Epigallocatechin gallate enhances human lens epithelial cell survival after UVB irradiation via the mitochondrial signaling pathway

Abstract

The aim of the present study was to explore the mechanism underlying the ultraviolet B (UVB) irradiation‑induced apoptosis of human lens epithelial cells (HLECs), and to investigate the protective effect of epigallocatechin gallate (EGCG) against the UVB‑induced apoptosis of HLECs. HLECs were exposed to different concentrations of EGCG plus UVB (30 mJ/cm²). Cell viability was determined using the MTT assay. Furthermore, mitochondrial membrane potential (Δψm) and apoptosis were assessed by flow cytometry with JC‑1 and Annexin V/PI staining, respectively. Moreover, the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH‑Px), as well as the levels of GSH, hydrogen peroxide (H₂O₂) and hydroxyl free radicals were determined using biochemical assay techniques. Reverse transcription‑quantitative PCR and western blotting were used to detect the mRNA and protein expression levels of Bcl‑2, Bax, cytochrome c, caspase‑9 and caspase‑3, respectively. The results revealed that UVB irradiation reduced the Δψm of HLECs and induced apoptosis. Notably, EGCG significantly attenuated the generation of H₂O₂ and hydroxyl free radicals caused by UVB irradiation in HLECs, and significantly increased CAT, SOD and GSH‑Px activities, however, the GSH levels were not significantly increased. EGCG also reduced UVB‑stimulated Bax, cytochrome c, caspase‑9 and caspase‑3 expression, and elevated Bcl‑2 expression, suggesting that EGCG may possess free radical‑scavenging properties, thus increasing cell viability. In conclusion, EGCG may be able to protect against UVB‑induced HLECs apoptosis through the mitochondria‑mediated apoptotic signaling pathway, indicating its potential application in clinical practice.

Keywords: apoptosis; epigallocatechin gallate; human lens epithelial cells; ultraviolet B.

Figure 1.

Schematic diagram of the molecular structure of catechins.

Figure 2.

Protective effect of EGCG against human lens epithelial cell damage induced by UVB irradiation. The cells were cultured in different concentrations of EGCG for 2 h before UVB irradiation (30 mJ/cm²), and the cell viability (%) was recorded at 24, 48 and 72 h. Cells that received neither irradiation nor EGCG treatment were used as the control group. Data are presented as the mean ± SD (n=3). At the same time point, *P<0.05 vs. the UVB irradiation group; at the same EGCG concentration, ^#P<0.05 vs. the 24 h time point. EGCG, epigallocatechin gallate; UVB, ultraviolet B.

Figure 3.

Protective effect of EGCG on the Δψm of HLECs. Δψm was measured by flow cytometry using JC-1 staining in the (A) control, (B) EGCG, (C) UVB and (D) EGCG + UVB groups. (E) Changes of Δψm relative to the control group. Data are presented as the mean ± SD (n=3). *P<0.05 vs. the UVB group; ^#P<0.05 vs. the control group. Δψm, mitochondrial membrane potential; EGCG, epigallocatechin gallate; LL, lower left; LR, lower right; UL, upper left; UR, upper right; UVB, ultraviolet B.

Figure 4.

Effect of EGCG on antioxidant enzyme activity and GSH concentration under UVB irradiation. (A) SOD activity, (B) CAT activity, (C) GSH-Px activity and (D) GSH concentration 24 h after UVB irradiation, with or without EGCG pretreatment. The results showed that EGCG significantly enhanced the activities of CAT, SOD and GSH-Px, but did not significantly increase the levels of GSH. Data are presented as the mean ± SD (n=3). *P<0.05 vs. the UVB group; ^#P<0.05 vs. the control group. CAT, catalase; EGCG, epigallocatechin gallate; GSH, glutathione; GSH-Px, GSH peroxidase; SOD, superoxide dismutase; UVB, ultraviolet B.

Figure 5.

Protective effect of EGCG on the hydrogen peroxide and hydroxyl free radical levels induced by UVB irradiation. Data are presented as the mean ± SD (n=3). *P<0.05 vs. the UVB group; ^#P<0.05 vs. the control group. EGCG, epigallocatechin gallate; UVB, ultraviolet B.

Figure 6.

Protective effect of EGCG against HLECs apoptosis under UVB irradiation. HLECs were treated with 50 µM EGCG for 2 h prior to UVB irradiation (30 mJ/cm²) and cultured for 16 h. Cell apoptosis was detected by flow cytometry using Annexin V/PI staining in the (A) control, (B) EGCG, (C) UVB and (D) EGCG + UVB groups. LL, viable non-stained cells; LR, early apoptotic Annexin V-FITC-stained cells; UR, late apoptotic/necrotic Annexin V-FITC and PI-stained cell; UL, dead PI-stained cells. (E) Proportion of early apoptotic and late apoptotic cells. Data are presented as the mean ± SD (n=3). *P<0.05 vs. the UVB group; ^#P<0.05 vs. the control group. EGCG, epigallocatechin gallate; LL, lower left; LR, lower right; UL, upper left; UR, upper right; UVB, ultraviolet B.

Figure 7.

Alterations in the mRNA expression levels of (A) Bcl-2, (B) Bax, (C) Cyt C, (D) Cas-9 and (E) Cas-3 following pretreatment with EGCG before UVB irradiation were assessed using RT-qPCR. EGCG inhibited the UVB irradiation-induced expression of Bax, Cyt C, Cas-9 and Cas-3, and promote Bcl-2 expression in HLECs. (F) RT-qPCR analysis of the Bcl-2/Bax ratio in HLECs. GAPDH was used as the internal control. *P<0.05 vs. the UVB group; ^#P<0.05 vs. the control group. Cas-3, caspase-3; Cas-9, caspase-9; Cyt C, cytochrome c; EGCG, epigallocatechin gallate; HLECs, human lens epithelial cells; RT-qPCR, reverse transcription-quantitative PCR; UVB, ultraviolet B.

Figure 8.

EGCG inhibits the UVB irradiation-induced expression of Cyt C, Cas-9 and Cas-3 and modulates the UVB irradiation-induced expression of Bcl-2 family proteins in human lens epithelial cells. (A) Protein expression levels of Bcl-2, Bax, Cyt C, Cas-9 and Cas-3 were measured by western blotting. (B) Histogram analysis of protein expression levels. Data are presented as the mean ± SD (n=3). *P<0.05 vs. the UVB group; ^#P<0.05 vs. the control group. Cas-3, caspase-3; Cas-9, caspase-9; Cyt C, cytochrome c; EGCG, epigallocatechin gallate; UVB, ultraviolet B.

Figure 9.

EGCG inhibits UVB-induced human lens epithelial cell apoptosis through the caspase-dependent pathway and caspase-independent pathway. Δψm, mitochondrial membrane potential; AIF, apoptosis-inducing factor; CAT, catalase; EGCG, epigallocatechin gallate; Endo G, endonuclease G; GSH, glutathione; GSH-Px, GSH peroxidase; SOD, superoxide dismutase; UVB, ultraviolet B.