AIM: To evaluate the antioxidant activity of naringenin in human retinal pigment epithelium (ARPE-19) cells andhuman umbilical vein endothelial cells (HUVEC).·METHODS: MTT assay was used to measure theviability and proliferation of ARPE-19 cells and HUVEC.·RESULTS: Three and 10mg/L naringenin significantlyincreased the proliferation of ARPE-19 cells by 10.8% and11.4%, respectively. Ten mg/L naringenin increasedhypoxia-, 0.3mmol/L NAN3-, and 200μmol/L H2O2- induced damage of ARPE-19 cells by 55.2%, 69.2%, and50.3%, respectively. One mg/L naringenin increased theviability of 50μmol/L t-BHP-, and 30mg/L NalO3-treatedARPE-19 cells by 20.2% and 30.4%, respectively. Thirtymg/L naringenin also increased the proliferation of50μmol/L t-BHP-treated ARPE-19 cells by 32.2%, and1mg/L naringenin increased the proliferation of 30, 100and 300 mg/L NalO3-treated ARPE-19 cells by 30.3%,10.3% and 18.5%, respectively. The reduction of HUVECwas 23.9%, 70.4% and 77.9% in the 3, 10 and 30mg/Lnaringenin-treated groups, respectively. Furthermore, 1and 3mg/L naringenin increased hypoxia-induced damagein HUVEC by 10.7% and 13.1%, and 300mg/L NalO3- induced damage in HUVEC by 41.2% and 37.7%. Threemg/L naringenin increased 200 and 400μmol/L H2O2-in-jured HUVEC by 20.1% and 21.5%, respectively.·CONCLUSION: Naringenin increases the proliferation ofARPE-19 cells and inhibites the growth of HUVEC, and haspotent antioxidant activity in ARPE-19 cells and HUVEC.

AIM: To investigate the antioxidant effect of hydralazine under hypoxia-induced damage on retinal pigment epithelial (ARPE-19) cells and the role of reactive oxygen species (ROS) in this effect.METHODS: Human retinal pigment epithelial (hRPE) cells were used to investigate the effect of hydralazine on oxidative stress, including tert-butyl hydroxyperoxide (t-BHP), H2O2, sodium azide (NaN3), and hypoxia induced cell damage. Cell viability was determined by MTT assay.RESULTS: When ARPE-19 cells were treated with oxidative stress induced by ROS, hydralazine showed concentration-dependent protection against t-BHP, H2O2 and hypoxia induced cell damage but not NaN3. Nitric oxide (NO) was not involved in this effect.CONCLUSION: Hydralazine showed antioxidant potential against oxidative stress induced damage in ARPE-19 cells. These effects might be caused through scavenger of ROS. Thus, hydralazine could be used for the treatment of age-related macular degeneration (AMD).

AIM: To investigate the effect of flavone on oxidation-induced injury in retinal pigment epithelium(RPE) cells. METHODS: In in vivo studies, NaIO3-induced RPE degeneration in rat eyes were treated with 5g/L flavone eye drops 3 times a day for 1 week before and 4 weeks after NaIO3injection. At the end of 2 and 4 weeks, all rats were measured c-wave by electroretinogram(ERG). In in vitro studies,ARPE-19 cells were treated with hypoxia, H2O2, NaN3and t-BHP to induce cell damages. MTT assay was used to measure the viable cells. RESULTS: The ERG c-wave results showed that flavone reversed NaIO3-induced injury at the end of 4 weeks. In vitro results showed flavone reversed the various oxidants-induced injuries in RPE cells.CONCLUSION: Flavone could prevent the RPE from oxidation-induced injury both in vivo and in vitro.

AIM:To investigate the effect of flavone on ocular blood flow in rabbit eyes and the formation of choroidal neovascularization(CNV)in rat model of age-related macular degeneration(AMD).METHODS:In in vivo studies,colored microsphere technique was used to determine the ocular blood flow in ocular hypertensive rabbit eyes.The rat eyes were treated with 5g/L flavone eye drops 3 times a day for 1 week before and 4 weeks after laser-induced injury of Bruch's membrane.The development of CNV was determined by fluorescein angiography(FA)performed on weeks 2 and 4.In in vitro studies,the effect of flavone on the viability of HUVECs was measured by MTT assay.RESULTS:The ocular blood flow in rabbit eyes was significantly increased after flavone instillation.Flavone significantly inhibited the formation of laser induced CNV.In vitro resultsshowedthat fiavoneinhibited the proliferation of HUVECs.CONCLUSION:Flavone could increase ocular blood flow and inhibit the formation of CNV.

