Biliverdin Reductase A Protects Lens Epithelial Cells against Oxidative Damage and Cellular Senescence in Age-Related Cataract

Abstract

Age-related cataract (ARC) is the common cause of blindness globally. Reactive oxygen species (ROS), one of the greatest contributors to aging process, leads to oxidative damage and senescence of lens epithelial cells (LECs), which are involved in the pathogenesis of ARC. Biliverdin reductase A (BVRA) has ROS-scavenging ability by converting biliverdin (BV) into bilirubin (BR). However, little is known about the protective effect of BVRA against ARC. In the present study, we measured the expression level of BVRA and BR generation in human samples. Then, the antioxidative property of BVRA was compared between the young and senescent LECs upon stress condition. In addition, we evaluated the effect of BVRA on attenuating H₂O₂-induced premature senescence in LECs. The results showed that the mRNA expression level of BVRA and BR concentration were decreased in both LECs and lens cortex of age-related nuclear cataract. Using the RNA interference technique, we found that BVRA defends LECs against oxidative stress via (i) restoring mitochondrial dysfunction in a BR-dependent manner, (ii) inducing heme oxygenase-1 (HO-1) expression directly, and (iii) promoting phosphorylation of ERK1/2 and nuclear delivery of nuclear factor erythroid 2-related factor 2 (Nrf2). Intriguingly, the antioxidative effect of BVRA was diminished along with the reduced BR concentration and repressed nuclear translocation of BVRA and Nrf2 in senescent LECs, which would be resulted from the decreased BVRA activity and impaired nucleocytoplasmic trafficking. Eventually, we confirmed that BVRA accelerates the G1 phase transition and prevents against H₂O₂-induced premature senescence in LECs. In summary, BVRA protects LECs against oxidative stress and cellular senescence in ARC by converting BV into BR, inducing HO-1 expression, and activating the ERK/Nrf2 pathway. This trial is registered with ChiCTR2000036059.

Figure 1

The relative mRNA expression of BVRA and BR concentration were decreased in LECs and lens cortex of ARC-N. The relative mRNA expression of BVRA (a) and BR concentration (b) in LECs and lens cortex among noncataract, ARC-C, ARC-N, and ARC-P were detected by qPCR and colorimetric method, respectively. n = 15 in each group. Data are shown as mean ± SEM, one-way ANOVA.

Figure 2

BVRA expression levels and BR production in young and senescent LECs under oxidative stress. The young and senescent LECs were exposed to 200 μM H₂O₂ for various time points. (a) The representative bands of BVRA protein were shown by western blot analysis and semiquantified with GAPDH as reference. (b) The relative mRNA expressions of BVRA were measured by qPCR. (c) The intracellular BR concentrations were assessed by colorimetric method. Data are shown as mean ± SEM, one-way ANOVA, n = 3. ^∗∗P < 0.01 and ^∗∗∗P < 0.001, compared with young LECs without H₂O₂ damage. ^ψP < 0.05, compared with senescent LECs without H₂O₂ damage. Student's t-test, ^#P < 0.05, compared with time-matched young LECs.

Figure 3

The effect of BVRA knockdown on intracellular ROS levels in young and senescent LECs upon stress conditions. (a) Young and senescent LECs were exposed to 200 μM H₂O₂ for 15 min, 30 min, 45 min, and 60 min. Then, cell extract was prepared and BVRA activity was evaluated by detecting the rate of BR converted from BV in an NADPH-dependent manner at pH 8.5. (b) After being transfected with BVRA siRNA or NC siRNA, the young and senescent LECs were exposed to 200 μM H₂O₂ for 24 h with or without 20 mM N-acetyl-L-cysteine (NAC). Intracellular ROS levels were detected by DCFH-DA staining. Data are shown as mean ± SEM, one-way ANOVA, n = 3. ^∗∗∗P < 0.001, compared with young LECs without H₂O₂ damage. ^ψP < 0.05, compared with senescent LECs without H₂O₂ damage. Student's t-test, ^#P < 0.05, compared with time-matched young LECs.

Figure 4

Failure of BVRA nuclear translocation led to hypoinduction of HO-1 in senescent LECs under oxidative stress. (a) After being transfected with BVRA siRNA or NC siRNA, the young and senescent LECs were exposed to 200 μM H₂O₂ for 1 h. The relative protein expressions of HO-1 were analyzed by western blotting. (b) The young and senescent LECs were exposed to 200 μM H₂O₂ for 4 h. Cells were immunostained with antibody against BVRA (red) and DAPI (blue). (c) The relative protein expressions of BVRA in the nucleus and cytoplasm were detected by western blot. Data are shown as mean ± SEM, n = 3, one-way ANOVA, ^∗∗P < 0.01 and ^∗∗∗P < 0.001, compared with young LECs. ^#P < 0.05, compared with senescent LECs. ^ψP < 0.05, compared with H₂O₂-treated young LECs. Bar 100 μm.

