[Hyperoside protects mouse spermatocytes GC-2 cells from oxidative damage by activating the Keap1/Nrf2/HO-1 pathway]

Abstract

Objective: To study the protective effect of hyperoside (Hyp) against ydrogen peroxide (H₂O₂)- induced oxidative damage in mouse spermatocytes GC-2 cells and explore the role of the Keap1/Nrf2/HO-1 pathway in this protective mechanism.

Methods: GC-2 cells were treated with 2.5 mmol/L azaacetylcysteine (NAC), 50, 100, and 200 μmol/L hyperoside, or the culture medium for 48 h before exposure to H₂O₂ (150 μmol/L) for 2 h. CCK-8 assay was used to detect the changes in cell viability, and cell apoptosis was analyzed using flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) activity and malondialdehyde (MDA) in the culture medium. Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of nuclear factor erythroid 2-related factor2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1); the nuclear translocation of Nrf2 was detected using immunofluorescence assay.

Results: Exposure to H₂O₂ significantly lowered the proliferation rate, reduced the activities of SOD, GSH and CAT, and obviously increased MDA content, cell apoptosis rate, and the expressions of Keap1 and Nrf2 mRNA and Keap1 protein in GC-2 cells (P < 0.05 or 0.01). Treatment of the cells prior to H₂O₂ exposure with either NAC or 200 μmol/L hyperoside significantly increased the cell proliferation rate, enhanced the activities of SOD, GSH-PX and CAT, and lowered MDA content and cell apoptosis rate (P < 0.05). Treatment with 200 μmol/L hyperoside significantly decreased the mRNA and protein expressions of Keap1 and increased the expressions of HO-1 mRNA and the protein expressions of Nrf2 and HO-1 (P < 0.05 or 0.01). Hyperoside also caused obvious nuclear translocation of Nrf2 in the cells (P < 0.05).

Conclusion: Hyperoside protects GC-2 cells against H₂O₂- induced oxidative damage possibly by activation of the Keap1/Nrf2/HO-1 signaling pathway.

Keywords: GC-2 cells; HO-1; Keap1; Nrf2; hyperoside; oxidative stress.

1

不同浓度H₂O₂处理2、4、6、8 h对GC-2细胞活力的影响 Effect of different concentrations of H₂O₂ on viability of GC-2 cells at 2, 4, 6, and 8 h (Mean±SD, n=3). ^##P < 0.01 vs control group.

2

金丝桃苷对GC-2细胞活力的影响 Effect of hyperoside (Hyp) on viability of GC-2 cells. A: Effect of different concentrations and treatment time of hyperoside on viability of GC-2 cells. B: Effect of hyperoside on viability of GC-2 cells damaged by H₂O₂. Data are presented as Mean±SD (n=3). ^#P < 0.05, ^##P < 0.01 vs control group; ^*P < 0.05, ^**P < 0.01 vs model group.

3

金丝桃苷对H₂O₂损伤GC-2细胞凋亡率的影响 Effects of different concentrations of hyperoside (Hyp) on GC-2 cell apoptosis (Mean±SD, n=3). ^##P < 0.01 vs control group; ^**P < 0.01 vs model group.

4

金丝桃苷对H₂O₂损伤GC-2细胞GSH、CAT、SOD活力及MDA含量的影响 Effect of hyperoside (Hyp) on the activity of GSH, CAT, SOD and MDA content in GC-2 cells with H₂O₂ exposure (Mean±SD, n=3). ^#P < 0.05, ^##P < 0.01 vs control group; ^*P < 0.05, ^**P < 0.01 vs model group.

5

金丝桃苷对H₂O₂损伤GC-2细胞Keap1、Nrf2、HO-1 mRNA表达影响 Effect of hyperoside (Hyp) on expressions of Keap1, Nrf2, HO-1 mRNA in GC-2 cells with H₂O₂ exposure (Mean±SD, n=3). ^#P < 0.05, ^##P < 0.01 vs control group; ^*P < 0.05, ^**P < 0.01 vs model group.

6

金丝桃苷对H₂O₂诱导GC-2细胞Keap1、NEs-Nrf2和HO-1蛋白的影响 Effects of hyperoside (Hyp) on Keap1, NEs-Nrf2 and HO-1 protein expressions in H₂O₂-exposed GC-2 cells (Mean±SD, n=3). ^#P < 0.05, ^##P < 0.01 vs control group; ^*P < 0.05, ^**P < 0.01 vs model group.

7

金丝桃苷对H₂O₂诱导GC-2细胞Nrf2核转位影响 Effect of hyperoside (Hyp) on nuclear translocation of Nrf2 in GC-2 cells induced by H₂O₂ (Scale bar=100 μm). Green represents Nrf2, and blue represents DAPI. Data are presented as Mean±SD (n=3). ^#P < 0.05, ^##P < 0.01 vs control group; ^*P < 0.05, ^**P < 0.01 vs model group.