4-Hydroxysesamin, a Modified Natural Compound, Attenuates Neuronal Apoptosis After Ischemic Stroke via Inhibiting MAPK Pathway

Abstract

Background: The 4-hydroxysesamin (4-HS, a di-tetrahydrofuran lignin) is a modified sesamin that was prepared in the laboratory. This preclinical study was designed to preliminarily investigate the neuroprotective properties of 4-HS.

Methods: In vitro, neuronal injury and inflammation were simulated by oxygen-glucose deprivation and lipopolysaccharide (LPS) exposure in mouse hippocampal neuronal HT22 cell line, and treated with 4-HS and/or metformin (MET, MAPK pathway activator for exploring mechanism). CCK-8, flow cytometry, and enzyme-linked immunosorbent assay were performed to evaluate cell viability, apoptosis, and inflammation. Apoptosis- and pathway-related proteins were detected by Western blotting. Middle cerebral artery occlusion (MCAO) was constructed as a stroke model and treated with 4-HS for in vivo confirmation. Histological staining was used for in vivo evaluation of 4-HS properties.

Results: The 4-HS showed similar anti-inflammatory activity to sesamin but did not affect the cell viability of HT22 cells. In vitro, 4-HS improved the cell viability, ameliorated neuronal apoptosis, along with the reversion of apoptotic proteins (Bax, cleaved-caspase 3/9, Bcl-2) expression and inflammatory cytokines (IL-6, TNF-α, IL-10) in LPS-treated HT22 cells. The 4-HS suppressed the phosphorylation of ERK, JNK, and p38 but the addition of MET reversed 4-HS-induced changes of phenotype and protein expression in LPS-treated cells. In vivo, 4-HS showed apparent improvement in cerebral infarction, brain tissue morphology, neuronal architecture, apoptosis, and inflammation of MCAO mice, and also showed inhibiting effects on the phosphorylation of ERK, JNK, and p38, confirming in vivo results.

Conclusion: In this first pre-clinical study on 4-HS, we preliminarily demonstrated the neuroprotective properties of 4-HS both in cell and animal models, and proposed that the underlying mechanism might be associated with the MAPK pathway.

Keywords: 4-hydroxysesamin; ischemic stroke; middle cerebral artery occlusion; neuronal apoptosis; sesamin.

Figure 1

4-HS attenuates the neuronal apoptosis and inflammation in LPS-treated hippocampal neuronal cells. (A) The 2D chemical structure and 3D conformer of 4-HS retrieved from the PubChem (CID: 5318999); (B) Compare the anti-inflammatory activity of 4-HS and sesamin by ELISA in OGD/LPS-treated cells; (C) Effects of 4-HS alone on cell viability of HT22 cells tested by CCK-8 assay; (D) Effects of 4-HS on cell viability of OGD/LPS-treated cells tested by CCK-8 assay; (E) Effects of 4-HS on neuronal apoptosis of OGD/LPS-treated cells examined by flow cytometry assay; (F) Western blotting for the apoptosis-related proteins and quantitative data of relative expression were shown; Data are expressed as mean ± SEM of three independent repeats. *** p<0.0001.

Figure 2

4-HS presents neuroprotective properties by inhibiting p38 MAPK pathway. (A) After the 4-HS and MET exposure, neuronal apoptosis of OGD/LPS-treated cells was examined by flow cytometry assay; (B) Western blotting for the apoptosis-related proteins and quantitative data of relative expression were shown; (C) After the 4-HS and MET exposure, the inflammation of OGD/LPS-treated cells tested by ELISA; (D) Western blotting for pathway-related proteins and quantitative data of relative expression were shown. Data are expressed as mean ± SEM three independent repeats. *** p<0.0001.

Figure 3

In vivo confirmation for the neuroprotective properties of 4-HS in MCAO model. (A) Representative TTC staining images for brain tissues and quantitative data of infarct volume percentage were shown (n=5 in each group); (B) Representative H&E and Nissl staining images for brain tissues were shown (n=5 in each group); (C) Representative TUNEL staining images for brain tissues and quantitative data were shown (n=5 in each group); (D) The in vivo anti-inflammatory activity of 4-HS in MCAO mice was verified by ELISA (n=5 in each group); (E) Western blotting for the apoptosis-related proteins and quantitative data of relative expression were shown (n=5 in each group); (F) Western blotting for pathway-related proteins in MCAO mice and quantitative data of relative expression were shown (n=5 in each group). Data are expressed as mean ± SEM of five independent repeats. *** p<0.0001; ** p<0.001.