Intracerebral haemorrhage (ICH) is a devastating subtype of stroke with high morbidity and mortality. It has been reported that paeonol (PAN) inhibits the progression of ICH. However, the mechanism by which paeonol mediates the progression of ICH remains unclear. To mimic ICH in vitro, neuronal cells were treated with hemin. An in vivo model of ICH was established to detect the effect of paeonol on ferroptosis in neurons during ICH. Cell viability was tested by MTT assay. Furthermore, cell injury was detected by GSH, MDA and ROS assays. Ferroptosis was examined by iron assay. RT-qPCR and western blotting were used to detect gene and protein expression, respectively. The correlation among HOTAIR, UPF1 and ACSL4 was explored by FISH, RNA pull-down and RIP assays. Paeonol significantly inhibited the ferroptosis of neurons in ICH mice. In addition, paeonol significantly reversed hemin-induced injury and ferroptosis in neurons, while this phenomenon was notably reversed by HOTAIR overexpression. Moreover, paeonol notably inhibited ferroptosis in hemin-treated neuronal cells via inhibition of ACSL4. Additionally, HOTAIR bound to UPF1, and UPF1 promoted the degradation of ACSL4 by binding to ACSL4. Furthermore, HOTAIR overexpression reversed paeonol-induced inhibition of ferroptosis by mediating the UPF1/ACSL4 axis. Paeonol inhibits the progression of ICH by mediating the HOTAIR/UPF1/ACSL4 axis. Therefore, paeonol might serve as a new agent for the treatment of ICH.

Ischemic stroke (IS) is a detrimental neurological disease with limited treatment options. Astragaloside IV (As-IV) was a promising bioactive constituent in the treatment of IS. However, the functional mechanism remains unclear. Here, IS cell and mouse models were established by oxygen glucose deprivation/re-oxygenation (OGD/R) and middle cerebral artery occlusion (MCAO). Quantitative reverse transcription PCR (RT-qPCR), Western blotting, or Immunofluorescence staining measured related gene and protein expression of cells or mice brain tissues, and the results revealed altered expression of acyl-CoA synthetase long-chain family member 4 (Acsl4), fat mass and obesity-associated (Fto), and activation transcription factor 3 (Atf3) after treatment with As-IV. Then, increased N 6 -methyladenosine (m 6 A) levels caused OGD/R or MCAO were reduced by As-IV according to the data from methylated RNA immunoprecipitation (MeRIP)-qPCR and dot blot assays. Moreover, through a series of functional experiments such as observing mitochondrial changes under transmission electron microscopy (TEM), evaluating cell viability by cell counting kit-8 (CCK-8), analyzing infract area of brain tissues by 2,3,5-triphenyltetrazolium chloride (TTC) staining, measuring levels of malondialdehyde (MDA), lactate dehydrogenase (LDH), Fe 2+ , solute carrier family 7 member 11 (Slc7a11) and glutathione peroxidase 4 (Gpx4) and concentration of glutathione (GSH), we found that Fto knockdown, Acsl4 overexpression or Atf3 knockdown promoted the viability of OGD/R cells, inhibited cell ferroptosis, reduced infract size, while As-IV treatment or Fto overexpression reversed these changes. In mechanism, the interplays of YTH N 6 -methyladenosine RNAbinding protein 3 (Ythdf3)/Acsl4 and Atf3/Fto were analyzed by RNA-pull down, RNA Zhenglong Jin and Wenying Gao are co-first authors.

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BackgroundIschemic stroke (IS) is a detrimental neurological disease with limited treatments options. Astragaloside IV (As-IV) was a promising bioactive constituent in the treatment of IS. However, the functional mechanism remains unclear. MethodsIS cell and mice models were established by oxygen glucose deprivation /re-oxygenation (OGD/R) and middle cerebral artery occlusion (MCAO). RT-qPCR, Western blotting or Immuno uorescence staining were used to measure the gene expression. The N6-methyladenosine (m6A) levels were measured by MeRIP-qPCR and dot blot assays. CCK-8 and TUNEL staining were used to evaluate cell viability and apoptosis. TTC staining analyzed the infract area of brain tissues. The interplays of YTHDF3/ACSL4 and ATF3/FTO were analyzed by RNA-pull down, RIP, ChIP and dual-luciferase reporter assay. ResultsAs-IV treatment promoted HT-22 and Neuro-2 cell viability and upregulated FTO levels in vitro and in vivo, as well as inhibited the levels of MDA, LDH, Fe2 + and ACSL4, while promoted the expression of GSH, SCL7A11 and GPX4. Knockdown of FTO, or overexpression of ACSL4 increased the infract size of brain tissues, neuron damage and the levels of MDA, LDH and Fe2+, while As-IV treatment reversed these changes. FTO regulated the m6A levels of ACSL4. YTHDF3 bound to ACSL4, and modulated its levels through m6A modi cation. ATF3 bound to FTO and positively regulated its levels. Knockdown of FTO or ATF3 increased the apoptosis of OGD/R cells, and promoted MDA, LDH and Fe2 + levels, while inhibited GSH expression. Knockdown of ACSL4, overexpression of FTO or treatment with As-IV reversed these effects. ConclusionAs-IV promoted the transcription of FTO by upregulating ATF3, resulting in the decreased m6A levels of ACSL4, thus improving neuronal injury in IS by inhibiting ferroptosis.