YTHDF2-regulated matrilin-3 mitigates post-reperfusion hemorrhagic transformation in ischemic stroke via the PI3K/AKT pathway

Abstract

Hemorrhagic transformation can complicate ischemic strokes after recanalization treatment within a time window that requires early intervention. To determine potential therapeutic effects of matrilin-3, rat cerebral ischemia-reperfusion was produced using transient middle cerebral artery occlusion (tMCAO); intracranial hemorrhage and infarct volumes were assayed through hemoglobin determination and 2,3,5-triphenyltetrazoliumchloride (TTC) staining, respectively. Oxygen-glucose deprivation (OGD) modeling of ischemia was performed on C8-D1A cells. Interactions between matrilin-3 and YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) were determined using RNA immunoprecipitation assay and actinomycin D treatment. Reperfusion after tMCAO modeling increased hemorrhage, hemoglobin content, and infarct volumes; these were alleviated by matrilin treatment. Matrilin-3 was expressed at low levels and YTHDF2 was expressed at high levels in ischemic brains. In OGD-induced cells, matrilin-3 was negatively regulated by YTHDF2. Matrilin-3 overexpression downregulated p-PI3K/PI3K, p-AKT/AKT, ZO-1, VE-cadherin and occludin, and upregulated p-JNK/JNK in ischemic rat brains; these effects were reversed by LY294002 (a PI3K inhibitor). YTHDF2 knockdown inactivated the PI3K/AKT pathway, inhibited inflammation and decreased blood-brain barrier-related protein levels in cells; these effects were reversed by matrilin-3 deficiency. These results indicate that YTHDF2-regulated matrilin-3 protected ischemic rats against post-reperfusion hemorrhagic transformation via the PI3K/AKT pathway and that matrilin may have therapeutic potential in ischemic stroke.

Keywords: Blood-brain barrier; Hemorrhagic transformation; Ischemic stroke; Matrilin-3; PI3K/AKT signaling pathway; YTH N6-methyladenosine RNA binding protein F2.

Figure 1.

Effects of matrilin-3 on hemorrhagic transformation in ischemic rat brains. (A) Ischemic stroke modeling was performed on rats injected with or without adenovirus-mediated matrilin-3 plasmids (Control, tMCAO, tMCAO+NC, and tMCAO+Matrilin-3 groups). Cerebral hemorrhage in rats from each group was observed. (B) Hemoglobin level was determined in each group to evaluate the severity of cerebral hemorrhage. (C) qRT-PCR was employed to detect the expression of matrilin-3 in each group. (D–G) Western blot was used to analyze expressions of p-PI3K/PI3K, p-AKT/AKT, and p-JNK/JNK in each group. GAPDH served as the loading control. The data are presented as the mean±SD of 3 independent experiments; ***p < 0.001 versus Control; ^p < 0.05, ^^^p < 0.001 versus tMCAO+NC. Abbreviations: qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; PI3K, phosphatidylinositol-3-kinase; p-PI3K, phosphorylated PI3K; AKT, protein kinase B; JNK, c-Jun NH2-terminal kinase; NC, negative control.

Figure 2.

PI3K inhibition reversed the protective role of matrilin-3 in hemorrhagic transformation and infarction in rat brains after tMCAO. (A) Ischemic stroke modeling was performed on rats who were injected with or without adenovirus-mediated matrilin-3 plasmids and LY294002 (Control, tMCAO, tMCAO+NC, tMCAO+Matrilin-3, and tMCAO+Matrilin-3 + LY294002 groups). Rat brain sample in each group were collected to observe cerebral hemorrhage. (B) Hemoglobin level was determined in each group to evaluate the severity of cerebral hemorrhage. (C, D) The infarction area in the TTC-stained brain sections in each group was investigated by observation and quantitative analysis. The data are presented as the mean±SD of 3 independent experiments; **p < 0.01, ***p < 0.001 versus Control; ^{^^}p < 0.01, ^{^^^}p < 0.001 versus tMCAO+NC; ^#p < 0.05, ^##p < 0.01 versus tMCAO+Matrilin-3. Abbreviation: TTC, 2,3,5-triphenyltetrazoliumchloride.

Figure 3.