AIM: To study the effects of naringenin eye drops on NaIO3-induced retinal pigment epithelium (RPE) degeneration and laser-induced choroidal neovascularization (CNV) in rat eyes.METHODS: The 35mg/kg NaIO3-induced RPE degeneration was prevented by 10g/L naringenin eye drops 3 times a day for 7 days in advance of NaIO3 injection, and then 2 to 4 weeks thereafter, RPE function was measured with C-wave of electroretinogram (ERG). The laser-induced CNV rats were treated with laser to break the Bruchs membrane and the CNV formation was prevented by 10g/L naringenin eye drops instilled 3 times a day for 2 to 4 weeks. The CNV formation was measured with fluorescein angiography (FA) and flat mount. RESULTS: Two weeks after NaIO3 injection, the amplitude of ERG C-wave fell markedly in NaIO3 group to 53% of normal group (P<0.01). No apparent difference was observed in naringenin+ NaIO3 group. Four weeks later, the NaIO3 group fell to 37% of normal group (P<0.01), while the naringenin+ NaIO3 group fell to only 57% of normal group (P<0.01). There is a 52% reversal of the ERG C-wave by naringenin as compared to NaIO3 treated group (P<0.05). Two weeks and four weeks after laser treatment, naringenin reduced the CNV formation to 53% and 49% of control group (100%) measured by FA (P<0.01). Four weeks after laser treatment, naringenin reduced the CNV formation by 47% as compared to control group measured with flat mount (P<0.01).CONCLUSION: Naringenin significantly protected RPE from NaIO3 induced degeneration and can also prevent CNV formation.

AIM: To investigate the effects of naringenin on laser-induced experimental choroidal neovascularization (CNV) in rat models,ocular blood flow in rabbit eyes and retinal function recovery after ischemic insults in rat eyes.membrane. Naringenin 10g/L(20mg/kg) was given once-daily through intraperitoneal injection for 4 weeks after laser treatment. The development of CNV was determined by fluorescein angiography(FA) performed on weeks 2 and 4. The colored microsphere technique and electroretinography method were used for the study of ocular blood flow and retinal function recovery,respectively.RESULTS: The choroidal blood flow in elevated intraocular pressure (IOP) rabbit eyes was significantly increased by 10g/L naringenin solution as compared to control group(P<0.05) . The retinal function recovery after ischemic insults in rat eyes indicated significant increase of b-wave recovery in treated group,as compared to control group(P<0.05).The intensity of fluorescein leakage from the photocoagulated lesions decreased significantly in treated group,compared to the control group(75.8%-95.0%,P<0.01). CONCLUSION: Naringenin could prevent the development of CNV on laser-induced experimental rat models,increase the choroidal blood flow in elevated IOP rabbit eyes and be beneficial on retinal function recovery in ischemic rat eyes.

·AIM: To study the effects of 10g/L hydralazine eye drops on 35mg/kg NaIO3-induced degeneration in rat eyes. · METHODS: Various doses of NalO3 and/or saline alone were injected into Brown Norway rats from hypoglossal vein. After 3, 7, 14 or 28 days of injection, ERG a-, b-, c- wave, fast oscillation (FO) and light peak (LP) were measured along with retinal colored pictures and fluorescein angiography taken. Some rats were chosen to study the histology of retinas by light microscopy and autofluorescence of retina flatmounts. Different concen- trations of NaIO3 were given to RPE-19 cells, and cell proliferation rate was measured. For hydralazine study, 35mg/kg NaIO3 was injected into Brown Norway rat from hypoglossal vein. NaIO3 group was treated with saline alone after NaIO3 injection, 10g/L hydralazine + NaIO3 group was treated with 10g/L hydralazine eyedrops after NaIO3 injection whereas normal group was treated with saline alone without NalO3 injection. All eyedrops were instilled locally 3 times a day for 4 weeks and ERG c-wave was measured at the end of 2 and 4 weeks.· RESULTS: After NaIO3 administration, the amplitude of all ERG waves fell markedly in large dose groups at 30, 40 or 60mg/kg NaIO3. Not many changes were observed in groups treated with < 30mg/kg NaIO3. Some retinal necrosis appeared from 3 days post-injection (PI) in 30mg/kg NaIO3 group, which became more serious in larger dose groups or longer treatment time, but no apparent change was found in smaller dose groups. Similarly, on the retina flatmount, RPE monolayer showed necrosis from 3 days PI in the 30mg/kg NaIO3 and larger dose groups. On histological examination, no significant change was seen in 30mg/kg NaIO3 and lower concentration groups. In cell culture experiment, changes were found in RPE-19 cells proliferation rate with a concentration of NaIO3 at 30mg/L or higher. In hydralazine experiments, 4 weeks after injection of NaIO3, ERG c-wave fell markedly in NaIO3 group to 31% of control group (P < 0.01). The ERG c-wave of hydra- lazine + NaIO3 group fell only to 50% of control group (P<0.05). This was a 61% reversal of the c-wave of NaIO3 treated group. · CONCLUSION: RPE degeneration induced by NaIO3 was both dose and time dependent. Around 30 to 40 mg/kg NaIO3 would be the optimal to be used as a non-exudative age-related macular degeneration rat model. Hydralazine may postpone the development of non-exudative age- related macular degeneration.

