Tea polyphenols alleviate hydrogen peroxide-induced oxidative stress damage through the Mst/Nrf2 axis and the Keap1/Nrf2/HO-1 pathway in murine RAW264.7 cells

Abstract

Tea polyphenols (TPs) are the major bioactive extract from green tea that have been extensively reported to prevent and treat oxidative stress damage. In previous studies, TPs have been demonstrated to protect cells against oxidative injury induced by hydrogen peroxide (H₂O₂). However, the underlying mechanism remains unclear. The aim of the current study was to investigate whether the protective and regulatory effects of TPs on oxidative stress damage were dependent on the mammalian STE20-like protein kinase (Mst)/nuclear factor (erythroid-derived 2)-like 2 (Nrf2) axis and the Kelch-like ECH-associated protein 1 (Keap1)/Nrf2/heme oxygenase 1 (HO-1) pathway in RAW264.7 cells, a murine macrophage cell line. Maintaining a certain range of intracellular reactive oxygen species (ROS) levels is critical to basic cellular activities, while excessive ROS generation can override the antioxidant capacity of the cell and result in oxidative stress damage. The inhibition of ROS generation offers an effective target for preventing oxidative damage. The results of the present study revealed that pretreatment with TPs inhibited the production of intracellular ROS and protected RAW264.7 cells from H₂O₂-induced oxidative damage. TPs was also demonstrated to attenuate the production of nitric oxide and malondialdehyde and increase the levels of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase). In addition, following TPs treatment, alterations in Mst1/2 at the mRNA and protein level inhibited the production of ROS and promoted the self-regulation of antioxidation. TPs-induced Keap1 gene downregulation also increased the expression of Nrf2 and HO-1. Collectively, the results of the present study demonstrated that TPs provided protection against H₂O₂-induced oxidative injury in RAW264.7 cells.

Keywords: Kelch-like ECH-associated protein 1/nuclear factor (erythroid-derived 2)-like 2/heme oxygenase 1 axis; hydrogen peroxide; mammalian STE20-like protein kinase/nuclear factor (erythroid-derived 2)-like 2 axis; oxidative stress damage; reactive oxygen species; tea polyphenols.

Figure 1

Effect of H₂O₂ and TPs on cell viability of RAW264.7 cells by CCK-8. (A) Chemical structures of major catechins in TPs. (B) RAW264.7 cells were treated with various concentrations of H₂O₂ (0-800 µM) for 12 h and (C) with 400 µM H₂O₂ in different times and cell viability was analyzed by CCK-8 assay. (D) RAW264.7 cells treated with TPs (0.1, 0.5, 1.0, 5.0 and 10.0 µg/ml) for 24 h. Data are the means ± SD from three independent experiments. ^*P<0.05, ^**P<0.01 vs. the control group. TPs, tea polyphenols.

Figure 2

Protective effects of TPs on H₂O₂-induced RAW264.7 cells injury. (A) Cell viability and (B) representative images (scale bars = 500 µm; Magnification, x10) of H₂O₂-induced RAW264.7 cells pretreated with different concentrations of TPs (0.1, 0.5 and 1.0 µg/ml) for 12 h. The results are expressed as the means ± SD of three independent experiments. ^**P<0.01 vs. Control, ^##P<0.01 vs. the H₂O₂ group. TPs, tea polyphenols.

Figure 3

TPs inhibited intracellular ROS generations of RAW264.7 cells under oxidative stress. (A) Flow cytometry of RAW264.7 cells stained for 30 min with ROS dye DCFH2-DA. RAW264.7 cells were incubated with or not for TPs (0.1, 0.5 and 1.0 µg/ml) for 12 h followed by 400 µM H₂O₂ for another 12 h. (B) Statistical analysis of the ROS per group. ^**P<0.01 vs. the control group, ^##P<0.01 vs. the H₂O₂ group. TPs, tea polyphenols.

Figure 4

Evaluation of NO level and activities of the antioxidant enzymes. The effects of TPs on the cellular concentrations of (A) NO and (B) MDA. The effects of TPs on the activities of (C) SOD, (D) CAT and (E) GSH-Px. The results are expressed as the mean ± SD of three independent experiments. ^*P<0.05 and ^**P<0.01 vs. the control group, ^#P<0.05 and ^##P<0.01 vs. the H₂O₂ group. NO, nitric oxide; TPs, tea polyphenols; SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase.

Figure 5

mRNA level changes after RAW264.7 cells were treated with TPs against oxidative damage. The (A) Mst1, (B) Mst2, (C) Keap1, (D) Nrf2 and (E) HO-1 mRNA expression levels were estimated using reverse transcription-quantitative PCR. Each bar represents the mean ± SD of three independent experiments. ^*P<0.05 or ^**P<0.01 vs. the control group, ^#P<0.05 or ^##P<0.01 vs. the H₂O₂ group. TPs, tea polyphenols; Mst, mammalian STE20-like protein kinase; Keap1, Kelch-like ECH-associated protein 1; Nrf2, nuclear factor (erythroid-derived 2)-like 2; HO-1, heme oxygenase 1.

Figure 6

Effects of TPs on the Mst-Nrf2 axis and Keap1/Nrf2/HO-1 pathway protein expression on RAW264.7 cells under oxidative damage. (A) The protein expression levels were tested using western blotting. (B) Keap1, (C) Mst2, (D) Mst1, (E) Nrf2 and (F) HO-1 proteins levels were quantified via densitometry and were normalized to β-actin. Each bar represents the mean ± SD of three independent experiments. ^*P<0.05 or ^**P<0.01 vs. the control group, ^##P<0.01 vs. the H₂O₂ group. TPs, tea polyphenols; Mst, mammalian STE20-like protein kinase; Nrf2, nuclear factor (erythroid-derived 2)-like 2; Keap1, Kelch-like ECH-associated protein 1; HO-1, heme oxygenase 1.

Figure 7

Molecular mechanism of TPs protection against H₂O₂-induced RAW264.7 cell injury. The excessive production of ROS in H₂O₂-injured RAW264.7 cells is alleviated and the balance of the oxidoreductase system is maintained by TPs. The expression of Nrf2 is directly affected by the change of intracellular ROS levels, following the Mst/Nrf2 axis maintenance of cellular redox balance and via Nrf2 binding to the ARE in the nucleus and thereby activating HO-1 protein expression. Induction via the Mst/Nrf2 axis and the Keap1/Nrf2/HO-1 pathway appears to represent the antioxidant mechanism of TPs. TPs, tea polyphenols; ROS, reactive oxygen species; Nrf2, nuclear factor (erythroid-derived 2)-like 2; Mst, mammalian STE20-like protein kinase; HO-1, heme oxygenase 1; Keap1, Kelch-like ECH-associated protein 1; ARE, antioxidant response element; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; CAT, catalase; NO, nitric oxide; MDA, malondialdehyde; Maf, musculoaponeurotic fibrosarcoma; ARE, antioxidant response element.