Association of DNA methylation/demethylation with the functional outcome of stroke in a hyperinflammatory state

Abstract

JOURNAL/nrgr/04.03/01300535-202410000-00024/figure1/v/2024-02-06T055622Z/r/image-tiff Inflammation is closely related to stroke prognosis, and high inflammation status leads to poor functional outcome in stroke. DNA methylation is involved in the pathogenesis and prognosis of stroke. However, the effect of DNA methylation on stroke at high levels of inflammation is unclear. In this study, we constructed a hyperinflammatory cerebral ischemia mouse model and investigated the effect of hypomethylation and hypermethylation on the functional outcome. We constructed a mouse model of transient middle cerebral artery occlusion and treated the mice with lipopolysaccharide to induce a hyperinflammatory state. To investigate the effect of DNA methylation on stroke, we used small molecule inhibitors to restrain the function of key DNA methylation and demethylation enzymes. 2,3,5-Triphenyltetrazolium chloride staining, neurological function scores, neurobehavioral tests, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot assay were used to evaluate the effects after stroke in mice. We assessed changes in the global methylation status by measuring DNA 5-mc and DNA 5-hmc levels in peripheral blood after the use of the inhibitor. In the group treated with the DNA methylation inhibitor, brain tissue 2,3,5-triphenyltetrazolium chloride staining showed an increase in infarct volume, which was accompanied by a decrease in neurological scores and worsening of neurobehavioral performance. The levels of inflammatory factors interleukin 6 and interleukin-1 beta in ischemic brain tissue and plasma were elevated, indicating increased inflammation. Related inflammatory pathway exploration showed significant overactivation of nuclear factor kappa B. These results suggested that inhibiting DNA methylation led to poor functional outcome in mice with high inflammation following stroke. Further, the effects were reversed by inhibition of DNA demethylation. Our findings suggest that DNA methylation regulates the inflammatory response in stroke and has an important role in the functional outcome of hyperinflammatory stroke.

Figure 1

The experimental design. Each behavioral testing and molecular biology experiment was repeated at least three times. Bobcat339: 4-Amino-1-[1,1′-biphenyl]-3-yl-5-chloro-2(1H)-pyrimidinone; ELISA: enzyme linked immunosorbent assay; qPCR: quantitative reverse transcription polymerase chain reaction; RG108: N-phthalyl-L-tryptophan; tMCAO: transient middle cerebral artery occlusion; TTC: 2,3,5-triphenyltetrazolium chloride staining; WB: western blot.

Figure 2

In a hyperinflammatory state, inhibition of DNA methyltransferase (DNMT3A) led to increased infarction volume, and inhibition of translocation methylcytosine dioxygenases 2 (TET2) showed the opposite result. (A) Representative coronal brain images with TTC staining for each group. (B) Graph of infarct volume percentage over whole brain volume (Sham, n = 8; Vehicle, n = 8; RG108, n = 8; Bobcat339, n = 8). (C) Body weight, n = 10 per group. (D) The weight of spleen, n = 10 per group. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance with Tukey’s multiple comparisons post hoc test for B and D; two-way analysis of variance with Tukey’s multiple comparisons post hoc test for C). All data were expressed as mean ± SEM with individual plots. Bobcat339: 4-Amino-1-[1,1′-biphenyl]-3-yl-5-chloro-2(1H)-pyrimidinone; RG108: N-phthalyl-L-tryptophan.

Figure 3

In a hyperinflammatory state, inhibition of DNMT3A resulted in poorer neurological function scores, and inhibition of TET2 elevated the scores. The Modified Garcia Scores in the different intervention groups at 24 hours after transient ischemia; n = 13 per group. Higher scores indicate better sensorimotor function performance. (A) Body proprioception. (B) Vibrissae touch. (C) Limb symmetry. (D) Lateral turning. (E) Forelimb walking. (F) Total neurological assessment score. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance with Tukey’s multiple comparisons post hoc test). All data were expressed as mean ± SEM with individual plots. Bobcat339: 4-Amino-1-[1,1′-biphenyl]-3-yl-5-chloro-2(1H)-pyrimidinone; RG108: N-phthalyl-L-tryptophan.

Figure 4

Motor behavioral deficits after cerebral ischemia in different intervention groups under a high inflammatory state. (A) Performance of each group in the foot misplacement apparatus; n = 10 mice per group. (B) Performance of each group in the rotarod test; n = 10 mice per group. (C) Performance of each group in the hanging test; n = 10 mice per group. (D–G) Performance of each group in the open field test (OFT) at 24 hours after stroke for the representative track plot (D), total distance in 30 minutes (E), mean velocity (F), and path length (G); n = 10 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance with Tukey’s multiple comparisons post hoc test for A, E, and F; two-way analysis of variance analysis with Tukey’s multiple comparisons post hoc test for B, C, and G). All data were expressed as mean ± SEM with individual plots. Bobcat339: 4-Amino-1-[1,1′-biphenyl]-3-yl-5-chloro-2(1H)-pyrimidinone; RG108: N-phthalyl-L-tryptophan.

