Synthesis, target analysis, and cerebroprotective effects of novel imide antioxidants via the Nrf2/HO-1 pathway in cerebral ischemia-reperfusion injury

Abstract

Background: Cerebral ischemia-reperfusion injury (CIRI) is a secondary brain injury that occurs after thrombolysis and is a primary cause of death in ischemic stroke patients. Antioxidants that effectively reduce oxidative stress are an efficient treatment approach for CIRI. Here, a novel diimide compound was synthesized using the chemical structure of previously designed anti-inflammatory skeletons.

Methods and results: The antioxidant activities of five compounds (Z1-Z5) were preliminarily evaluated using the hydrogen peroxide-induced PC12 cell damage model, of which Z3 exhibited the best antioxidant effect, even exceeding that of the positive control (tert-butylhydroquinone). Enrichment analysis using network targeting and network pharmacology methods predicted seven candidate core target genes of Z3 in CIRI. Of these targets, computer molecular docking analysis predicted that Z3 has the strongest binding affinity for nuclear factor erythroid 2-related factor (Nrf2). MTT and colony formation assays, reactive oxygen species analysis, immunofluorescence, and immunoblotting experiments verified that Z3 reduced reactive oxygen species to play a protective antioxidant role via the Nrf2/hemoxygenase 1 (HO-1) pathway. The protective effect of Z3 in vivo was explored through TTC staining and neurobehavioral scoring of CIRI model mice.

Conclusion: This study provides a new drug development strategy and candidate drug for the treatment of CIRI, offering ideas for the design of new antioxidants.

Keywords: Nrf2 signaling pathway; antioxidant; cerebral ischemia-reperfusion injury; molecular docking; network pharmacology; oxidative stress.

FIGURE 1

Typical nitrogen-containing antioxidants.

SCHEME 1

Synthesis and structure of novel diimide derivatives Z1–Z5. (a) CDI, NaH, DCM, r.t.

FIGURE 2

Protective antioxidant effect of the Z1–Z5 family of compounds. MTT assays of cell viability of PC12 cells grown for 1 day on 96-well plates, treated with the indicated concentrations of each candidate compound for 18 h, and stimulated with H₂O₂ (500 μM) for 24 h; *P < 0.05, **P < 0.01.

FIGURE 3

Cytoprotective effects of Z3 against H₂O₂-induced PC12 cell damage. (A,B) MTT (A) and LDH (B) assays to detect the cytoprotective effect of Z3 against H₂O₂-induced PC12 cells treated with Z3, TBHQ for 24 h, then exposed to H₂O₂ for another 24 h. (C) Colony formation assay of PC12 cells pretreated with Z3 for 24 h, and then exposed to H₂O₂. Cells were stained with crystal violet. (D) Inverted microscopy showing the morphological improvements in crystal violet-stained PC12 cells pretreated with Z3, then stimulated with H₂O₂ compared with DMSO-treated cells. Data represented as means ± SD (n = 3); ^#### P < 0.0001; **P < 0.01, ***P < 0.001.

FIGURE 4

Venn diagram (A) and PPI action network diagram (B) of common gene targets between Z3 and active components of CIRI.

FIGURE 5

Results of GO enrichment analysis of common target genes between Z3 and CIRI. The top 13 biological processes related to CIRI are shown.

FIGURE 6

Antioxidant activity of Z3 in PC12 cells. (A) Representative fluorescence images showing the reduction in the accumulation of intracellular ROS in cells pretreated with Z3 or DMSO control for 24 h, then stimulated with H₂O₂ for 2 h, followed by incubation with DCFH-DA for 30 min. (B) Reduction in MDA in cells pretreated with Z3 or DMSO for 24 h, then stimulated with H₂O₂ for 3 h. Data expressed as means ± SD (n = 3); ^#### P < 0.0001; ^** P < 0.01, ^*** P < 0.001, ^**** P < 0.0001.

FIGURE 7

Molecular docking diagrams of Z3 with Nrf2 (PDB:2FLU).

FIGURE 8

Z3-mediated activation of the sestrin 2/p62/Nrf2/HO-1 signaling pathway in PC12 cells. (A) Immunofluorescence of cells pre-incubated with 10 μmol·L⁻¹ Z3 or TBHQ for 6 h and stained with Nrf2 antibody (red) and DAPI (blue), showing the nuclear translocation of Nrf2. (B,C) Western blotting of sestrin 2, p62, and HO-1 in cells treated with Z3 for 24 h (D) MTT viability assays of cells pretreated with ZnPP for 1 h prior to the addition of Z3 and stimulation with H₂O₂. Data expressed as means ± SD (n = 3); ^#### P < 0.0001; ^** P < 0.01, ^*** P < 0.001, ^**** P < 0.0001.

FIGURE 9

Protective effect of Z3 on MCAO-induced CIRI. (A) TTC staining analysis of representative brain slices from model mice treated with 15 mg·kg⁻¹ Z3. (B) Quantitative analysis of brain infarction was performed by calculating the ratio of infarct area to whole brain area in individual mice. (C) Quantitative analysis of neurobehavioral scores in MCAO mice with intraperitoneal injection of normal saline (NS), vehicle, Z3, or ED. Data expressed as means ± SD (n = 5–8); ^## P < 0.01, ^#### P < 0.0001; ^**** P < 0.0001.