Pterostilbene improves neurological dysfunction and neuroinflammation after ischaemic stroke via HDAC3/Nrf1-mediated microglial activation

Abstract

Background: Stroke is a type of acute brain damage that can lead to a series of serious public health challenges. Demonstrating the molecular mechanism of stroke-related neural cell degeneration could help identify a more efficient treatment for stroke patients. Further elucidation of factors that regulate microglia and nuclear factor (erythroid-derived 2)-like 1 (Nrf1) may lead to a promising strategy for treating neuroinflammation after ischaemic stroke. In this study, we investigated the possible role of pterostilbene (PTS) in Nrf1 regulation in cell and animal models of ischaemia stroke.

Methods: We administered PTS, ITSA1 (an HDAC activator) and RGFP966 (a selective HDAC3 inhibitor) in a mouse model of middle cerebral artery occlusion-reperfusion (MCAO/R) and a model of microglial oxygen‒glucose deprivation/reperfusion (OGD/R). The brain infarct size, neuroinflammation and microglial availability were also determined. Dual-luciferase reporter, Nrf1 protein stability and co-immunoprecipitation assays were conducted to analyse histone deacetylase 3 (HDAC3)/Nrf1-regulated Nrf1 in an OGD/R-induced microglial injury model.

Results: We found that PTS decreased HDAC3 expression and activity, increased Nrf1 acetylation in the cell nucleus and inhibited the interaction of Nrf1 with p65 and p65 accumulation, which reduced infarct volume and neuroinflammation (iNOS/Arg1, TNF-α and IL-1β levels) after ischaemic stroke. Furthermore, the CSF1R inhibitor PLX5622 induced elimination of microglia and attenuated the therapeutic effect of PTS following MCAO/R. In the OGD/R model, PTS relieved OGD/R-induced microglial injury and TNF-α and IL-1β release, which were dependent on Nrf1 acetylation through the upregulation of HDAC3/Nrf1 signalling in microglia. However, the K105R or/and K139R mutants of Nrf1 counteracted the impact of PTS in the OGD/R-induced microglial injury model, which indicates that PTS treatment might be a promising strategy for ischaemia stroke therapy.

Conclusion: The HDAC3/Nrf1 pathway regulates the stability and function of Nrf1 in microglial activation and neuroinflammation, which may depend on the acetylation of the lysine 105 and 139 residues in Nrf1. This mechanism was first identified as a potential regulatory mechanism of PTS-based neuroprotection in our research, which may provide new insight into further translational applications of natural products such as PTS.

Keywords: HDAC3; Ischaemic stroke; Neuroinflammation; Nrf1 acetylation; PTS.

Fig. 1

PTS improves motor behaviour and tissue infarction after I/R. PTS (1 mg/kg, 5 mg/kg or 10 mg/kg) was administered via intraperitoneal injection immediately after MCAO/R, and neurological tests were conducted twice before surgery and 1 day, 2 day and 3 day after MCAO/R, including assessment of the neurological deficit score (A) and performance in the hidden platform trial of the MWM (B). The data are presented as the means ± SEMs (n = 8). C Representative images of TTC staining at 24 h after MCAO/R and quantitative analysis of the hemispheric infarct ratio. Total HDAC activation (D) and the levels of the inflammatory factors TNF-α and IL-1β (E) in ischaemic brain tissue at 1 d after MCAO/R. The data are presented as the means ± standard error of the means (SEMs; n = 5). *p < 0.05, versus the sham group; #p < 0.05, versus the MCAO/R group. L, PTS 1 mg/kg; M, PTS 5 mg/kg; H, PTS 10 mg/kg

Fig. 2

HDAC3 regulates Nrf1 expression and iNOS activation after I/R. After MCAO/R mice were treated with the HDAC activator ITSA1 and the HDAC3 inhibitor RGFP966, we conducted qRT‒PCR analysis of HDAC1, HDAC2, HDAC3 and Nrf1 mRNA levels (A); WB analysis of HDAC1, HDAC2 and HDAC3 expression (B); WB analysis of Nrf1, iNOS and Arg1 expression (C); and ELISA analysis of iNOS activation (D) in total cell lysates of ischaemic brain tissue 24 h after MCAO/R. The data are presented as the means ± SEMs (n = 5). *p < 0.05, versus the sham group; #p < 0.05, versus the MCAO + vehicle group; &p < 0.05, versus the MCAO/R + PTS group

Fig. 3

PTS induces Nrf1 and reduces IbA1 after I/R. At 24 h after MCAO/R, IF analysis of IbA1 expression (A) and HDAC3 and Nrf1 expression (B) in the hippocampus and cortex. Nuclei were stained with DAPI, n = 5; scale bar: 20 μm