Matrilin-3 was involved in the restoration of the BBB via the PI3K/AKT signaling pathway. (A–E) Ischemic stroke modeling was performed on rats who were injected with or without adenovirus-mediated matrilin-3 plasmids and LY294002 (Control, tMCAO, tMCAO+NC, tMCAO+Matrilin-3, and tMCAO+Matrilin-3 + LY294002 groups). Western blot was used for quantitative analysis of p-PI3K/PI3K, p-AKT/AKT, ZO-1, VE-cadherin, and occludin protein expressions in each group. GAPDH served as the loading control. The data are presented as the mean±SD of 3 independent experiments; ***p < 0.001 versus Control; ^{^^^}p < 0.001 versus tMCAO+NC; ^###p < 0.001 versus tMCAO+Matrilin-3. Abbreviations: ZO-1, zonula occludens-1; VE-cadherin, vascular endothelial cadherin.

Figure 4.

Expressions of YTHDF2 and matrilin-3 in C8-D1A cells after OGD. (A, B) Western blot was used to detect the protein expression of YTHDF2 in the brains of rats received with or without ischemic stroke modeling. (C–E) C8-D1A cells in glucose-free medium were induced by normoxia or hypoxia for 3, 6, 9, and 12 hours before subjected to Western blot for detecting YTHDF2 and matrilin-3 protein expressions. GAPDH served as the loading control. The data are presented as the mean±SD of 3 independent experiments; ^***p < 0.001 versus Control; ^##p < 0.01, ^###p < 0.001 versus Normoxia. Abbreviation: YTHDF2, YTH N6-methyladenosine RNA binding protein F2.

Figure 5.

YTHDF2 bound to matrilin-3 and YTHDF2 knockdown reduced the inflammation of OGD-induced C8-D1A cells. (A, B) QRT-PCR was employed to detect YTHDF2 and matrilin-3 expressions in C8-D1A cells transfected with or without shNC/shYTHDF2. (C) RNA immunoprecipitation assay was performed to verify the binding of YTHDF2 and matrilin-3. (D) C8-D1A cells transfected with or without shNC/shYTHDF2 were subjected to 5 μg/mL ACTD treatment for 0, 1, 2, and 3 hours, followed by qRT-PCR for detecting the changes of matrilin-3 decay. (E, F) C8-D1A cells transfected with or without shNC/shYTHDF2 were subjected to hypoxic induction in glucose-free medium for obtaining OGD models (Control, OGD, OGD+shNC, and OGD+shYTHDF2 groups). TNF-α and IL-6 expressions in each group were determined using qRT-PCR. GAPDH served as the loading control. The data are presented as the mean±SD of 3 independent experiments; ^**p < 0.01, ^***p < 0.001 versus shNC; ^###p < 0.001 versus Control; ^{^^^}p < 0.001 versus OGD+shNC. Abbreviations: shYTHDF2, shRNA targeting YTHDF2; shNC, scramble shRNA; ACTD, Actinomycin D; OGD, oxygen-glucose deprivation; TNF-α, tumor necrosis factor alpha; IL-6, interleukin 6.

Figure 6.

YTHDF2 knockdown suppressed inflammation, the PI3K/AKT signaling pathway and BBB-related proteins in C8-D1A cells by regulating matrilin-3. (A) QRT-PCR was employed to detect the expression of matrilin-3 in C8-D1A cells transfected with or without shNC/shMatrilin-3. (B–C) C8-D1A cells transfected with shMatrilin-3 or/and shYTHDF2 (Control, shMatrilin-3, shYTHDF2, and shMatrilin-3 + shYTHDF2 groups). TNF-α and IL-6 expressions in each group were determined using qRT-PCR. (D–H) Western blot was used for quantitative analysis of p-PI3K/PI3K, p-AKT/AKT, ZO-1, VE-cadherin, and occludin protein expressions in each group. GAPDH served as the loading control. The data are presented as the mean±SD of 3 independent experiments; **p < 0.01, ***p < 0.001 versus shNC; ^##p < 0.01, ^###p < 0.001 versus shMatrilin-3; ^{^^^}p < 0.001 versus shYTHDF2. Abbreviation: shMatrilin-3, shRNA targeting matrilin-3.