Soluble epoxide hydrolase inhibition enhances anti-inflammatory and antioxidative processes, modulates microglia polarization, and promotes recovery after ischemic stroke

Abstract

Background: Ischemic stroke triggers inflammatory responses and oxidative stress in the brain, and microglia polarization affects the degree of neuroinflammation. It has been reported that the inhibition of soluble epoxide hydrolase (sEH) activity protects brain tissue. However, the anti-inflammatory and antioxidative effects of sEH inhibition in the ischemic brain are not fully understood. This study aimed to investigate the effects of a selective sEH inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), after ischemic stroke.

Methods: Adult male rats with middle cerebral artery occlusion (MCAO) were administered with AUDA or a vehicle. Behavioral outcome, infarct volume, microglia polarization, and gene expression were assessed.

Results: Rats treated with AUDA showed better behavioral outcomes and smaller infarct volumes after MCAO. After AUDA treatment, a reduction of M1 microglia and an increase of M2 microglia occurred at the ischemic cortex of rats. Additionally, there was an increase in the mRNA expressions of antioxidant enzymes and anti-inflammatory interleukin-10, and pro-inflammatory mediators were decreased after AUDA administration. Heme oxygenase-1 was mainly expressed by neurons, and AUDA was found to improve the survival of neurons.

Conclusion: The results of this study provided novel and significant insights into how AUDA can improve outcomes and modulate inflammation and oxidative stress after ischemic stroke.

Keywords: anti-inflammation; antioxidant; ischemic stroke; microglia polarization; middle cerebral artery occlusion; soluble epoxide hydrolase.

Figure 1

sEH inhibitor improved recovery of neurological function at 1 and 3 days after MCAO. Notes: The sensorimotor function of MCAO rats treated with or without AUDA was examined in (A) beam walk score (n=6/group) and (B) footslips percentage in beam walk test (n=6/group). Data are presented as mean ± SEM. *P<0.05, **P<0.01. Abbreviations: IS, ischemia; AUDA, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid; MCAO, middle cerebral artery occlusion; sEH, soluble epoxide hydrolase; SEM, standard error of the mean.

Figure 2

sEH inhibition decreased the infarct volume after MCAO. Notes: (A and B) Representative brain slices with TTC staining from rats of sham-operated, vehicle treatment and AUDA treatment at 1 and 3 days after MCAO. The pale region indicates the infarct area. (C) Quantification of infarct volume of rats subjected to vehicle treatment and AUDA treatment at 1 and 3 days after MCAO. The sham-operated rats revealed no infarction. n=6/group. Data are presented as mean ± SEM.*P<0.01. Abbreviations: AUDA, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid; MCAO, middle cerebral artery occlusion; sEH, soluble epoxide hydrolase; SEM, standard error of the mean; TTC, triphenyltetrazolium chloride.

Figure 3

sEH inhibition modulated microglia/macrophages polarization after MCAO. Notes: (A) Representative immunofluorescent staining of CD86 (green) and Iba-1 (red) in the non-ischemic hemisphere, the ischemic border of rats in vehicle and AUDA treatment groups. Scale bar represents 100 μm. (B) Representative immunofluorescent staining of CD206 (green) and Iba-1 (red) in the non-ischemic hemisphere, the ischemic border of rats in vehicle and AUDA treatment group. Scale bar represents 100 μm. (C) Statistical analysis of CD86⁺/Iba-1⁺ or CD206⁺/Iba-1⁺ double positive microglia/macrophages in the ischemic border is expressed as cells/mm, n=3/group. Data are presented as mean ± SEM.*P<0.05 and **P<0.01. Abbreviations: AUDA, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid; Iba-1, ionized calcium binding adaptor molecule 1; MCAO, middle cerebral artery occlusion; sEH, soluble epoxide hydrolase; SEM, standard error of the mean.

Figure 4

Flow cytometry analysis revealed that the sEH inhibitor suppresses M1 microglia and promotes M2 microglia polarization after MCAO. Notes: (A) Gating strategy to detect M1 microglia (CD11⁺CD45^intCD86⁺) and M2 microglia (CD11⁺CD45^intCD206⁺). In the histograms, the pink regions represented unstained cells (negative control). The green regions on the right of the vertical black lines represented APC-positive (M1) or FITC-positive (M2) cells. (B) The percentage of M1 and M2 microglia of rats in vehicle and AUDA treatment groups, n=4/group. Data are presented as mean ± SEM.*P<0.05. Abbreviations: APC, allophycocyanin; AUDA, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid; FITC, fluorescein isothiocyanate; MCAO, middle cerebral artery occlusion; PE, phycoerythrin; sEH, soluble epoxide hydrolase; SEM, standard error of the mean.

Figure 5

Inhibition of sEH regulated mRNA expression of pro- and anti-inflammatory cytokines and antioxidant mediators at 1 day after MCAO. Notes: (A) AUDA-regulated mRNA expression of pro- and anti-inflammatory cytokines (IL-1β, IL-6, IL-10, iNOS, MCP-1, MMP-9). (B) AUDA-regulated mRNA expression of antioxidant mediators (CAT, HO-1, Nrf2, PPARγ, SOD-1). Reverse-transcription polymerase chain reaction was performed using total RNA extracted from ischemic cortex tissue (n=4/group) and microglia (n=3‒4/group). Data are presented as mean ± SEM. *P<0.05. Abbreviations: AUDA, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid; CAT, catalase; HO-1, heme oxygenase-1; IL, interleukin; iNOS, inducible nitric oxidase synthase; MCAO, middle cerebral artery occlusion; MCP-1, monocyte chemoattractant protein-1; MMP-9, matrix metalloproteinase-9; Nrf2, nuclear factor erythroid 2-related factor 2; PPARγ, peroxisome proliferator-activated receptor-γ; sEH, soluble epoxide hydrolase; SEM, standard error of the mean; SOD-1, superoxide dismutase-1.

Figure 6

sEH inhibition modulated HO-1 and NeuN expression in neuronal cells at 1 day after MCAO. Notes: (A) Representative immunofluorescence staining of HO-1 (green) and Iba-1 (red) in the non-ischemic hemisphere, the ischemic border of rats in vehicle and AUDA treatment groups. Scale bar: 100 μm. (B) Representative immunofluorescence staining of HO-1 (green) and NeuN (red) in the non-ischemic hemisphere, the ischemic border of rats in vehicle and AUDA treatment groups. Scale bar: 100 μm. (C) Statistical analysis of HO-1⁺/Iba-1⁺ in microglia and HO-1⁺/NeuN⁺ in neuronal cells in the ischemic border expressed as cells/mm² n=3/group. (D) The mRNA expression of NeuN in the ischemic cortex, n=4/group. Data are presented as mean ± SEM. *P<0.05. Abbreviations: AUDA, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid; HO-1, heme oxygenase-1; Iba-1, ionized calcium binding adaptor molecule 1; MCAO, middle cerebral artery occlusion; NeuN, neuronal nuclei; sEH, soluble epoxide hydrolase; SEM, standard error of the mean; SOD-1, superoxide dismutase-1.