Figure 5

Failure of Nrf2 nuclear localization in senescent LECs. (a) The young and senescent LECs were transfected with BVRA siRNA or NC siRNA, followed by 200 μM H₂O₂ exposure for 1 h. The effect of BVRA knockdown on the relative expression of phosphorylated-ERK1/2 and Nrf2 was assessed by western blot. (b) The young and senescent LECs were exposed to 200 μM H₂O₂ for 4 h. The binding capacity of BVRA and ERK2 in LECs was determined by CoIP assay. Data are shown as mean ± SEM, n = 3, one-way ANOVA, ^∗∗P < 0.01 and ^∗∗∗P < 0.001, compared with young LECs.

Figure 6

The impaired nucleocytoplasmic trafficking in senescent LECs under oxidative stress. The young and senescent LECs were exposed to 200 μM H₂O₂ for 4 h. (a) The nucleocytoplasmic trafficking gene expressions were measured with qPCR. (b) NPCs (red arrows) in young and senescent LECs under oxidative stress were observed by a transmission electron microscope. Data are shown as mean ± SEM, n = 3, one-way ANOVA, ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001, compared with young LECs. ^#P < 0.05, compared with senescent LECs. ^ψP < 0.05, compared with H₂O₂-treated young LECs.

Figure 7

Mitochondrial dysfunction induced by BVRA depletion under oxidative stress was rescued by BR. LECs were transfected with BVRA siRNA. Then, cells were pretreated with 20 μM BR for 2 h before exposure to 200 μM H₂O₂ for 24 h. (a) The representative diagram of mitochondrial membrane potential determined by JC-1 staining. (b) Mitochondrial ROS levels were detected by MitoSOX probe. (c) The apoptotic rates of LECs were assessed by Annexin V-FITC assay. Data are shown as mean ± SEM. One-way ANOVA, ^∗∗P < 0.01 and ^∗∗∗P < 0.001, compared with the control group. ^#P < 0.05 compared with the BVRA siRNA-transfected cells exposed to H₂O₂.

Figure 8

BVRA protects LECs against oxidative stress-induced cellular senescence. (a) After overexpressing or knocking down BVRA, LECs were incubated with H₂O₂ for 96 h. The morphological changes of young LECs were observed at 48 h and 96 h by a microscope. The cell proliferation was determined by CCK-8 assay at 48 h and 96 h. (b) BVRA overexpressed or silenced LECs were incubated with 100 μM H₂O₂ for 7 days. The percentage of senescent cells was analyzed by SA-β-gal staining at day 7. (c) The effects of BVRA on the relative expression of cell cycle regulators (p21, p16^INK4α, cyclin D1, and CDK4) at day 4 were determined by western blot. (d) The effects of BVRA on cell cycle progression in different groups at day 4. Data are shown as mean ± SEM. One-way ANOVA, ^∗∗P < 0.01 and ^∗∗∗P < 0.001, compared with the control group. ^#P < 0.05 compared with the BVRA siRNA-transfected cells exposed to H₂O₂. ^ψP < 0.05 compared with the pcDNA3.1-BVRA-transfected cells exposed to H₂O₂. Bar 200 μm.

Figure 9

The effect of BVRA on preventing cellular senescence against oxidative stress in LECs. Cellular senescence, an important risk factor for ARC formation, is closely associated with redox imbalance in LECs. Cataractogenic stresses give rise to intracellular ROS accumulation, antioxidant depletion, and cell aging in LECs. To prevent cellular senescence against oxidative stress, the BVRA-mediated antioxidative defense system takes effect in three manners. First, BVRA protects LECs from mitochondrial dysfunction and eliminates ROS in a BR-dependent manner by converting BV into BR. Second, enhanced nuclear trafficking of BVRA directly promotes HO-1 expression upon oxidation. Third, BVRA activates ERK/Nrf2 signaling by promoting phosphorylation of ERK1/2 and Nrf2 nuclear localization. However, the antioxidative effect of BVRA was diminished in senescent LECs, which would be resulted from the decreased enzymatic activity of BVRA and the repressed nucleocytoplasmic trafficking.