Fig 1.
Lutein rescued rMC-1 cells from CoCl2-induced hypoxic injury.
rMC-1 cells were exposed to CoCl2 (300μM) with or without lutein for various periods. Representative photographs of rMC-1 cells (A-C) normal control, (D-F) hypoxia with vehicle (0.01% DMSO), (G-I) hypoxia with lutein (20μM). (J) Percentage of cell viability. Treatment of lutein only without hypoxia did not affect the viability when compared with the normal control. Lutein-treated rMC-1 cells showed higher cell viability when compared with the vehicle-treated group at 24 hours. (K) Percentage of lactate dehydrogenase (LDH) release from damaged cells. Lutein attenuated LDH release after CoCl2-induced injury when compared with that in vehicle-treated group at 24 hours. n = 5 in each group. **P< 0.01, ***P<0.001 versus normal control group; #P< 0.05, ##P<0.01 versus vehicle-treated group. Scale bar, 100 μm.
Fig 2.
Number of apoptotic nuclei was attenuated in lutein-treated cells.
Apoptotic nuclei were revealed by TUNEL assay after CoCl2 treatment at different time points. (A) Representative images of TUNEL-positive nuclei (green) and DAPI-stained nuclei (blue) in rMC-1 cells. (B) Quantification of TUNEL-positive cells. n = 5 in each group. Lutein administration significantly reduced the number of TUNEL-stained nuclei. Scale bar, 100 μm. *P<0.05, **P< 0.01, ***P<0.001 versus normal control group; #P< 0.05 versus vehicle-treated group.
Fig 3.
Anti-apoptotic effects of lutein in rMC-1 cells upon CoCl2-induced hypoxia.
rMC-1 cells were exposed to CoCl2 (300μM) with various concentration of lutein for 24 hours. (A-D) Protein levels of different apoptotic-related proteins including Bcl-2, Bcl-XL, Bax, and cleaved caspase 3 were measured by Western blotting (normalized by β-actin) and quantified by densitometry. 20μM of Lutein was able to up-regulate protein expression of Bcl-2 and protein level of cleaved caspase 3 was inhibited by both 10μM and 20μM of lutein. (E) Densitometry analysis of ratio of Bax and Bcl-2 protein expression with different concentration of lutein. Lutein improved rMC-1 cell survival by decreasing Bax/Bcl-2 ratio. n = 5 in each group. *P<0.05, ***P<0.001 versus normal control group; #P< 0.05, ##P<0.01 versus vehicle-treated group. N, Normal Control; Veh, Vehicle (0.01% DMSO).
Fig 4.
The length of lutein treatment in rMC-1 cells upon CoCl2-induced hypoxia.
rMC-1 cells were exposed to CoCl2 (300μM) with or without lutein (20μM) for different periods of time. (A-D) Protein levels of different apoptotic-related proteins including Bcl-2, Bcl-XL, Bax, and cleaved caspase 3 were measured by Western blotting (normalized by β-actin) and quantified by densitometry. Lutein treatment was able to rescue cells upon CoCl2-induced cell death by up-regulating protein expression of Bcl-2 and suppressing cleaved caspase 3 at 24 hours. (E) Densitometry analysis of ratio of Bax and Bcl-2 protein expression at different time points. Lutein improved rMC-1 cell survival by decreasing Bax/Bcl-2 ratio at 24 hours. n = 5 in each group. *P<0.05, **P< 0.01, ***P<0.001 versus normal control group; #P< 0.05, ###P<0.001 versus vehicle-treated group.
Fig 5.
Lutein protected rMC-1 cells from CoCl2-induced autophagy.
Western blot analysis of expression of an autophagic marker, LC3II, along with the densitometric quantification (normalized by β-actin). (A) rMC-1 cells were exposed to CoCl2 (300μM) together with various concentration of lutein for 24 hours. LC3II expression was up-regulated upon CoCl2-induced hypoxia and attenuated by 20μM of lutein. (B) Expression of LC3II was up-regulated 2 hours after CoCl2 treatment and continued to increase in the remaining time points. Lutein was able to attenuate LC3II expression at 24 hours after hypoxic challenge. (C) Autophagosome formation was monitored in rMC-1 cells 24 hours after CoCl2 treatment. Both normal control and treated cells were stained with Cyto-ID® green dye and Hoechst 33342. Representative fluorescence microscopy images exhibited a decreased number of green fluorescence-labeled autophagosomes in lutein-treated cells when compared with that in vehicle-treated cells. Rapamycin and chloroquine co-treatment in rMC-1 cells were used as the positive control to identify the presence of autophagosome in the cells. (D) Quantification of Cyto-ID® green positive cells. Data was presented as the percentage of Cyto-ID® green positive cells over the total number of cells counted. n = 4 in each group. **P< 0.01, ***P<0.001 versus normal control group; #P<0.05, ##P<0.01 versus vehicle-treated group. Scale bar, 50 μm.
Fig 6.
Lutein protected rMC-1 cells from CoCl2 –induced autophagy through reduction of autophagic flux.
rMC-1 cells were treated with CoCl2 in the presence or absence of an autophagic flux inhibitor, ammonium chloride (NH4Cl), for 24 hours. (A) Western blotting and the densitometric quantification revealed that LC3II expression was activated after CoCl2 treatment in the presence of NH4Cl (lane 4) while lutein was able to attenuate this LC3II accumulation (lane 5). (B) Representative fluorescence microscopy images showed an increased number of LC3 punctate cells in CoCl2 and NH4Cl co-treated cells. Administration of lutein alleviated the puncate cells upon CoCl2 treatment. (C) Quantification of number of LC3 puncate cells. Data was presented as the percentage of LC3 puncate cells over the total number of cells counted. n = 5 in each group. *P< 0.05, ***P<0.001 versus normal control group; ##P< 0.01 versus NH4Cl and CoCl2 co-treatment group; #P< 0.05, versus vehicle-treated group. Scale bar, 50 μm.
Fig 7.
Anti-autophagic property of lutein was involved in mTOR-mediated autophagy pathway.
(A) Western blotting and the densitometric analysis showed that the phosphorylated AMPK was up-regulated in CoCl2-treated cells. (B-D) Protein levels of mTOR-associated proteins including P-mTOR, P-p70S6K and P-ULK1 (Ser757) were measured by Western blotting (normalized by β-actin) and quantified by densitometry. 20μM of lutein was able to restore the phosphorylation levels of P-mTOR and P-p70S6K upon CoCl2-induced hypoxia. (E) Rapamycin was used to induce autophagy and chloroquine was also added to block the formation of autolysosome upon rapamycin-mediated autophagy. Densitometry analysis showed that LC3II protein expression was up-regulated in rapamycin-induced autophagy and accumulated in the presence of chloroquine (lane 6). Lutein treatment was able to decrease LC3II expression in rMC-1 cells upon rapamycin and chloroquine co-treatment (lane 7). n = 5 in each group. *P< 0.05, **P<0.01, ***P<0.001 versus normal control group; #P< 0.05, ##P<0.01 versus Lutein-treated group; #P< 0.05, rapamycin and chloroquine co-treatment group.