(±)-5-bromo-2-(5-fluoro-1-hydroxyamyl) Benzoate Protects Against Oxidative Stress Injury in PC12 Cells Exposed to H2O2 Through Activation of Nrf2 Pathway

Abstract

Background: Oxidative stress is associated with the pathogenesis of ischemic stroke (±)-5-bromo-2-(5-fluoro-1-hydroxyamyl) benzoate (BFB) is a novel compound modified by dl-3-n-butylphthalide (NBP). Here, we hypothesized that BFB may protect the PC12 cells against H₂O₂-induced oxidative stress injury through activation of the Nrf2 pathway. Methods: We measured the cell viability and levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) to determine the construction of the H₂O₂-induced models of oxidative stress in PC12 cells. Additionally, apoptotic cell death, mitochondrial membrane potential, and cellular morphology were examined to determine the effect of BFB on oxidative stress injury in H₂O₂-treated PC12 cells. The expression levels of Nrf2-related and autophagy-related genes and proteins were detected using real time quantative PCR (RT-qPCR), Western Blot, and immunofluorescence analyses. Results: Our study showed that BFB treatment reduced the elevated levels of MDA, LDH, and ROS, and decreased cell viability and GSH in H₂O₂-treated PC12 cells. We also observed the elevated expression of Nrf2 pathway-related factors and intranuclear transitions and found that Nrf2 inhibitors (ML385) could block the protective effect of BFB. The inhibitory effect of BFB on oxidative stress may be partially regulated by Nrf2 activation, and the initiation and induction of autophagy. Conclusion: BFB inhibited H₂O₂-induced oxidative stress injury in PC12 cells by activating the Nrf2 pathway, initiating and inducing autophagy, suggesting that BFB may be a promising therapeutic agent in treating neurological disorders like cerebral ischemia.

Keywords: BFB; H2O2; Nrf2; PC12 cell; autophagy.

FIGURE 1

BFB’s role in H₂O₂-induced PC12 cell viability (A) H₂O₂ at different doses (0, 200, 300, 400, 500, 600 μm) was added to treat PC12 cells for a 24-h period, then CCK-8 assays were conducted to assess cell viability (B) BFB (0.1, 1, 10, 50, 100 μm)’s cytotoxicity to PC12 cells was analyzed by CCK-8 assays for a 24-h period (C) Bright-field images revealed H₂O₂-induced cell loss and alterations in cell morphology, which were remarkably abolished by administering BFB (scale bar = 125 μm) (D) Concentration-response curve for H₂O₂-induced PC12 cell viability. 400 μm H₂O₂ and low-glucose DMEM were added to treat PC12 cells for a 24-h period, with pretreatment with BFB (0.1–50 μm) or NBP (15 μm) for 3 h respectively. Data were analyzed by mean ± SD from 3 separate assays. *p < 0.05, **p < 0.01, versus control, ^# p < 0.05, ^## p < 0.01, versus H₂O₂ group.

FIGURE 2

BFB alleviated OS characteristics in H₂O₂-exposed PC12 cells. H₂O₂ (400 μm) was added to treat PC12 cells for a 24-h period with elevating concentrations of BFB (1, 15, 30 μm) or NBP (15 μm) for a 3-h period, followed by measurement of oxidative stress-associated indicators (A,B) ROS generation analyzed by DCFH-DA through FCM (C) Fluorescence microscopy images of the protective effects of BFB against H₂O₂. Administration of 400 µm H₂O₂ significantly increased ROS accumulation relative to Control. BFB significantly decreased ROS compared with 400 µm H₂O₂ group, and the reduction in ROS expression increased with the BFB concentration. Cell GSH (D) LDH leakage (E) MDA (F) GSH contents were determined using commercially available kits. Data were analyzed by mean ± SD from 3 separate assays. *p < 0.05, **p < 0.01, versus control, ^# p < 0.05, ^## p < 0.01, versus H₂O₂ group. ^▲ p < 0.05, ^▲▲ p < 0.01, versus NBP group.

FIGURE 3

BFB inhibited apoptosis and abolished mitochondrial alterations of H₂O₂-induced PC12 cells (A,B) FCM on apoptotic rate using Annexin V/PI staining (C) WB assay regarding Bax, Bcl-2, capase-3, cleaved-capase three expression within PC12 cells exposed to 400 µm H₂O₂ and 1, 15, 30 µm BFB or NBP (15 μm). Bcl-2 protein expression was significantly upregulated and Bax, capase-3 and cleave-capase 3 decreased within BFB-treated cells relative to H₂O₂-treated cells (D,E) WB assay on Bax/Bcl-2 and cleaved-capase 3/capase-3 ratio. Ratio of H₂O₂ group increased compared with Control group, but BFB partially recovered this expression ratio from upregulation (F) Typical images displaying mitochondrial staining under diverse conditions by using fluorescence microscopy (scale bar = 100 µm) (G) Bar graph presenting fluorescence intensity, which indicated MMP, by using fluorescence microplate reader (H) Typical images showing ultrastructure of mitochondria through TEM (scale bar = 1.0 µm). Arrows stand for mitochondria. The data were represented by mean ± SD of three separate assays. *p < 0.05, **p < 0.01, versus control, ^# p < 0.05, ^## p < 0.01, versus H₂O₂ group. ^▲ p < 0.05, ^▲▲ p < 0.01, versus NBP group.

