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Stimulator of IFN genes mediates neuroinflammatory injury by suppressing AMPK signal in experimental subarachnoid hemorrhage.

ABSTRACT:

Background

Neuroinflammation is closely associated with the poor prognosis in subarachnoid hemorrhage (SAH) patients. This study was aimed to determine the role of stimulator of IFN genes (STING), an essential regulator to innate immunity, in the context of SAH.

Methods

A total of 344 male C57BL/6 J mice were subjected to endovascular perforation to develop a model of SAH. Selective STING antagonist C-176 and STING agonist CMA were administered at 30 min or 1 h post-modeling separately. To investigate the underlying mechanism, the AMPK inhibitor compound C was administered intracerebroventricularly at 30 min before surgery. Post-SAH assessments included SAH grade, neurological test, brain water content, western blotting, RT-PCR, and immunofluorescence. Oxygenated hemoglobin was introduced into BV2 cells to establish a SAH model in vitro.

Results

STING was mainly distributed in microglia, and microglial STING expression was significantly increased after SAH. Administration of C-176 substantially attenuated SAH-induced brain edema and neuronal injury. More importantly, C-176 significantly alleviated both short-term and persistent neurological dysfunction after SAH. Meanwhile, STING agonist CMA remarkably exacerbated neuronal injury and deteriorated neurological impairments. Mechanically, STING activation aggravated neuroinflammation via promoting microglial activation and polarizing into M1 phenotype, evidenced by microglial morphological changes, as well as the increased level of microglial M1 markers including IL-1β, iNOS, IL-6, TNF-α, MCP-1, and NLRP3 inflammasome, while C-176 conferred a robust anti-inflammatory effect. However, all the mentioned beneficial effects of C-176 including alleviated neuroinflammation, attenuated neuronal injury and the improved neurological function were reversed by AMPK inhibitor compound C. Meanwhile, the critical role of AMPK signal in C-176 mediated anti-inflammatory effect was also confirmed in vitro.

Conclusion

Microglial STING yielded neuroinflammation after SAH, while pharmacologic inhibition of STING could attenuate SAH-induced inflammatory injury at least partly by activating AMPK signal. These data supported the notion that STING might be a potential therapeutic target for SAH.

SUBMITTER: Peng Y

PROVIDER: S-EPMC7247752 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundInnate immunity can facilitate early brain injury (EBI) following subarachnoid hemorrhage (SAH). Numerous studies suggest that pyroptosis could exacerbate extracellular immune responses by promoting secretion of inflammatory cytokines. Transforming growth factor-β-activated kinase 1 (TAK1) is a quintessential kinase that positively regulates inflammation through NF-κB and MAPK signaling cascades. However, the effects of TAK1 on neuroinflammation in EBI following SAH are largely unknown.MethodsTwo hundred and forty-six male C57BL/6J mice were subjected to the endovascular perforation model of SAH. A selective TAK1 inhibitor, 5Z-7-oxozeaenol (OZ) was administered by intracerebroventricular (i.c.v) injection at 30 min after SAH induction. To genetic knockdown of TAK1, small interfering RNA (siRNA) was i.c.v injected at 48 h before SAH induction. SAH grade, brain water content, BBB permeability, neurological score, western blot, real-time PCR, ELISA, transmission electron microscope, and immunofluorescence staining were performed. Long-term behavioral sequelae were evaluated by the rotarod and Morris water maze tests. Furthermore, OZ was added to the culture medium with oxyhemoglobin (OxyHb) to mimic SAH in vitro. The reactive oxygen species level was detected by DCFH-DA staining. Lysosomal integrity was assessed by Lyso-Tracker Red staining and Acridine Orange staining.ResultsThe neuronal phosphorylated TAK1 expression was upregulated following SAH. Pharmacologic inhibition of TAK1 with OZ could alleviate neurological deficits, brain edema, and brain-blood barrier (BBB) disruption at 24 h after SAH. In addition, OZ administration restored long-term neurobehavioral function. Furthermore, blockade of TAK1 dampened neuronal pyroptosis by downregulating the N-terminal fragment of GSDMD (GSDMD-N) expression and IL-1β/IL-18 production. Mechanistically, both in vivo and in vitro, we demonstrated that TAK1 can induce neuronal pyroptosis through promoting nuclear translocation of NF-κB p65 and activating nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) inflammasome. TAK1 siRNA treatment mitigated SAH-induced neurobehavioral deficits and restrained phosphorylated NF-κB p65 expression and NLRP3 inflammasome activation. TAK1 blockade also ameliorated reactive oxygen species (ROS) production and prevented lysosomal cathepsin B releasing into the cytoplasm.ConclusionsOur findings demonstrate that TAK1 modulates NLRP3-mediated neuronal pyroptosis in EBI following SAH. Inhibition of TAK1 may serve as a potential candidate to relieve neuroinflammatory responses triggered by SAH.