A novel compound DBZ ameliorates neuroinflammation in LPS-stimulated microglia and ischemic stroke rats: Role of Akt(Ser473)/GSK3β(Ser9)-mediated Nrf2 activation

Abstract

Microglia-mediated neuroinflammation plays a crucial role in the pathophysiological process of multiple neurological disorders such as ischemic stroke, yet lacks effective therapeutic agents. Previously, we discovered one novel synthetic compound, tanshinol borneol ester (DBZ), possesses anti-inflammatory and anti-atherosclerotic activities, whereas little is known about its effects in CNS. Therefore, the present study aims to explore the effects and potential mechanism of DBZ on neuroinflammation and microglial function. Our studies revealed that DBZ significantly inhibited NF-κB activity, suppressed the production of pro-inflammatory mediators meanwhile promoted M2 mediators expression in LPS-stimulated BV2 cells and mouse primary microglia cells. DBZ also exhibited antioxidant activity by enhancing Nrf2 nuclear accumulation and transcriptional activity, increasing HO-1 and NQO1 expression, and inhibiting LPS-induced ROS generation in BV2 cells. Importantly, the anti-neuroinflammatory and antioxidant effects of DBZ above were reversed by Nrf2 knockdown. Additionally, DBZ ameliorated sickness behaviors of neuroinflammatory mice induced by systemic LPS administration, and significantly reduced infract volume, improved sensorimotor and cognitive function in rats subjected to transient middle cerebral artery occlusion (tMCAO); besides, DBZ restored microglia morphological alterations and shifted the M1/M2 polarization in both murine models. Mechanistically, DBZ-induced Nrf2 nuclear accumulation and antioxidant enzymes expression were accompanied by increased level of p-Akt(Ser473) (activation) and p-GSK3β(Ser9) (inactivation), and decreased nuclear level of Fyn both in vitro and in vivo. Pharmacologically inhibiting PI3K or activating GSK3β markedly increased nuclear density of Fyn in microglia cells, which blocked the promoting effect of DBZ on Nrf2 nuclear accumulation and its antioxidant and anti-neuroinflammatory activities. Collectively, these results indicated the effects of DBZ on microglia-mediated neuroinflammation were strongly associated with the nuclear accumulation and stabilization of Nrf2 via the Akt(Ser473)/GSK3β(Ser9)/Fyn pathway. With anti-neuroinflammatory and antioxidant properties, DBZ could be a promising new drug candidate for prevention and/or treatment of cerebral ischemia and other neuroinflammatory disorders.

Keywords: Antioxidant; Functional recovery; Microglia polarization; Neuroinflammation; Nrf2; Stroke.

Fig. 1

DBZ suppressed pro-inflammatory mediator production, NF-κB (p65) activation, and promoted anti-inflammatory M2 markers expression in LPS-stimulated BV2 microglia cells. BV2 microglia cells were pretreated with the indicated concentrations of DBZ for 1 h followed by stimulation with 1 μg·mL⁻¹ LPS for another 24 h (A-D, I), 12 h (E-G), 2 h (J-L), or 6 h (M), respectively. (A-D) The media were collected and the concentrations of IL-1β, IL-6, TNF-α and NO were determined using ELISA kits or Griess regent. (E) DBZ suppressed the iNOS mRNA expression and (F, G) promoted the mRNA expression of M2 microglial markers (CD206 and IL-10) in LPS-stimulated BV2 cells as determined by qRT-PCR. (H) BV2 cells were incubated with various concentrations of DBZ (0.5–10 μM) for 48 h to investigate the cytotoxicity. (I) Effect of DBZ on LPS-induced iNOS protein expression was determined by Western blotting assay and quantified by densitometry. (J, K) The effect of DBZ on LPS-induced nuclear translocation of NF-κB (p65) in BV2 cells was determined by Western blot analysis and immunofluorescent assay. The scale bar represents 50 μm. (L) A commercially available NF-κB ELISA kit was used to test the nuclear extracts and determine the NF-κB binding activity. (M) BV-2 cells were transfected with a plasmid containing NF-κB-luciferase reporter construct and pretreated with DBZ for 1 h before the addition of LPS. After 6 h of LPS treatment, cell lysates were assayed for luciferase activity measured as the fold induction by normalizing the transfection efficiency and dividing values of each experiment relative to the control. Results are expressed as means ± SD (n = 5 independent experiments for A-H; n = 3 independent experiments for I–K; n = 4 independent experiments for L and M). *p < 0.05 compared with untreated control group, ^#p < 0.05 compared with LPS group. Statistical analyses were performed by one-way ANOVA followed by Bonferroni's post hoc test or Kruskal-Wallis test with Dunn's post hoc test.

