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

Abstract

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), Fe 2+ 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 Guanfeng Xie and Yubing Liang contributed equally to this work.

FERREG: ferroptosis-based regulation of disease occurrence, progression and therapeutic response

“…Advancements in ferroptosis research have led to the discovery of a series of approved drugs (e.g. sulfasalazine [ 69 ], artesunate [ 70 ], sorafenib [ 71 ], acetaminophen [ 72 ]) that could induce ferroptosis. Moreover, there is an increasing emphasis on utilizing ferroptosis targets and regulators as the basis for large-scale screening of small-molecule drugs or developing larger-molecule drugs, such as specific antibodies.…”

Artemisinin and its derivatives as promising therapies for autoimmune diseases

“…Similar to artemisinin, artesunate (a culture containing 0–60 μM artesunate for 24 h) inhibited the expression of TLR4 and MyD88, and the activation of NF-κB via blocking the degradation of IκB, suggested that artesunate might inhibit the production of pro-inflammatory mediators by suppressing TLR4/MyD88/NF-κB signaling pathway [ 84 ]. What's more, artesunate ameliorated intracerebral haemorrhage (ICH) secondary injury in vitro (at 2, 5, 10, and 15 μM was added 24 h after Hb treatment) and in vivo (rats received artesunate at 20, 50 or 70 mg/kg/day via i. p. injection for 3 consecutive days before ICH induction) by inducing microglial cell iron death and further inhibiting inflammation mainly through the AMPK/mTORC1/GPX4 pathway [ 85 ]. This finding may provide a new target for the treatment of ICH.…”

Recent advances in potential therapeutic targets of ferroptosis‑associated pathways for the treatment of stroke (Review)

“…AMPK is an energy sensor widely present in various tissues and cells. For instance, when it is present in brain tissue and microglial cells, it can alleviate secondary damage from cerebral haemorrhage ( 92 ). It regulates cellular energy metabolism balance ( 93 ), and participates in the regulation of multiple biological processes, including glycogen synthesis ( 94 ), fatty acid synthesis ( 95 ) and mitochondrial biogenesis ( 96 ).…”

“…In vitro experiments showed a phosphorylation reaction of AMPK after initial intervention with AMPK inhibitors in BV2 cells. Subsequently, pharmacological intervention led to upregulation of ROS and lipid peroxidation levels (positive regulators of ferroptosis) in BV2 cells, and silenced GPX4 (a key negative regulator of ferroptosis) levels through the AMPK signaling pathway ( 92 ). In in vivo experiments, iron deposition at the site of brain injury was observed through Perl's staining ( 92 ).…”

“…Subsequently, pharmacological intervention led to upregulation of ROS and lipid peroxidation levels (positive regulators of ferroptosis) in BV2 cells, and silenced GPX4 (a key negative regulator of ferroptosis) levels through the AMPK signaling pathway ( 92 ). In in vivo experiments, iron deposition at the site of brain injury was observed through Perl's staining ( 92 ). So, both in vitro and in vivo experiments have demonstrated the occurrence of ferroptosis during cerebral hemorrhage.…”