FIGURE 4

BFB’s role in cellular P62-Keap1-Nrf2 pathway-related factors expression level. H₂O₂ (400 μm) was added to treat PC12 cells with/without BFB (1, 5, 10 μm) or NBP (15 μm) for a 3-h period. RT-qPCR was conducted to analyze mRNA expression (A) p62 (B) keap1 (C) Nrf2 (D) HO-1 (E) CAT (F) SOD. Nrf2 suppression alleviated BFB’s inhibition against oxidative stress. 5 μm ML385 was added to pretreat PC12 cells for a 0.5-h period, followed by H₂O₂ (400 μm) stimulation with/without BFB (30 μm) or NBP (30 μm) (G)Nrf2 (H) HO-1(I) CAT (J) SOD. Data were represented by mean ± SD from 3 separate assays. *p < 0.05, **p < 0.01, versus Control, ^# p < 0.05, ^## p < 0.01, versus H₂O₂ group. ^▲ p < 0.05, ^▲▲ p < 0.01, versus NBP group. ^△ p < 0.05, ^△△ p < 0.01, versus ML385 group.

FIGURE 5

BFB’s role in cellular P62-Keap1-Nrf2 pathway-related factors protein expression level. 400 μM H₂O₂ treatment in PC12 cells with/without BFB (1, 5, 10 μm) or NBP (15 μm) for a 3-h period (A) p62, keap1, HO-1 and total Nrf2 protein levels were examined by WB (B) Nuclear and cytoplasmic Nrf2 protein fractions were analyzed through WB (C–H) Quantification of relative band intensities of p62, keap1, total Nrf2, HO-1, nuclear Nrf2 and cytoplasmic Nrf2, and β-actin or Histone H3 served as loading control. Nrf2 suppression mitigated BFB’s inhibition on oxidative stress. 5 μm ML385 was added to pretreat PC12 cells for a 0.5-h period, followed by stimulation using H₂O₂ (400 μm) with/without BFB (30 μm) or NBP (30 μm) (I) Total Nrf2 and HO-1 protein expression determined through WB assay (J) Nuclear and cytoplasmic Nrf2 protein determined by Western Blot (K–N) Quantification of relative band intensities of total Nrf2, HO-1, nuclear Nrf2 and cytoplasmic Nrf2, with β-actin or Histone H3 being the endogenous reference. The values were represented by mean ± SD from 3 separate assays. *p < 0.05, **p < 0.01, versus Control, ^# p < 0.05, ^## p < 0.01, versus H₂O₂ group. ^▲ p < 0.05, ^▲▲ p < 0.01, versus NBP group. ^△ p < 0.05, ^△△ p < 0.01, versus ML385 group.

FIGURE 6

BFB promoted Nrf2 nuclear translocation. Nrf2 protein localization was detected via immunofluorescence staining (A) Representative immunofluorescence images showing BFB promoted distribution of Nrf2 in nuclear fraction (B) Nrf2 inhibition blocked cellular Nrf2 distribution induced by BFB in nuclear fraction.

FIGURE 7

Nrf2 suppression mitigated BFB’s inhibition on oxidative stress. 5 μm ML385 pretreatment of PC12 cells for a 0.5-h period, followed by H₂O₂ (400 μm) stimulation with/without BFB (30 μm) or NBP (30 μm) (A,B) FCM on apoptotic rate using Annexin V/PI staining (C,D) FCM on ROS production using DCFH-DA (E) Typical images presenting mitochondrial staining under diverse conditions by using fluorescence microscopy (scale bar = 100 µm) (F) Bar graph presenting fluorescence intensity, which indicated MMP, by using fluorescence microplate reader. Data were represented by mean ± SD from 3 separate assays. *p < 0.05, **p < 0.01, versus Control, ^# p < 0.05, ^## p < 0.01, versus H₂O₂ group. ^▲ p < 0.05, ^▲▲ p < 0.01, versus NBP group. ^△ p < 0.05, ^△△ p < 0.01, versus ML385 group.

FIGURE 8

BFB inhibits oxidative stress by initiating and inducing autophagy of H₂O₂-mediated PC12 cells (A–C) Autophagy-associated gene levels (Atg 4, Atg 5, Rab 5) were tested respectively via RT-qPCR (D,E) The autophagy-associated proteins expression (LC3, Beclin-1) was measured via WB. Data were represented by mean ± SD of 3 separate assays. *p < 0.05, **p < 0.01, versus Control, ^# p < 0.05, ^## p < 0.01, versus H₂O₂ group. ^▲ p < 0.05, ^▲▲ p < 0.01, versus NBP group.