·AIM: Impairment of choroidal perfusion was found in AMD patients. We postulated that vasoactive agents,which can reduce choroidal blood flow resistance, might prevent the development of choroidal neovascularization (CNV). D-Timolol and L-Timolol are hypotensive agents used in cardiovascular and glaucoma therapy. Their effects on laser-induced experimental CNV rat model and human umbilical vein endothelial cells (HUVEC) were thus evaluated.·METHODS: Male Brown Norway rats were anesthetized to receive Nd:YAG laser to break the Bruch's membrane. D-Timolol and L-Timolol were given once daily through intraperitoneal injection after laser treatment for 4wk. Fluorescein angiography was performed on 2wk and 4wk. HUVEC were tested by proliferation assay and adhesion assay with D-Timolol and L-Timolol at different concentrations.· RESULTS: D-Timolol reduced the fluorescein leakage to 83% of the control group in laser-induced rat's CNV model at a dosage of 15mg/(kg·d). L-Timolol had no effect on CNV formation even at a higher dosage of 20mg/(kg·d). D-Timolol inhibited the endothelial cells proliferation significantly by 300mg/L. L-Timolol also significantly inhibited the cell proliferation at 1 000mg/L. But at a lower dose such as 300mg/L, no significant inhibitory effect was found. Both drugs showed no effect on cell adhesion function in cell culture experiments.· CONCLUSION: D-Timolol was found to prevent CNV development in laser-induced model in vivo and inhibit vascular endothelial cells proliferation in vitro. L-Timolol had no effect on cell proliferation at the same dose, and neither on rat CNV model. The results indicate these two isomers have different functions on rat's CNV prevention and on HUVEC cell proliferation.

AIM: To study the effects of Tetramethylpyrazine (TMP) on retinal pigment epithelium (RPE) degeneration, choroidal blood flow and oxidative stress of RPE cells.METHODS: The 35mg/kg NaIO3-induced RPE degeneration rat eyes was given 25μg 1% TMP eye drops 3 times a day for 7 days before NaIO3 injection, and then 2 to 4 weeks after NaIO3 injection. RPE function was measured with c-wave of electroretinogram (ERG). Colored microsphere technique was used for in vivo experiments to determine the choroidal blood flow in ocular hypertensive (40mmHg) rabbit eyes. Methylthiazoltetrazolium (MTT) assay was used to study in vitro effect of TMP on various oxidants induced injury in the hRPE (ARPE-19 (ATCC, Manassas, VA, USA)) . RESULTS: Two weeks after NaIO3 injection, the amplitude of ERG c-wave fell markedly in NaIO3 group to 36% of control group (P<0.01). No apparent difference was observed in TMP+NaIO3 group. Four weeks later, the NaIO3 group fell to 46% of control group (P<0.01), while the TMP+NaIO3 group fell to only 77% of control group (P<0.01). There was a 67% reversal of the ERG c-wave by TMP as compared to NaIO3 group (P<0.01). The choroidal blood flow was significantly increased at all time points (at 30, 60 and 120 minutes after TMP instillation) as compared with corresponding controls. TMP had no effect on hypoxia-(1%O2), t-BHP- and H2O2-induced damage in RPE cells. 10μg/mL TMP could reverse 1 and 3mmol/L NaN3-induced loss of viability of RPE by 18.5% (P<0.01) and 23% (P<0.01), respectively. 30μg/mL TMP could reverse 30 and 100mmol/L NaIO3 induced loss of viability of RPE by 18.1% (P<0.05) and 16.8% (P<0.01), respectively.CONCLUSION: TMP can significantly protect RPE from NaIO3 induced degeneration in vivo and oxidative stress in vitro and can increase choroidal blood flow markedly in vivo .

AIM: To investigate the mechanism of proliferation effect induced by (R,R)-XY-10 and (S,S)-XY-10 on retinal pigmented epithelial cells(ARPE-19).METHODS: Human retinal pigmented epithelial cells(ARPE-19) and human umbilical vein endothelial cells (HUVECs) were used to investigate the effect of (R,R)-XY-10 and (S,S)-XY-10 on cell growth,and their mechanisms of proliferative action by using ERK、 AKT、PI3K、Protein kinase C (PKC)and Nitric oxide synthase (NOS) inhibitors.RESULTS: (R,R)-XY-10 and (S,S)-XY-10 dose-dependently increased ARPE-19 cell proliferation,but not on HUVECs. When treated with proliferative inhibitors,H7(5μmol/L)、hypericin(20μmol/L)、PD98059(2μmol/L)、LY294002(50μmol/L)、SH-5 (10μmol/L) and L-NAME (100μmol/L),the proliferative effect was reduced by H7、hypericin、PD98059 and LY294002,but not by SH-5 and L-NAME.CONCLUSION: (R,R)-XY-10 and (S,S)-XY-10 can induce cell proliferation through MAPK and PI3K dependent pathway. KEYWORDS: age-related macular degeneration; (R,R)-XY-10; (S,S)-XY-10; ARPE-19 cells; human umbilical vein endothelial cells; proliferation