Inhibitory effect of astaxanthin on oxidative stress damage in lens epithelial cells and its mechanism

Authors: Li Yong,  Liu Zhaochen,  Peng Huacong
DOI: 10.3760/cma.j.cn115989-20200624-00448
Published 2023-05-10
Cite as Chin J Exp Ophthalmol, 2023, 41(5): 428-435.

Abstract                              [Download PDF] [Read Full Text]

Objective

To investigate the regulatory effect of astaxanthin on oxidative stress injury induced by hydrogen peroxide (H2O2) in lens epithelial cells and its possible mechanism.

Methods

The HLEB-3 cells were cultured with different concentrations (0, 50, 100, 200, 500, 750 μmol/L) of H2O2.The cell inhibition rate was detected by the methyl thiazolyl tetrazolium (MTT) method, and the 50%inhibiting concentration (IC50) was calculated.HLEB-3 cells were cultured with different concentrations (0, 5, 10, 20, 50 μmol/L) of astaxanthin.The cell survival rate was detected by the MTT method.HLEB-3 cells were divided into four groups for 24-hour culture, namely normal control group cultured with complete medium, oxidative stress group cultured with 250 μmol/L H2O2, 10 μmol/L astaxanthin group cultured with 10 μmol/L astaxanthin and 250 μmol/L H2O2, and 20 μmol/L astaxanthin group cultured with 20 μmol/L astaxanthin and 250 μmol/L H2O2.The cell apoptosis rate was determined by flow cytometry.The nitric oxide (NO) concentration, superoxide dismutase (SOD) activity, glutathione (GSH) activity and malondialdehyde (MDA) content were detected by ELISA.The protein expressions of nuclear factor erythroid-2 related factor 2 (Nrf2) in nuclei, cytoplasmic Nrf2, heme oxygenase-1 (HO-1) and NAD (P) H, quinine oxidoreductase 1 (NQO1) were detected by Western bolt.The cells were divided into four groups, namely normal control-small interfering RNA (NC-siRNA) group, Nrf2-siRNA group, NC-siRNA+ astaxanthin group and Nrf2-siRNA+ astaxanthin group.The cells were transfected with NC-siRNA or Nrf2-siRNA accordingly.The cells were co-cultured for 24 hours with 0/10 μmol/L astaxanthin and 250 μmol/L H2O2 24 hours after transfection, respectively.The cell apoptosis rate was determined by flow cytometry.The NO concentration, SOD activity, GSH activity and MDA content were detected by ELISA.

Results

With the increase of H2O2 concentration, the inhibition rate of HLEB-3 cells increased.There were significant differences in the inhibition rate of HLEB-3 cells treated with different concentrations of H2O2 (F=12.358, P<0.05). The IC50 value of H2O2 on HLEB-3 cells was 264.20 μmol/L.The survival rates of HLEB-3 cells treated with 0, 5, 10, 20 and 50 μmol/L astaxanthin were (100.00±0.00)%, (102.20±1.34)%, (109.50±3.60)%, (115.40±4.13)%, (93.60±2.59)%, respectively.Then 10 μmol/L and 20 μmol/L were chosen as the experimental dose.The cell apoptosis rate of oxidative stress group was (38.50±2.38)%, which was higher than (9.20±0.24)% of normal control group, with a statistically significant difference (P<0.05). The cell apoptosis rate of 10 μmol/L astaxanthin group was (27.60±4.33)%, which was lower than (38.50±2.38)% of oxidative stress group, but higher than (14.90±1.23)% of 20 μmol/L astaxanthin group and (9.20±0.24)% of normal control group, showing statistically significant differences (all at P<0.05). The NO and MDA contents were higher and the SOD and GSH concentrations were lower in oxidative stress group than in normal control group, 10 μmol/L astaxanthin group and 20 μmol/L astaxanthin group, and the differences were statistically significant (all at P<0.05). The NO and MDA contents were higher and the SOD and GSH concentrations were lower in 10 μmol/L astaxanthin group than in normal control group and 20 μmol/L astaxanthin groups, and the differences were statistically significant (all at P<0.05). There were significant differences in the relative expression levels of nuclear Nrf2, cytoplasmic Nrf2, HO-1 and NQO1 proteins among normal control group, oxidative stress group, 10 μmol/L astaxanthin group and 20 μmol/L astaxanthin group (F=43.512, 20.381, 31.014, 23.435; all at P<0.001). The relative expression of nuclear Nrf2 protein gradually decreased, and the relative expression of nuclear Nrf2, HO-1 and NQO1 proteins increased gradually in normal control group, oxidative stress group, 10 μmol/L astaxanthin group and 20 μmol/L astaxanthin group, and there were significant differences when compared in pairs (all at P<0.05). The apoptosis rates of Nrf2-siRNA group and Nrf2-siRNA+ astaxanthin group were higher than those of NC-siRNA group and NC-siRNA+ astaxanthin group, and the differences were statistically significant (all at P<0.05). The cell apoptosis rate was higher in NC-siRNA group than in NC-siRNA+ astaxanthin group, showing a statistically significant difference (P<0.05). There was no significant difference in the apoptosis rate between Nrf2-siRNA+ astaxanthin group and Nrf2-siRNA group (P>0.05). The NO and MDA concentrations were higher and the SOD and GSH activities were lower in Nrf2-siRNA group than in the NC-siRNA group, with statistically significant differences (all at P<0.05). The NO and MDA concentrations were lower and the SOD and GSH activities were higher in NC-siRNA+ astaxanthin group than in NC-siRNA group and Nrf2-siRNA+ astaxanthin group, and the differences were statistically significant (all at P<0.05). There was no significant difference in NO and MDA concentrations or the SOD and GSH activities between Nrf2-siRNA+ astaxanthin group and Nrf2-siRNA group (all at P>0.05).

Conclusions

Astaxanthin enhances the resistance of lens epithelial cells to H2O2-induced oxidative stress damage, which may be achieved by activating the Nrf2-related signaling pathway.

Key words:

Lens epithelial cells; Astaxanthin; Oxidative stress; Nrf2 signaling pathway

Contributor Information

Li Yong

Xiaogan Aier Eye Hospital, Xiaogan 432000, China

Liu Zhaochen

Department of Ophthalmology, Xiaogan Central Hospital, Xiaogan 432000, China

Peng Huacong

Wuhan Aige Eye Hospital, Wuhan 430019, China

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