Fig. 2

DBZ inhibited ROS production, up-regulated antioxidant enzymes expression and activated Nrf2 signaling in BV2 microglia cells. (A-C) Effects of DBZ on LPS-induced cellular ROS production and antioxidant enzymes expression. BV2 microglia cells were pretreated with the indicated concentrations of DBZ for 1 h followed by stimulation with 1 μg·mL⁻¹ LPS for another 12 h, then the cells were (A, B) incubated with DCFH-DA for intracellular ROS measurement, or (C) lysed for Western blot and densitometry quantification analysis. The scale bar represents 100 μm. Results are expressed as means ± SD (n = 5 independent experiments for A and B; n = 3 independent experiments for C). *p < 0.05 compared with untreated control group, ^#p < 0.05 compared with LPS group. Statistical analyses were performed by one-way ANOVA followed by Bonferroni's post hoc test. (D-I) BV2 cells were treated with DBZ for 12 h (D), 6 h (E), or 2 h (F–I), respectively. The direct effects of DBZ on HO-1 and NQO1 expression were detected by (D) Western blotting and (E) qRT-PCR assays. (F, G) Effect of DBZ on Nrf2 nuclear accumulation was determined by immunofluorescent and Western blot assay. The scale bar represents 50 μm. (H) A commercially available Nrf2 ELISA kit was used to test the nuclear extracts and determine the Nrf2 binding activity. (I) Cells transiently transfected with ARE-luciferase or control vector were incubated for 2 h with the indicated concentrations of DBZ. Cell lysates were assayed for luciferase activity measured as the fold induction by normalizing the transfection efficiency and dividing values of each experiment relative to the control. Results are expressed as means ± SD (n = 3 independent experiments for D and E; n = 4 independent experiments for F–I). *p < 0.05 compared with untreated control group. Statistical analyses were performed by one-way ANOVA followed by Bonferroni's post hoc test.

Fig. 3

Nrf2 nuclear accumulation by DBZ was critical for its antioxidant and anti-inflammatory activity in LPS-stimulated BV2 microglia cells (A) BV2 microglia cells were transfected with Nrf2 siRNA or NC siRNA for 24 h, then treated with DBZ (10 μM) for 12 h. Protein levels were determined by Western blotting assay and quantified by densitometry. (B–H) Cells transfected with Nrf2 siRNA or NC siRNA for 24 h, were treated with DBZ (10 μM) for 1 h, followed by exposure to LPS (1 μg·mL⁻¹) for 12 h (B, G), 24 h (C–F) or 2 h (H). (B) Cells were incubated with DCFH-DA for intracellular ROS measurement. (C–F) The culture media were collected and the concentrations of IL-1β (C), IL-6 (D), TNF-α (E) and NO (F) were determined using ELISA kits or Griess regent. (G) The mRNA expression of M2 microglial marker (CD206) was determined by qRT-PCR. (H) A commercially available NF-κB ELISA was used to test nuclear extracts and determine the degree of NF-κB binding. Results are expressed as means ± SD (n = 5 independent experiments for A-G; n = 4 independent experiments for H). *p < 0.05 compared with the NC + DBZ group. Statistical analyses were performed by two-way ANOVA followed by Bonferroni's post hoc test. NC, negative control.

