¡¡¡¡RESULTS

¡¡¡¡Cytotoxicity of Naringenin  Naringenin significantly improved the formation of formazan on ARPEª²19 cells in a concentrationª²dependent manner (Figure 1A). If ARPEª²19 cells were rinsed with PBS before adding MTT, 3 and 10mg/L naringenin still significantly improved the proliferation of ARPEª²19 cells by 10.8% and 11.4%, respectively (Figure 1B). However, naringenin significantly inhibited the proliferation of HUVEC. The reduction of the endothelial cells was 23.9%, 70.4% and 77.9% in the 3, 10 and 30mg/L naringeninª²treated groups, respectively (Figure 1C).

¡¡¡¡Effect of Naringenin on Hypoxiaª²induced Damage in Cells

¡¡¡¡Naringenin significantly increased the hypoxiaª²induced damage in RPE cells in a concentrationª²dependent manner. Ten mg/L naringenin increased the viabilily of ARPEª²19 cell by 55.2% in hypoxic condition (Figure 2A). However, if the cells were rinsed with PBS before adding MTT solution, the result showed that naringenin had no effect on the proliferation of ARPEª²19 cells in hypoxic condition (data not shown). Naringenin increased hypoxiaª²induced damage by 10.7% and 13.1% at the concentrations of 1 and 3mg/L naringeninª²treated groups as compared with vehicle control group in HUVEC (Figure 2B).

¡¡¡¡Figure 1Effect of naringenin on proliferation of cells inª²cubated for 72h  (aP<0.05,bP<0.01 vs vehicle control, ¡Às, n=6)£¨ÂÔ£©

¡¡¡¡Figure 2  Effect of naringenin on hypoxiaª²induced injury in cells for 72h  (aP<0.05,bP<0.01 vs vehicle control, ¡Às,n=6)£¨ÂÔ£©

¡¡¡¡Effect of Naringenin on NaN3ª²induced Injury in Cells  Naringenin significantly increased 0.3, 1 and 3mmol/L NaN3ª²induced injury in ARPEª²19 cells concentrationª²dependently. Ten mg/L naringenin increased the viability of 0.3mmol/L NaN3ª²injuried ARPEª²19 cells by 69.2%( Figure 3). Hoª²wever, if the cells were rinsed with PBS before adding MTT solution, the result showed that naringenin had no effect on the proliferation of NaN3ª²injured ARPEª²19 cells (data not shown). Naringenin had no effect on NaN3ª²induced injury in HUVEC either (data not presented).

¡¡¡¡Effect of Naringenin on tª²BHPª²induced Injury in Cells  Naringenin significantly improved the viability of 50 and 100mol/L tª²BHPª²treated ARPEª²19 cells concentrationª²dependently (Figure 4A). At the concentration of 1mg/L, naringenin increased the 50  mol/L tª²BHPª²induced damage by 20.2%. If the cells were rinsed with PBS before adding MTT solution, the result showed that naringenin still increased the proliferation of tª²BHPª²treated ARPEª²19 cells. At higher concentration of 30mg/L, naringenin increased the prolifeª²ration of ARPEª²19 cells by 32.2% (Figure 4B). Naringenin had no effect on tª²BHPª²induced injury in HUVEC (data not shown).

¡¡¡¡Effect of Naringenin on H2O2ª²induced Injury in Cells  Naringenin significantly increased the H2O2ª²induced damage in ARPEª²19  cells in a concentrationª²dependent manner. Ten mg/L naringenin increased the viability of H2O2ª²injured ARPEª²19 cells by 50.3% (Figure 5A). However, if the cells were rinsed with PBS before adding MTT solution, the result showed that naringenin had no effect on the proliferation of H2O2ª²treated ARPEª²19 cells (data not shown). Naringenin also increased H2O2ª²induced injury in HUVEC. At the coª²ncentration of 3mg/L, naringenin increased the viability of 400mol/L H2O2ª²injured HUVEC by 20.1% and 21.5% as compared with 200 and 400mol/L H2O2ª²treated groups, respectively (Figure 5B).

