MLKL regulates Cx43 ubiquitinational degradation and mediates neuronal necroptosis in ipsilateral thalamus after focal cortical infarction.

Necroptosis is known to play an important role in the pathophysiology of cerebral ischemia; however, its role in the occurrence of secondary thalamic injury after focal cerebral infarction and the mechanism about how mixed lineage kinase domain-like (MLKL) executes necroptosis in this pathophysiology are still unclear. In this study, Sprague-Dawley rats were subjected to distal branch of middle cerebral artery occlusion (dMCAO). The expression of MLKL, connexin 43 (Cx43) and Von Hippel-Lindau (VHL) in vitro and in vivo were assessed by Western blot. Bioinformatic methods were used to predict the potential binding sites where MLKL interacted with Cx43, and the ubiquitination degradation of Cx43 regulated by VHL. The interactions among MLKL, Cx43, VHL, and Ubiquitin were assessed by immunoprecipitation. Dye uptake assay were used to examine the Cx43 hemichannels. Intracellular Ca2+ concentration was measured using Fluo-4 AM. Overexpression and site-directed mutagenesis studies were used to study the mechanisms by which MLKL regulates Cx43 ubiquitinational degradation to mediate neuronal necroptosis. We found that MLKL and Cx43 were upregulated in the ventral posterolateral nucleus (VPN) of the ipsilateral thalamus after dMCAO. In the in vitro experiments MLKL and Cx43 were upregulated after TSZ-mediated necroptosis in SH-SY5Y cells. The interaction between MLKL and Cx43 inhibited the K48-linked ubiquitination of Cx43 in necroptotic SH-SY5Y cells. VHL is an E3 ubiquitin ligase for Cx43, and MLKL competes with VHL for binding to Cx43. Interaction of MLKL Ser454 with Cx43 can trigger the opening of Cx43 hemichannels, causing increased intracellular Ca2+, and cell necroptosis. This innovative study at animal models, cellular, and molecular levels is anticipated to clarify the roles of MLKL and Cx43 in thalamic damage after focal cortical infarction. Our findings may help identify novel targets for neurological recovery after cortical infarction.

Artesunate alleviates intracerebral haemorrhage secondary injury by inducing ferroptosis in M1-polarized microglia and suppressing inflammation through AMPK/mTORC1/GPX4 pathway.

Intracerebral haemorrhage (ICH) is a catastrophic subtype of stroke with severe morbidity and mortality. However, little progress has been made in the subsequent secondary injury. Artesunate, a water-soluble semi-synthetic derivative of artemisinin, exhibits remarkable pharmacological effects on anti-neuroinflammation. However, the effects of artesunate on ICH remain unknown. In the present study, haemoglobin (Hb) treatment in BV2 cell and collagenase type IV intracerebroventricular injection in Sprague-Dawley rats were used to establish in vitro and in vivo ICH models, respectively. For in vivo, the neurological scores, haematoma volume, brain oedema, inflammatory factors and iron deposition were evaluated. Besides, lipopolysaccharide (LPS) was used in in vitro to polarize BV2 cell to M1 phenotype. Cell viability, cellular reactive oxygen species (ROS), Fe2+ concentration, and lipid peroxidation levels, ferroptosis-associated proteins and mRNA, morphological of mitochondria were measured in vitro. Additionally, the AMP-activated protein kinase (AMPK)/mammalian/mechanistic target of rapamycin (mTOR) pathway were measured by western blot and immunofluorescence staining. The present in vivo results indicated that artesunate significantly ameliorated neurological deficits, haematoma volume and brain oedema in ICH rats. Besides, artesunate suppressed the M1-microglia relative inflammatory factors and up-regulated iron deposition. For in vitro, artesunate significantly selectively decreased the viability of LPS-stimulated BV2 cell. Furthermore, ROS and lipid peroxidation levels were up-regulated. And the glutathione peroxidase 4 (GPX4) were silenced via the AMPK/mTORC1 axis. Our finding supports that artesunate ameliorates the ICH secondary injury both in vitro and in vivo by inducing ferroptosis in microglia and further inhibiting inflammation mainly through the AMPK/mTORC1/GPX4 pathway. This finding may provide a novel target for ICH treatment.

Identification of vasospasm biomarkers for cerebral hemorrhage via bio-informatics analysis.

BACKGROUND: The incidence of cerebral hemorrhage has rapidly increased over time, and vascular dysfunction has a significant influence on the pathogenesis and outcome of these patients. This is also the case for vasospasm in cerebral hemorrhage, but there is no method to assess this. We conducted this study to find molecular biomarkers of vasospasm in cerebral hemorrhage patients. METHODS: Raw data of GSE37924 was downloaded from the Gene Expression Omnibus (GEO) database, including 66 samples with cerebral vasospasm and 62 samples without cerebral vasospasm. Differentially expressed genes (DEGs) between samples with cerebral vasospasm and those without cerebral vasospasm were analyzed using the limma package in R software. To determine the functions of DEGs, we conducted functional enrichment analysis of DEGs through the clusterProfiler package in R. The protein-protein interaction (PPI) network of DEGs was constructed through STRING (https://string-db.org/) and generated via Cytoscape software. To understand the correlation between DEGs and immune-related genes, immune-related cerebral vasospasm genes were obtained via intersecting immune-related genes and cerebral vasospasm DEGs. We also compared the infiltration of 28 immune cells between cases with cerebral vasospasm and those without cerebral vasospasm. Finally, we constructed a model to perform the validation experiments. RESULTS: Of the DEGs, there were 24 upregulated and 21 downregulated genes in the vasospasm samples compared to the no-vasospasm samples. Functional enrichment analysis showed that these genes play key roles in several biological processes and signaling pathways such as the bone morphogenetic protein (BMP) signaling pathway, cellular response to BMP stimulus, natural killer cell chemotaxis, negative regulation of transmembrane receptor protein serine/threonine kinase signaling pathway, MHC protein complex binding, and receptor ligand activity, among others. CCL4, HLA-DQA1, IGF2, NTS, and so on were the significant immune-related genes. Furthermore, the immune cell infiltration results showed that there were differences between patients with vasospasm and those without vasospasm. Finally, we found that CCL4 had significantly higher expression in patients with vasospasm than those without vasospasm. CONCLUSIONS: CCL4 is an important regulator of vascular dysfunction in cerebral hemorrhage.