Fig. 4

The Akt(Ser473)/GSK3β(Ser9)/Fyn pathway was involved in DBZ-induced Nrf2 nuclear accumulation and stabilization, antioxidant enzyme expression and anti-neuroinflammatory activity in BV2 microglia cells. (A-C) BV2 cells were incubated with (A) indicated concentrations of DBZ for 12 h or with (B, C) 10 μM DBZ for indicated time periods (15 min, 60 min and 120 min) respectively. The direct effects of DBZ on protein levels of (A) total Nrf2, (B, C) p-Akt(Ser473), p-GSK3β(Ser9) and nuclear Fyn were detected by Western blotting assay and quantified by densitometry. Data are presented as mean ± SD (n = 4 independent experiments for A-C). *P < 0.05 vs. untreated control. Statistical analyses were performed by one-way ANOVA followed by Bonferroni's post hoc test or Kruskal–Wallis test with Dunn's post hoc test. (D-K) Effects of Akt inhibition or GSK3β activation on protein levels of p-Akt(Ser473), p-GSK3β(Ser9), antioxidant enzymes (HO-1, NQO1), and nuclear protein level of Fyn and Nrf2. To directly define the role of Akt/GSK3β/Fyn signaling pathway in Nrf2 activation by DBZ, BV2 cells were pre-treated with (D-G) a PI3k inhibitor (LY294002, 10 μM), or (H–K) a GSK3β activator [sodium nitroprusside (SNP), 2 mM] for 30 min before the addition of DBZ (10 μM). After incubation for another 60 min (D, F, H, J) or 2 h (E, I for nuclear Nrf2), or 12 h (E, I for HO-1 and NQO1), the levels of indicated proteins were detected by Western blotting assay and quantified by densitometry. Data are presented as mean ± SD (n = 3 independent experiments for D-K). *p < 0.05 compared with control group, ^#p < 0.05 compared with DBZ group. Statistical analyses were performed by one-way ANOVA followed by Bonferroni's post hoc test. (L, M) Cells pretreated with LY294002 (10 μM) or SNP (2 mM) for 30 min, were treated with DBZ (10 μM) for 60 min, followed by exposure to LPS (1 μg mL⁻¹) (L) for 12 h to examine ROS generation, or (M) for 24 h to examine NO production. Results are expressed as means ± SD (n = 5 independent experiments for L and M). *p < 0.05 compared with the LPS group, ^#p < 0.05 compared with DBZ + LPS group. Statistical analyses were performed by one-way ANOVA followed by Bonferroni's post hoc test.

Fig. 5

DBZ ameliorated depressive behaviors, regulated M1/M2 polarization and affected Akt(Ser473)/GSK3β(Ser9)/Fyn-Nrf2 signal axis in the cerebral cortex of LPS-treated mice. (A) Experimental design is illustrated schematically. (B-E) Effects of DBZ (5, 20 mg·kg⁻¹) on locomotor activity of mice administrated with LPS. The total distance, central time, and number of rearing were investigated by open field test. (F, G) DBZ reduced the increased number of Iba1⁺ cells and restored the microglia morphological alterations in neuroinflammatory mice induced by LPS. (F) The average number of Iba1⁺ cells in frontal cortex. (G) Representative images of labeling for Iba1 are shown. White arrows indicate the enlarged insert of a representative cell. The scale bar represents 100 μm. (H-M) DBZ inhibited LPS-induced pro-inflammatory M1 genes expression and increased anti-inflammatory M2 genes expression in mice cortex. The mRNA levels of IL-1β, IL-6, TNF-α, iNOS, CD206 and IL-10 were determined by qRT-PCR. (N, O) The protein levels of p-Akt, p-GSK3β, nuclear Fyn, nuclear Nrf2, HO-1 and NQO1 were determined by Western blotting assay and quantified by densitometry. Results are expressed as means ± SD (n = 12 mice per group for B-E; n = 5 mice per group for F and G; n = 4 mice per group for H-M; n = 3 mice per group for N and O). *p < 0.05 compared with the VEH + SAL group, ^#p < 0.05 compared with the VEH + LPS group. Statistical analyses were performed by one-way ANOVA followed by Bonferroni's post hoc test or Kruskal–Wallis test with Dunn's post hoc test. VEH: vehicle; SAL: saline.

