of 5g/L tetracaine was given for surface anesthesia before recording. All rats were kept warm during ERG measurement. DCERG recording methods by Peachey et al[11] were followed. Briefly, the 1mm diameter glass capillary tube with filament (Sutter Instruments, Novato, CA, USA) which was filled with Hanks balanced salt solution (Invitrogen, Carlsbad, CA, USA) was used to connect with the Ag/AgCl wire electrode with the attached connector. The capillary tube was contacted with rats corneal surface completely. Another similar electrode placed on the surface of the other eye served as a reference lead. Responses were amplified (dc100 Hz; gain=1000X; DP31, Warner Instruments, Hamden, CT, USA) and digitized at 10Hz or 1000Hz. Data were analyzed by iWORX LabScribe Data Recording Software (iWorx0CB Sciences, Dover, NH). Light stimuli were derived from an optical channel using the fiberlite high intensity illuminator (DolanJenner Industries, Inc., MA, USA) with neutral density filters (Oriel, Stratford, CT, USA) placed in the light path to adjust stimulus luminance. The stimulus luminance used in this experiment was 3.22 log cd/m2 and stimulated for 4 minutes. Luminance calibration was made by the Minolta (Ramsey, NJ, USA) LS110 photometer focused on the output side of the fiber optic bundle where the rat eye was located.
Cell Culture ARPE19 cells were grown in DMEM/F12 medium supplemented with 10% FBS, 100 units/mL penicillin G, and 100μg/mL streptomycin sulfate. Cells were incubated in a humidified incubator at 37℃ under 50mL/L CO2 and 950mL/L air.
Effect of Flavone on the Viability of ARPE19 Cells MTT assay was used to measure the viability of ARPE19 cells. 1×105 cells were seeded in 96well plates (100μL/well) and allowed to grow overnight. Negative control was prepared by adding 100μL medium without cells. The cells were then treated with fresh medium with flavone (flavone was dissolved in 30% HPβCD, the final concentration of HPβCD in cells is less than 0.3%) and/or oxidizing agents (H2O2, NaN3 and tBHP) for 12, 24, or 72 hours (200μL/well). The vehicle control group was treated with 30% HPβCD solution with fresh medium (the final concentration of HPβCD in cells is less than 0.3%). 20μL MTT (5g/L) was added to wells, and incubated for another 4 hours. After incubation, the medium was discarded and 100μL DMSO was added to solubilize formazan produced from MTT by the viable cells. Absorbance was measured at 570nm using a microplate reader (BioRad Laboratories, Inc., CA). Cells viability was calculated according to the following formula: Viability of cells (%)=(absorbance in tested sample absorbance in negative control)/(absorbance in vehicle control absorbance in negative control)×100%.
Hypoxia Treatment Cells were allowed to attach overnight, and then exposed to flavone or vehicle under hypoxic condition for 72 hours. Hypoxic conditions (10mL/L O2, 50mL/L CO2
Figure 1Representative ERG wave form at 4 weeks after NaIO3 injection A: vehicle control group; B: NaIO3 group; C: flavone+NaIO3 group.
and 940mL/L N2) were maintained by using a temperature and humidity controlled environmental Cchamber by O2 and CO2 controllers (Proox Model 110 and Pro CO2 Model 120, Bio Spherix Ltd., Redfield, NY, USA) with N2 and CO2 gas sources.
Statistical Analysis All data were expressed as mean±SEM. Statistical analysis was performed using the Students t test. P<0.05 was considered to be statistically significant.
RESULTS
Effect of Flavone on NaIO3induced RPE Degeneration in Rats Eyes Four weeks after NaIO3 injection, the amplitude of ERG cwave was (0.330±0.036)Volts in the control group, (0.029±0.005)Volts in the NaIO3 group, and (0.070±0.006)Volts in the flavone+NaIO3 group. There was a significant reversal of the ERG cwave by flavone as compared with NaIO3 group (P<0.05, Figure 1).
Cytotoxicity of Flavone in ARPE19 Cells The results showed that flavone did not affect cell growth of ARPE19 up to the concentration of 10μg/mL. However, the proliferation of ARPE19 cells was significantly inhibited at the concentrations of 30 and 100μg/mL (P<0.01, Figure 2).
Figure 2Effect of flavone on proliferation of ARPE19 cells ARPE19 cells were incubated with flavone for 72 hours.n=6 in each group; bP<0.01 vs vehicle control group.
