Figure 1.

(A) Curcumin protected hLECs against UVB exposure, optimized ROS expression and reduced apoptotic cell death. Increase in survival of curcumin-treated hLECs exposed to UVB. Cells were treated with 5 μM of curcumin or dimethyl sulfoxide (DMSO) (a control vehicle). After 12 h, cells were submitted to UVB (200 J/m²), and effects on cell growth and viability were determined after 24, 48 and 72 h by MTS assay. (B) Effect of curcumin on lowering ROS expression. Cells were treated with DMSO or 5 μM of curcumin and after 12 h were exposed to UVB (200 J/m²). ROS expression was measured at 24, 48 and 72 h by replacing the medium with Hank's medium containing 10 μM H2-DCF-DA at Ex485/Em530 nm. (C, left panel) hLECs 7 × 10⁵ were cultured and pretreated with DMSO (a) or curcumin (Cur; b) and then exposed to UVB (200 J/m²). After 48 h, cells were photomicrographed. Arrows indicate dead cells. (Right panel) Cells were trypsinized and the percentage of apoptotic cells was monitored by Annexin V-FITC staining, followed by fluorescence-activated cell sorter (FACS) analysis. A representative photomicrograph (left panel) and FACS analysis of Annexin V-FITC and PI staining were provided. Results are expressed as mean±S.D. for three replicate determinations for each treatment group, and were significant (P<0.001) compared with solvent (DMSO) control indicated by an asterisk (^*). (D) Curcumin enhanced cell viability and blunted ROS expression and apoptotic cell death. hLECs 2 × 10⁴ were cultured in 48-well plate and treated with curcumin (5 μM) or DMSO (control vehicle). Cells were subjected to H₂O₂ (200 μM). Cell viability was tested with MTS assay at 24, 48 and 72 h. (E) Curcumin attenuation of ROS induction by H₂O₂. hLECs 1 × 10⁴ were cultured in 96-well plate, treated with curcumin (5 μM) and exposed to H₂O₂ (200 μM).⁷ ROS expression was quantified with H2-DCF-DA fluorescence dye at Ex485/Em530 nm with plate reader; results are presented as a histogram. (F) Curcumin negatively regulated the vulnerability of hLECs to H₂O₂-evoked apoptotic cell death. DMSO- or curcumin-treated hLECs were cultured and exposed to H₂O₂ (200 μM). After 48 h, cells were trypsinized and the percentage of apoptotic cells was monitored by Annexin V binding assay, followed by FACS analysis. (Left panel) Representative photomicrograph of H₂O₂-induced apoptosis in untreated (a) and curcumin-treated (b) cells. Arrow denotes dead cell. (Right panel) Representative FACS analysis of Annexin V-FITC and PI staining with control vehicle DMSO (c) and curcumin (d). Experiments were performed in triplicate and repeated at least three times, and the results are expressed as means±S.D. (^*P<0.001). (G) Curcumin potentiated hLECs viability by blocking ROS generation and progression of apoptosis against paraquat-induced insults. Curcumin- or DMSO-treated hLECs cultured in 48-well plates containing medium were subjected to paraquat (1 mM), an oxidative stressor. Cell viability assay (MTS assay) was performed at 24, 48 and 72 h. The histogram is representative of three experiments. (H) Curcumin limited paraquat-induced ROS expression in LECs. hLECs cultured in 96-well plate were treated with DMSO (vehicle) or curcumin (5 μM), and submitted to H2-DCF-DA dye, ROS expression assay (24, 48 and 72 h) following paraquat addition as described earlier. The histogram is representative of fluorescence, which is directly proportional to intracellular ROS levels. (I) Curcumin protected hLECs from apoptotic cell death caused by paraquat-induced oxidative damage. Photomicrograph represents hLECs treated with DMSO (a) and curcumin (b) following paraquat addition. Arrow indicates white rounded dead cell (left panel). (Right panel) Representative FACS analysis of Annexin V-FITC and PI staining. hLECs were cultured and pretreated with curcumin (Cur) for 12 h and then exposed to paraquat (1 mM). After 48 h, cells were trypsinized and the percentage of apoptotic cells was monitored by Annexin V binding assay, followed by FACS analysis. Data were derived from three experiments and expressed as means±S.D. ^*P<0.001. (J) or (K) or (L) Curcumin delivery to cultured hLECs attenuated UVB-, H₂O₂- or paraquat-induced LPO process in hLECs. Cells were either treated with DMSO (a control vehicle) or curcumin (5 μM), or then exposed to stressors, UVB (J) or H₂O₂ (K) or paraquat (L) to generate oxidative stress. After 48 h, cells were assessed for levels of LPO using LPO assay as ascribed in commercial kit. Histogram values are mean±S.D. of three independent experiments. Asterisks indicate statistically significant difference (P<0.001 versus control). The results were derived from three experiments