Fig. 6

DBZ reduced infract volume and improved sensorimotor and cognitive function in rats subjected to tMCAO. (A) Experimental design is illustrated schematically. When administered at 1 h before, 4 h after tMCAO, and then injected daily for another seven days, (B, C) DBZ reduced cerebral infarct volume at 72 h after tMCAO as assessed by TTC staining and (D) improved the neurological deficit as evaluated by Longa's test; (E–I) DBZ treatment improved sensorimotor recovery as evaluated by (E) corner test, (F) cylinder test, (G) limb-placing test, (H) adhesive-removal test and (I) the grip test at 3 and 8 days after tMCAO. Results are expressed as means ± SD (n = 5 rats per group for B and C; n = 10–12 rats per group for D-I). *p < 0.05 compared with the VEH + Sham group, ^#p < 0.05 compared with the VEH + tMCAO group. Statistical analyses were performed by Kruskal–Wallis test with Dunn's post hoc test or two-way repeated measures ANOVA followed by Bonferroni's post hoc test. (J–M) Spatial learning and memory were assessed at 4–8 d after injury by Morris water maze test. (J) Representative traces indicating the sample paths of the rats from the maze latency trials (learning) and the swimming traces from probe trials (memory). (K) The latency until the rats located the submerged platform as tested on days 4–8 (defined as spatial learning). (L, M) Spatial memory was evaluated on day 8 by measuring (L) the time spent swimming in the target quadrant and (M) the number of crossing original platform after the platform was removed. (N) Effect of DBZ on cognitive functions in rats subjected to tMCAO was also assessed by radial-arm maze test on day 8 after tMCAO. Results are expressed as means ± SD (n = 10–12 rats per group for J-N). *p < 0.05 compared with the VEH + Sham group, ^#p < 0.05 compared with the VEH + tMCAO group. Statistical analyses were performed by two-way repeated measures ANOVA followed by Bonferroni's post hoc test or one-way ANOVA followed by Bonferroni's post hoc test. VEH: vehicle.

Fig. 7

DBZ attenuated microglia accumulation and cell apoptosis, regulated M1/M2 polarization, enhanced Nrf2 nuclear retention and antioxidant enzymes expression in the ischemic hemisphere via Akt(Ser473)/GSK3β(Ser9)/Fyn pathway. (A, B) Effects of DBZ treatment on microglia/macrophage recruitment and cell apoptosis. Representative images and quantitative data of (A) Iba1-positive microglia/macrophage number and (B) TUNEL-positive cell number in penumbra region of rats at 3 days after stroke. White arrows indicate the enlarged insert of a representative cell. The scale bar represents 200 μm. (C) The mRNA levels of IL-1β, IL-6, TNF-α, iNOS, CD206 and IL-10 were determined by qRT-PCR. (D, E) The protein levels of p-Akt, p-GSK3β, nuclear Fyn, nuclear Nrf2, HO-1 and NQO1 were determined by Western blotting assay and quantified by densitometry. Results are expressed as means ± SD (n = 5 rats per group for A and B; n = 3 rats per group for C; n = 3 rats per group for D and E). *p < 0.05 compared with the VEH + Sham group, ^#p < 0.05 compared with the VEH + tMCAO group. Statistical analyses were performed by one-way ANOVA followed by Bonferroni's post hoc test. VEH: vehicle; Ipsil: ipsilateral side; Contra: contralateral side.

Fig. 8

Scheme summarizing the proposed mechanisms for the anti-neuroinflammatory and neuroprotective effects of DBZ in LPS-stimulated microglia and ischemic stroke rats. The mechanisms involve the promotion of antioxidant enzymes expression, inhibition of ROS production and NF-κB activation, transition of M1/M2 polarization in microglia, and the role of Akt(Ser473)/GSK3β(Ser9)-mediated Nrf2 activation.