Isolation of the hemeoxygenase-1 inducer from rice-derived peptide

Abstract

Bioactive peptides with various health benefits have been reported from rice protein hydrolysates. We previously showed that rice-derived peptides (RP) increased intracellular glutathione levels and induced the expression of γ-glutamylcysteine synthetase, which is regulated by nuclear transcription factor-erythroid 2-related factor 2 (Nrf2). Heme oxygenase-1 (HO-1) is an important Nrf2 downstream antioxidant enzyme that protects against oxidative stress. This study aimed to investigate the protective effects of RP on hydrogen peroxide (H₂O₂)-induced oxidative stress in human hepatoblastoma cell line HepG2 and identified HO-1 induced peptides from RP. Pretreatment of cells with RP reduced the cytotoxicity caused by H₂O₂ in a dose-dependent manner. Moreover, RP induced HO-1 expression in a concentration- and time-dependent manner. Next, we attempted to isolate the HO-1 inducer from RP by bioactivity-guided fractionation. Purification of the active peptides using a Sep-Pak C18 cartridge and reversed-phase HPLC, followed by sequence analysis by mass spectrometry, led to the identification of the three peptides. These peptides effectively reduced H₂O₂-induced oxidative stress. Among them, only P3 (peptide sequence: RSAVLLSH) increased HO-1 protein expression. Additionally, the knockdown of Nrf2 suppressed the induction of HO-1 expression by P3. Our results indicated that P3 identified from RP induced HO-1 by activating the Nrf2 signaling pathway.

Keywords: cytoprotection; heme oxygenase-1; nuclear transcription factor-erythroid 2-related factor 2; reverse-phase high-performance liquid chromatography; rice-derived peptides.

Fig. 1.

Protective effect of RP against cell damage caused by oxidative stress in HepG2 Cells. HepG2 cells were treated with RP (0, 2.5, 5, and 10 mg/ml) for 24 h and subsequently exposed to H₂O₂ (150 or 300 μM) for 24 h. The cytotoxicity was determined by measurement of LDH activity released from damaged cells into the medium. Values are the means ± SEM (n = 6). **p<0.01 vs 0 mg/ml.

Fig. 2.

Effects of RP treatment on HO-1 and Nrf2.tif expression levels. Protein extracts from HepG2 cells were analyzed by SDS-PAGE and immunoblotting using antibodies against the target proteins. (A) Effect of RP on HO-1 expression levels. The cells were treated with the indicated concentrations of RP for 24 h. Values are mean ± SEM (n = 6). *p<0.05 and **p<0.01 vs 0 mg/ml. (B) Time-dependent effects of RP on HO-1 and Nrf2 expression levels. The cells were treated with 5 mg/ml RP for the indicated times. Values are mean ± SEM (n = 3). *p<0.05 and **p<0.01 vs 0 h.

Fig. 3.

Effects of HO-1 protein expression by different fractions of RP. Protein extracts from HepG2 cells were analyzed by SDS-PAGE and immunoblotting using antibodies against the target proteins. (A) Fractionation of HO-1 inducers by the Sep-Pak cartridge. Cells were treated with fractions at a concentration of 2.5 mg/ml for 24 h. (B) HPLC chromatogram for the first purification of the active fraction separated using a TSKgel ODS-100V column. (C) Effects of HO-1 protein expression on the fraction of the first HPLC purification. Cells were treated with fractions at a concentration of 0.1 mg/ml for 24 h. (D) HPLC chromatogram for the second purification of the active fraction separated using a TSKgel ODS-100V column. (E) Effect of HO-1 protein expression on the fractions collected during the second HPLC purification. The cells were treated with each fraction for 24 h. Values are mean ± SEM (n = 3). *p<0.05 and **p<0.01 vs control.

Fig. 4.

Protective effect of peptides identified from RP against cell damage caused by oxidative stress in HepG2 Cells. Cells were treated with each peptides (6 μM) for 24 h and subsequently exposed to H₂O₂ (300 μM) for 24 h. The cytotoxicity was determined by measurement of LDH activity released from damaged cells into the medium. Values are mean ± SEM (n = 6). *p<0.05 and **p<0.01 vs control.

Fig. 5.

Effects of peptides identified from RP on HO-1 expression levels. Protein extracts from HepG2 cells were analyzed by SDS-PAGE and immunoblotting using antibodies against the target proteins. Cells were treated with peptides at a concentration of 6 μM for 24 h. Values are mean ± SEM (n = 3). *p<0.05 vs control.

Fig. 6.

Effects of Nrf2 knockdown on HO-1 expression levels. HepG2 cells were transfected with control or Nrf2.tif siRNA and incubated for 48 h. After further incubation in fresh medium with or without P3 (6 μM) for 24 h. Protein extracts from cells were analyzed by SDS-PAGE and immunoblotting using antibodies against the target proteins. Values are mean ± SEM (n = 3). *p<0.05 vs control siRNA without P3.