¡¡¡¡Effect of Naringenin on NaIO3ª²induced Injury in Cells  As shown in Figure 6A, naringenin increased the viability of 30, 100 and 300mg/L NaIO3ª²induced injuries in ARPEª²19 cells concentrationª²dependently. One mg/L naringenin increased the viability of 30, 100 and 300mg/L NaIO3ª²treated ARPEª²19 cells by 30.4%, 26.6%, and 20.7%, respectively. Naringenin also increased NaIO3ª²induced reducª²tion of ARPEª²19 cells in a concentrationª²dependent manner. One mg/L naringenin increased the proliferation of 30, 100 and 300mg/L NaIO3ª²treated ARPEª²19 cells by 30.3%, 10.3% and 18.5%, respectively (Figure 6B). Naringenin only increased 300mg/L NaIO3ª²induced injuries in HUVEC. One mg/L naringenin increased the viablility of HUVEC that injured by 300mg/L NaIO3 by 41.2% (Figure 6C).

¡¡¡¡DISCUSSION

¡¡¡¡The present data demonstrated that naringenin differed in its cytotoxicity and antioxidative capacity toward different oxidants and cell lines. Naringenin increased the viability of ARPEª²19 cells alone and hypoxiaª², NaN3ª², tª²BHPª², H2O2ª², and NaIO3ª²treated ARPEª²19 cells in a concentrationª²dependent manner. The results indicate that naringenin could prevent the oxidative damage of RPE in AMD patients. The mechanism of significant increase of formazan in ARPEª²19 cells requires further study.Naringenin improved the proliferation of ARPEª²19 cells, and tª²BHPª² and NaIO3ª²treated ARPEª²19 cells. These results indicate that naringenin could reverse the reduction of RPE in AMD patients.

¡¡¡¡Figure 3  Effect of naringenin on NaN3ª²induced injury in ARPEª²19 cells for 72h  (aP<0.05,bP<0.01 vs model control, cP<0.05,dP<0.01 vs vehicle control, ¡Às, n=6)£¨ÂÔ£©

¡¡¡¡Figure 4  Effect of naringenin on tª²BHPª²induced injury in ARPEª²19 cells for 12h  A: not rinsed with PBS before adding MTT; B: washed with PBS before adding MTT (aP<0.05, bP<0.01 vs model control, cP<0.05,dP<0.01 vs vehicle control, ¡Às, n=6)£¨ÂÔ£©

¡¡¡¡Figure 5  Effect of naringenin on H2O2ª²induced injury in cells for 24h  (aP<0.05, bP<0.01 vs model control, cP<0.05,dP<0.01 vs vehicle control, ¡Às, n=6)£¨ÂÔ£©

¡¡¡¡Figure 6  Effect of naringenin on NaIO3ª²induced injury in cells for 72h  (aP<0.05, bP<0.01 vs model control, cP<0.05,dP<0.01 vs vehicle control. ¡Às, n=6)£¨ÂÔ£©

¡¡¡¡Endothelial cells played an important role in the process of CNV development [14]. 3, 10, and 30mg/L naringenin inhibited the proliferation of HUVEC, which suggested that naringenin might inhibit the development of CNV in vivo. These results indicate that naringenin might be useful to prevent wet AMD. It is interesting to find out that 3mg/L naringenin increased hypoxiaª², H2O2ª², and NaIO3ª²induced damage in HUVEC. These results indicate that naringenin alone could inhibit the formation of CNV, and could also protect the endothelial cells of blood vessel from oxidative stress if the endothelial cells were attacked by some oxidants. The result of antioxidant effect of naringenin on H2O2ª²inª²duced damage in HUVEC was consistent with previous publication [15] which reported that naringenin was effective as an inhibitor of H2O2ª²induced oxidative stress in HUVEC and could protect cell viability with ¡Ý85% viable cells compared with the control without apparent nuclear condensation or DNA fragmentation.

¡¡¡¡In conclusion, the present study showed that naringenin could reverse hypoxiaª², NaN3ª², tª²BHPª², H2O2ª², and NaIO3ª²induced injury in ARPEª²19 cells, and improve the proliferation of ARPEª²19 cells. Furthermore, naringenin could inhibit the proliferation of HUVEC and increased hypoxiaª², H2O2ª², and NaIO3ª²induced damage at the same time. These results indicate that naringenin is not only useful to treat dry AMD but also the wet AMD. Thus, naringenin might become a promising candidate for treating AMD in the future.

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