| ObjectiveNaringenin has various ophthalmological activities. Primary studies showed that topical application of 1% naringenin eye drops prevented N-methyl-N-nitr6sourea (MNU)-induced photoreceptor cell apoptosis in rats. The aim of this study was to investigate the ocular pharmacokinetics of 1% naringenin eye drops following topical administration to rabbits, and the effect of naringenin eye drops on MNU-induced photoreceptor cell apoptosis in rats. Antiapoptotic mechanisms was studied in light- and H2O2-induced 661W cells in vitro.Methods(1) Ocular pharmacokinetics of naringenin eye drops:One drop (50μL) of 1% naringenin eye drops was instilled into each eye of each rabbit. The animals were sacrificed at predetermined intervals after dosing, and ocular tissues and plasma were then collected. Concentrations of naringenin were analyzed using specific electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.(2) Protective effect of naringenin eye drops on MNU-induced photoreceptor cell apoptosis in rats:Photoreceptor cell apoptosis was induced by single intraperitoneal injection of MNU (60 mg/kg) in rats. Both eyes of all animals were instilled with one drop of vehicle,0.5% or 1% naringenin eye drops three times per day from 7 days before to 17 days after MNU injection. Effects of naringenin on MNU-induced photoreceptor cell apoptosis were evaluated by electrophysiological, behavior performance and histological analyses.(3) Mechanism of naringenin on light-induced 661W cells apoptosis:661W cells were incubated with 0.5-100 mg/L naringenin for 24 h after light exposure for 8 h. Cell viability was detected by MTT assay, and apoptotic rate was observed by flow cytometry. Intracellular ROS in 661W cells was evaluated using H2DCFDA probe.(4) Mechanism of naringenin on H2O2-induced 661W cells injury:661W cells were incubated with 0.5-100 mg/L naringenin for 24 h after 5 μmol H2O2 exposure for 4 h. Cell viability was detected by MTT assay, and apoptotic rate was observed by flow cytometry. Intracellular ROS in 661W cells was evaluated using H2DCFDA probe.Results(1) Ocular pharmacokinetics of naringenin eye drops:The LC-MS/MS method has been proved to be sensitive, specific, precise, and suitable for determination of naringenin in ocular tissues and plasma of rabbits. Ocular exposure to naringenin, based on AUC(0-t), was highest in cornea, followed by aqueous humor, retina and vitreous body. The Cmax of naringenin in cornea, aqueous humor, vitreous body and retina were 67945.30±4109.34 ng/g,1325.69±239.34 ng/mL,160.52±38.78 ng/mL and 1927.08±660.77 ng/g at 0.083, 0.75,0.083 and 0.083 h after topical administration, respectively. The half-life for these tissues were 9.37,0.65,1.17 and 4.62 h, respectively. There was no significant difference between free naringenin and total naringenin in plasma based on Cmax and Tmax. Cmax of total naringenin in plasma at 0.083 h was 35.12±0.54 ng/mL.(2) Protective effect of naringenin eye drops on MNU-induced photoreceptor cell apoptosis in rats:Flash electroretinography (FERG) and oscillatory potentials (OPs) recordings showed that the vehicle control group had remarkable reduction of amplitudes and prolongation of latency times. FERG and OPs responses were significantly reversed in MNU-induced rats instilled with 0.5% or 1% naringenin eye drops compared with the vehicle control. Moreover, Morris water maze experiment showed that either 0.5% or 1% naringenin eye drops increased the numbers of platform-site crossover (P<0.05 vs. vehicle control). The retinal morphological results showed that naringenin dose-dependently preserved the outer nuclear layer (ONL), outer retina (OR) and total retina (TR).(3) Mechanism of naringenin on light-induced 661W cells apoptosis:Although 100 mg/L naringenin had no influence on proliferation in normal 661W cells, it significantly increased light-induced cell viability. Moreover,10 mg/L naringenin significantly reduced apoptotic cells and intracellular ROS in light-induced 661W cells.(4) Mechanism of naringenin on H2O2-induced 661W cells injury:Cell viability was improved by 5-100 mg/L naringenin in H2O2-induced 661W cells. Ten mg/L naringenin significantly reversed the increase of apoptotic cells and intracellular ROS in 661W cells.Conclusion(1) Measurable concentrations of naringenin were achieved in ocular tissues after topical application in rabbits. Topical instillation of naringenin may be an effective approach in the treatment of posterior section diseases.(2) Topical treatment with naringenin eye drops prevented retinal neurons from MNU-induced structural and functional damages.(3) Naringenin protected 661W cells apoptosis from light-induced damage. Reducing ROS contributed to the protective effect of naringenin.(4) Naringenin reduced H2O2-induced apoptosis in mouse cone cells. One of the mechanisms may be reducing intracellular ROS in 661W cells. |
PDF Full Text Request