Resveratrol and 4-hydroxynonenal act in concert to increase glutamate cysteine ligase expression and glutathione in human bronchial epithelial cells

Abstract

Resveratrol has been shown to protect against oxidative stress through modulating antioxidant capacity. In this study, we investigated resveratrol-mediated induction of glutathione (GSH) and glutamate cysteine ligase (GCL), and the combined effect of resveratrol and 4-hydroxynonenal (HNE) on GSH synthesis in cultured HBE1 human bronchial epithelial cells. Resveratrol increased GSH and the mRNA contents of both the catalytic (GCLC) and modulatory subunit (GCLM) of GCL. Combined HNE and resveratrol treatment increased GSH content and GCL mRNAs to a greater extent than either compound did alone. Compared to individual agent, combining exposure to HNE and resveratrol also showed more protection against cell death caused by oxidative stress. These effects of combined exposure were additive rather than synergistic. In addition, Nrf2 silencing significantly decreased the combined effect of HNE and resveratrol on GCL induction. Our data suggest that resveratrol increases GSH and GCL gene expression and that there is an additive effect on GSH synthesis between resveratrol and HNE. The results also reveal that Nrf2-EpRE signaling was involved in the combined effects.

Fig. 1

Structure of resveratrol. Resveratrol (3,5,4′-trihydroxystilbene) is a polyphenolic phytoalexin existing as two geometric isomers: cis- and trans-form. Trans-isomer of resveratrol as shown in the figure is used in current study.

Fig. 2

Resveratrol increased GSH content. HBE1 cells were treated with 0, 0.5, 1, 2, 5, and 10 µM resveratrol for different time as indicated and the total cellular GSH was determined. N = 3, *P < 0.05 compared with control.

Fig. 3

Resveratrol increased GCL mRNAs at transcription level. (A and B) Resveratrol increased the mRNA contents of GCLC (A) and GCLM (B). HBE1 cells were treated with different concentrations of resveratrol for indicated time and the mRNA levels of GCLC and GCLM were determined using RT-real-time PCR assay. N = 3, * P < 0.05 compared with vehicle control. (C and D) The effect of resveratrol on the decay rate of the mRNAs of GCLC (C) and GCLM (D). HBE1 cells were pretreated with 100 ng/ml actinomycin D (ActD) for 1 h and then treated with vehicle control (ethanol) or 5 µM resveratrol for indicated time. The mRNA levels of GCLC and GCLM were determined. The value is shown as percentage of control or the experiment starting point (0 h), N = 3.

Fig. 4

Combined effects of HNE and resveratrol on increasing GSH synthesis. (A) Combined effect on the GSH content. Cells were sequentially exposed to HNE for 1 h and then to resveratrol for 24 h, the cellular GSH level was measured. (B) Combined effect on GCLC mRNA expression. Cells were pretreated with HNE for 1 h and then treated with/without resveratrol for 12 h. RNA was extracted and the GCLC mRNA was determined. N = 3, *P < 0.05 compared with vehicle control; #P < 0.05 compared with the same concentration of HNE; ^$P < 0.05 compared with the same concentration of resveratrol.

Fig. 5

Combined exposure of HNE and resverarol exhibits enhancing protective effect against cell death. Cells were sequentially treated with HNE for 1 h and resveratrol for 24 h, then cells were exposed to DMNQ for 2 h and finally the MTT assay was performed to determine cell survival. N = 3, *P < 0.05 compared with vehicle control; ^#P < 0.05 compared with the same concentration of HNE or resveratrol.

Fig. 6

Effect of Nrf2 silencing on GCL induction. Nrf2 silencing reduced resveratrol-mediated mRNA induction of GCLC (A) and GCLM (B), and the combined effect of HNE and resveratrol on GCLC mRNA induction (C). HBE1 cells were transfected with 50 nM of siRNA-Nrf2 or siRNA-control. Twenty-four hours after the transfection, cells were treated with 5 µM of resveratrol or sequentially with 2 µM of HNE and 2 µM of resveratrol as described above for 12 h. N = 3, *P < 0.05 compared with vehicle control of siRNA- control; ^#P < 0.05 compared with resveratrol treatment of siRNA-control.