Figure 3Effect of flavone on hypoxiainduced injury in ARPE19 cells ARPE19 cells were incubated with flavone for 72 hours. Control group treated with vehicle (30% HPβCD solution) under normal condition (50mL/L CO2 and 950mL/L air) for 72 hours; model group treated with vehicle under hypoxic condition (10mL/L O2, 50mL/L CO2 and 940mL/L N2) for 72 hours. n=6 in each group; aP<0.05 and bP<0.01 vs model group.
Effect of Flavone on Hypoxiainduced Injury in ARPE19 Cells Flavone significantly decreased the viability of ARPE19 cells in hypoxic condition at concentration of 1 and 3μg/mL (Figure 3). However, at the concentration of 100μg/mL, flavone significantly increased the viability of ARPE19 cells by 38% (P<0.01) in hypoxic condition.
Effect of Flavone on H2O2induced Injury in ARPE19 Cells At 0.3, 30 and 100μg/mL, flavone reversed 200μmol/L H2O2induced injury by 35%, 15% and 56% respectively in ARPE19 cells. And 30μg/mL flavone reversed 400μmol/L H2O2induced injury by 25% in ARPE19 cells (Figure 4).
Effect of Flavone on NaN3induced Injury in ARPE19 Cells Flavone significantly reversed 0.3, 1 and 3mmol/L NaN3induced injury in ARPE19 cells by 34%, 86% and 72% (P<0.01), respectively, at the concentration of 100μg/mL (Figure 5).
Effect of Flavone on tBHPinduced Injury in ARPE19 Cells At the concentrations of 30 and 100μg/mL, flavone significantly increased the viability of 50 and 100μmol/L tBHP treated ARPE19 cells (Figure 6).
DISCUSSION
AMD is the leading cause of blindness in the elderly worldwide, affecting 3050 million individuals. It is particularly prevalent in the United States and European countries.
Figure 4Effect of flavone on H2O2induced injury in ARPE19 cells ARPE19 cells were incubated with flavone and H2O2 for 24 hours. n=6 in each group; aP<0.05 and bP<0.01 vs model group.
Figure 5Effect of flavone on NaN3induced injury in ARPE19 cells ARPE19 cells were incubated with flavone and NaN3 for 72 hours. n=6 in each group; aP<0.05 and bP<0.01 vs model group.
Figure 6Effect of flavone on tBHPinduced injury in ARPE19 cells ARPE19 cells were incubated with flavone and tBHP for 12 hours. n=6 in each group; aP<0.05 and bP<0.01 vs model group.
It is generally accepted that the impairment of RPE cell function is an early and crucial event in the molecular pathways leading to clinically relevant AMD[12,13]. RPE serves a variety of metabolic and supportive functions that are of vital importance for retinal photoreceptors, including maintenance of the bloodretinal barrier, participation in the visual cycle, and phagocytic uptake and degradation of constantly shed apical photoreceptor outer segments[14]. The direct toxic effect of NaIO3 on RPE cells with secondary effects on photoreceptors and the choriocapillaries in vivo is well known[15]. The results of this research showed that flavone reversed NaIO3induced injury in RPE by 141% at the end of 4 weeks. This result might indicate that flavone had protective effect against NaIO3 induced RPE degeneration in rat eyes and might slow down the development of AMD.
RPE cells are particularly susceptible to oxidative stress because of high concentration of polyunsaturated fatty acids in the outer segments[16] and exposure to visible light[1720]. Different types of oxidative stress results in different patterns of oxidative damage to proteins in RPE cells and different patterns of loss of viability[21]. In addition to this indirect evidence in support of oxidative stress as a pathogenic factor in AMD, clinical data provide direct validation of the antioxidant approach to AMD treatment. It is a general consensus that oxidative damage plays an important role in pathogenesis of dry AMD[22]. Clearly, antioxidants are needed for dry AMD treatment. In this study, H2O2, NaN3 and tBHP was used as oxidants to induce injuries in RPE cells. The results showed flavone reversed the various oxidants induced injuries in RPE cells. In other words, flavone could prevent the oxidative injury of RPE in AMD.
In conclusion, flavone acts as an antioxidant to protect the RPE from damage by oxidative stress. Thus, flavone might be a promising candidate for the treatment of AMD.
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