Obstructive sleep apnea aggravates neuroinflammation and pyroptosis in early brain injury following subarachnoid hemorrhage via ASC/HIF-1α pathway

Abstract

Obstructive sleep apnea can worsen the prognosis of subarachnoid hemorrhage. However, the underlying mechanism remains unclear. In this study, we established a mouse model of subarachnoid hemorrhage using the endovascular perforation method and exposed the mice to intermittent hypoxia for 8 hours daily for 2 consecutive days to simulate sleep apnea. We found that sleep apnea aggravated brain edema, increased hippocampal neuron apoptosis, and worsened neurological function in this mouse model of subarachnoid hemorrhage. Then, we established an in vitro HT-22 cell model of hemin-induced subarachnoid hemorrhage/intermittent hypoxia and found that the cells died, and lactate dehydrogenase release increased, after 48 hours. We further investigated the underlying mechanism and found that sleep apnea increased the expression of hippocampal neuroinflammatory factors interleukin-1β, interleukin-18, interleukin-6, nuclear factor κB, pyroptosis-related protein caspase-1, pro-caspase-1, and NLRP3, promoted the proliferation of astrocytes, and increased the expression of hypoxia-inducible factor 1α and apoptosis-associated speck-like protein containing a CARD, which are the key proteins in the hypoxia-inducible factor 1α/apoptosis-associated speck-like protein containing a CARD signaling pathway. We also found that knockdown of hypoxia-inducible factor 1α expression in vitro greatly reduced the damage to HY22 cells. These findings suggest that sleep apnea aggravates early brain injury after subarachnoid hemorrhage by aggravating neuroinflammation and pyroptosis, at least in part through the hypoxia-inducible factor 1α/apoptosis-associated speck-like protein containing a CARD signaling pathway.

Keywords: apoptosis associated speck like protein containing a CARD; early brain injury; hypoxia-inducible factor 1α; neuroinflammation; nucleotide-binding domain and leucine-rich repeat protein 3; obstructive sleep apnea; pyroptosis; subarachnoid hemorrhage.

Figure 1

Mortality and SAH grade after SAH and OSA. (A) Schematic of experimental design for modeling SAH and OSA in vitro and in vivo. (B) Mortality at 48 hours after surgery. (C) SAH grade at 48 hours after surgery. Higher scores indicate better neurological function. Data are expressed as mean ± SEM (n = 15). *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). OSA: Obstructive sleep apnea; SAH: subarachnoid hemorrhage.

Figure 2

OSA aggravates EBI following SAH in vivo. (A, B) OSA worsens brain edema (A) and neurological behavior scores (B) after SAH. Data are expressed as mean ± SEM (n = 5). *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). (C) OSA increases hippocampal tissue damage at 48 hours after SAH. Red arrows indicate dead cells. Scale bar: 50 μm. EBI: Early brain injury; OSA: obstructive sleep apnea; SAH: subarachnoid hemorrhage.

Figure 3

OSA increases hemin-induced HT-22 neuronal cell injury in vitro. (A) MTT assay showing that OSA increases HT-22 neuronal cell death 48 hours following hemin-induced SAH in vitro. (B) LDH release assay showing that OSA increases HT-22 neuronal cell death 48 hours following hemin-induced SAH in vitro. Data are expressed as mean ± SEM (n = 5). *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). (C) Live/dead assay showing that OSA aggravates hemin-induced HT-22 neuronal cell death. Red arrows indicate dead cells. Red fluorescence indicates dead cells, and green fluorescence indicates live cells. Scale bars: 100 μm. LDH: Lactate dehydrogenase; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; OSA: obstructive sleep apnea; SAH: subarachnoid hemorrhage.

Figure 4

OSA aggravates neuroinflammation after SAH in vivo. (A–D) OSA significantly increases IL-1β (A), IL-18 (B), IL-6 (C), and NF-κB (D) levels in the hippocampus 48 hours following SAH. Data are expressed as mean ± SEM (n = 5). *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). (E) Immunofluorescence shows that GFAP (green, marked by Alexa Fluor® 488) expression and astrocyte proliferation increased after SAH, and that these effects were aggravated by OSA. Red arrows indicate astrocytes. Scale bar: 50 μm. DAPI: 4′,6-Diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; IL: interleukin; NF-κB: nuclear transcription factor-κB; OSA: obstructive sleep apnea; SAH: subarachnoid hemorrhage.

Figure 5

OSA aggravates pyroptosis after SAH in vivo. (A) Detection of NLRP3, pro-caspase-1 p20, and caspase-1 by western blot assay. (B–D) Expression of pro-caspase-1 (B), caspase-1 (C), and NLRP3 (D) (normalized to β-actin) relative to the control. OSA increases pro-caspase-1 p20, caspase-1, and NLRP3 expression following SAH in mice. Data are expressed as mean ± SEM (n = 5). *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). NLRP3: Nucleotide-binding domain leucine-rich repeat pyrin domain containing 3; OSA: obstructive sleep apnea; SAH: subarachnoid hemorrhage.

Figure 6

OSA aggravates pyroptosis after SAH in vivo via the HIF-1α/ASC signaling pathway. (A, B) OSA increases HIF-1α (A) and ASC (B) mRNA expression of in the left cortex, as detected by real-time polymerase chain reaction. (C) Detection of HIF-1α and ASC in left cortex samples by western blot. (D, E) HIF-1α (D) and ASC (E) expression (normalized to β-actin) relative to the control. OSA increases HIF-1α expression following SAH in mice. Data are expressed as mean ± SEM (n = 5). *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). ASC: Apoptosis associated speck like protein containing a CARD; HIF-1α: hypoxia-inducible factor-1α; OSA: obstructive sleep apnea; SAH: subarachnoid hemorrhage.

Figure 7

HIF-1α knockdown alleviates pyroptosis after SAH/OSA in vitro. (A) Detection of NLRP3, Pro-caspase-1 p20, and caspase-1 in HT-22 neuronal cells by western blot. (B–D) Pro-caspase-1 (B), caspase-1 (C), and NLRP3 (D) expression (normalized to β-actin) relative to the control. (E) MTT assay showing that HT-22 neuronal cell death decreased significantly after HIF-1α knockdown 48 hours following hemin-induced SAH. (F) LDH release assay showing that HT-22 neuronal cell death decreased significantly after HIF-1α knockdown 48 hours after hemin-induced SAH in vitro. Data are expressed as mean ± SEM (n = 5). *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). (G) Live/dead assay of HT-22 cells exposed to hemin or OSA. HIF-1α knockdown alleviated hemin-induced HT-22 neuronal death. Red arrows indicate dead cells. Scale bar: 100 μm. HIF-1α: Hypoxia-inducible factor-1α; LDH: lactate dehydrogenase; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NLRP3: nucleotide-binding domain leucine-rich repeat pyrin domain containing 3; OSA: obstructive sleep apnea; SAH: subarachnoid hemorrhage.

Figure 8

HIF-1α knockdown alleviates EBI after SAH and OSA in vivo. (A) HIF-1α knockdown improved neurological dysfunction after SAH/OSA. (B) HIF-1α knockdown decreased brain edema after SAH/OSA. Data are expressed as mean ± SEM (n = 5)*P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). (C) HIF-1α knockdown decreased hippocampal tissue damage at 48 hours after SAH, as determined by TUNEL staining (red arrows indicate dead cells). Scale bar: 50 μm. DAPI: 4′,6-Diamidino-2-phenylindole, dihydrochloride; EBI: early brain injury; HIF-1α: hypoxia-inducible factor-1α; OSA: obstructive sleep apnea; SAH: subarachnoid hemorrhage; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.