Figure 5

In a hyperinflammatory state, inhibition of DNMT3A function results in changes of DNA methylation level. (A) Dnmt3a mRNA expression level of each group; n = 5 mice per group. (B) Representative western blot images of DNMT3A and graph of quantified band gray scale values of DNMT3A of each group; n = 10 mice per group. (C) Dnmt1 mRNA expression level of each group; n = 5 mice per group. (D) Representative western blot images of DNMT1 and graph of quantified band gray scale values of DNMT1 of each group; n = 10 mice per group. (E) Dnmt3b mRNA expression level of each group; n = 5 mice per group. (F) Representative western blot images of DNMT3B and graph of quantified band gray scale values of DNMT3B of each group; n = 10 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance with Tukey’s multiple comparisons post hoc test). All data were expressed as mean ± SEM with individual plots. Bobcat339: 4-Amino-1-[1,1′-biphenyl]-3-yl-5-chloro-2(1H)-pyrimidinone; DNMT: DNA methyltransferase; RG108: N-phthalyl-L-tryptophan.

Figure 6

In a hyperinflammatory state, inhibition of TET2 function results in changes of DNA demethylation level. (A) Tet2 mRNA expression level of each group; n = 5 mice per group. (B) Representative western blot images of TET2 and graph of quantified band gray scale values of TET2 of each group; n = 10 mice per group. (C) Tet1 mRNA expression level of each group; n = 5 mice per group. (D) Representative western blot images of TET1 and graph of quantified band gray scale values of TET1 of each group; n = 10 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance with Tukey’s multiple comparisons post hoc test). All data were expressed as mean ± SEM with individual plots. Bobcat339: 4-Amino-1-[1,1′-biphenyl]-3-yl-5-chloro-2(1H)-pyrimidinone; RG108: N-phthalyl-L-tryptophan; TET: translocation methylcytosine dioxygenases.

Figure 7

Changes in levels of inflammatory markers in ischemic brain tissue. (A) IL-1β mRNA expression level of each group; n = 5 mice per group. (B) Representative western blot images of IL-1β and graph of quantified band gray scale values of IL-1β of each group; n = 10 mice per group. (C) IL-6 mRNA expression level of each group; n = 5 mice per group. (D) Representative western blot images of IL-6 and graph of quantified band gray scale values of IL-6 of each group; n = 10 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance with Tukey’s multiple comparisons post hoc test). All data were expressed as mean ± SEM with individual plots. Bobcat339: 4-Amino-1-[1,1′-biphenyl]-3-yl-5-chloro-2(¹H)-pyrimidinone; RG108: N-phthalyl-L-tryptophan.

Figure 8

Changes in DNA methylation and demethylation and inflammation levels in peripheral blood. (A–B) Enzyme linked immunosorbent assay of DNA 5-mc (A) and 5-hmc (B) in the peripheral blood cell samples of each group; n = 10 mice per group. (C) Enzyme linked immunosorbent assay of IL-6 level of each group; n = 10 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance with Tukey’s multiple comparisons post hoc test). All data were expressed as mean ± SEM with individual plots. 5-hmc: 5-Hydroxymethylcytosine; 5-mc: 5-methylcytosine; Bobcat339: 4-amino-1-[1,1′-biphenyl]-3-yl-5-chloro-2(1H)-pyrimidinone; RG108: N-phthalyl-L-tryptophan.

Figure 9

In a hyperinflammatory state, DNA methylation and demethylation changes affect the NF-κB pathway. (A) mRNA expression levels of five key factors in inflammatory pathways; n = 5 mice per group. (B) Representative western blot images of p-NF-κB p65/NF-κB p65. (C) Graph of quantified band gray scale values of p-NF-κB p65/NF-κB p65 of each group; n = 10 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance with Tukey’s multiple comparisons post hoc test). All data were expressed as mean ± SEM with individual plots. Akt: Akt kinase; Bobcat339: 4-amino-1-[1,1′-biphenyl]-3-yl-5-chloro-2(1H)-pyrimidinone; MAPK: mitogen-activated protein kinase; mTOR: mammalian target of rapamycin; NF-κB: nuclear factor kappa B; Nrf2: nuclear factor erythroid 2-related factor 2; p-NF-κB: phosphorylated nuclear factor kappa B; RG108: N-phthalyl-L-tryptophan.