Fig. 4

Microglia elimination with the CSF1R antagonist PLX5622 attenuated the therapeutic effects of PTS following I/R in vivo. A Overview of the timeline of in vivo experiments. Before MCAO/R, the mice were fed a PLX5622 (PLX) AIN-76A diet or AIN-76A chow for 14 days. B IF analysis of Iba1 staining in the brain after MCAO/R; the cell nuclei are shown in blue (DAPI). Scale bar = 20 μm, n = 5. C Representative images of TTC staining at 24 h after MCAO/R and quantitative analysis of the hemispheric infarct ratio (n = 5). Neurological tests, including assessment of the neurological deficit score (D) and performance in the hidden platform trial of the MWM (E), were conducted twice before surgery and at 1, 2 and 3 days after MCAO/R (n = 8). The data are presented as the means ± SEMs (n = 5). p < 0.05, versus the sham group; *p < 0.05, versus the MCAO + vehicle group; #p < 0.05, versus the MCAO/R + PTS group

Fig. 5

HDAC3 inhibition improves motor behaviour and tissue infarction after I/R. After PTS, ITSA1 and/or RGFP966 were administered via intraperitoneal injection, MCAO/R mice underwent neurological tests twice before surgery and at 1, 2 and 3 days after MCAO/R; testing included assessment of the neurological deficit score (A) and performance in the hidden platform trial of the MWM (B). The data are presented as the means ± SEMs (n = 8). C Representative images of TTC staining at 24 h after MCAO/R and quantitative analysis of the hemispheric infarct ratio. HDAC activation (D) and the levels of the inflammatory factors TNF-α and IL-1β (E) in ischaemic brain tissue 1 day after MCAO/R. The data are presented as the means ± SEMs (n = 5). *p < 0.05, versus the sham group; #p < 0.05, versus the MCAO/R + vehicle group; &p < 0.05, versus the MCAO/R + ITSA1 group

Fig. 6

PTS improves Nrf1 acetylation and microglial activation by inhibiting HDAC3. A Nrf1 acetylation in the cell nucleus of ischaemic brain tissue was detected by a co-IP assay 24 h after MCAO/R. The data are presented as the means ± SEMs (n = 5). *p < 0.05, versus the sham group; #p < 0.05, versus the MCAO/R + vehicle group; p < 0.05, versus the MCAO/R + ITSA1 group. After OGD/R, microglia were immediately treated with PTS, ITSA1 + PTS, or vehicle for 24 h in vitro. Then, the acetylation of Nrf1 in the cell nuclei of microglia was detected by co-IP (B). Total HDAC activation (C), iNOS activation (D) and the levels of the inflammatory factors TNF-α and IL-1β were determined by ELISA (E), and WB analysis of iNOS and Arg1 expression (F) was performed using total cell lysates from OGD/R-induced microglia. The data are presented as the means ± SEMs (n = 3). *p < 0.05, versus the control group; #p < 0.05, versus the OGD/R + vehicle group; p < 0.05, versus the OGD/R + PTS group

Fig. 7

Nrf1 acetylation affects Nrf1 stability and microglial activation. A Lysine mutation design of Nrf1. B Luciferase activity was measured in microglia transfected with a murine p65 promoter–luciferase reporter constructed along with WT Nrf1 or Nrf1 mutants. HDAC3 was co-expressed as indicated. The p65 promoter reporter activity was normalized to Renilla luciferase activity and empty vector (NC) activity. Luciferase activity was measured and analysed. An empty vector was used as a control, and pairwise comparisons were made between the glutamate and arginine mutants for each amino acid site (n = 5 per group). C Protein stability in microglia transfected with the K105Q, K105R, K139Q and K139R mutants after treatment with 100 μg/ml CHX. Samples were obtained 0 and 90 min after CHX treatment (n = 3 per group). In microglia with OGD/R-induced injury, the influence of the Nrf1 K105R and/or K139R mutations on the improvement observed after PTS treatment were evaluated by WB analysis of iNOS and Arg1 (D) and ELISA analysis of iNOS activation (E). The data are presented as the means ± SEMs (n = 3). *p < 0.05, versus the control group; #p < 0.05, versus the OGD/R + vehicle group; p < 0.05, versus the OGD/R + PTS group

Fig. 8

Nrf1 acetylation mediates microglial injury and TNF-α and IL-1β release. After treatment with PTS or ITSA1, a CCK8 assay (A) and an LDH release assay (B) were used to assess microglial injury in the OGD/R-induced microglial injury model; *p < 0.05, versus the control group; #p < 0.05, versus the OGD/R + vehicle group; p < 0.05, versus the OGD/R + PTS group. After treatment with PTS or RGFP966, a CCK8 assay (C) and an LDH release assay were performed (D), and the levels of the inflammatory factors TNF-α and IL-1β (E) were detected in OGD/R-induced microglia with the Nrf1 K105R and/or K139R mutation. F Model of microglial inflammation induced by PTS-mediated HDACA/Nrf1 after ischaemic stroke. *p < 0.05, versus the OGD/R group; #p < 0.05, versus the PTS group. The data are presented as the means ± SEMs (n = 3)