Astragaloside IV alleviates neuronal ferroptosis in ischemic stroke by regulating fat mass and obesity-associated-N6-methyladenosine-acyl-CoA synthetase long-chain family member 4 axis.

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 N6 -methyladenosine (m6 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), Fe2+ , 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 N6 -methyladenosine RNA-binding protein 3 (Ythdf3)/Acsl4 and Atf3/Fto were analyzed by RNA-pull down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assay. Fto regulated the m6 A levels of Acsl4. Ythdf3 bound to Acsl4, and modulated its levels through m6 A modification. Atf3 bound to Fto and positively regulated its levels. Overall, As-IV promoted the transcription of Fto by upregulating Atf3, resulting in decreased m6 A levels of Acsl4, thus, improving neuronal injury in IS by inhibiting ferroptosis.

Psoralen inhibits the inflammatory response and mucus production in allergic rhinitis by inhibiting the activator protein 1 pathway and the downstream expression of cystatin­SN.

Psoralen (PSO) exerts anti­inflammatory pharmacological effects and plays an important role in a variety of inflammatory diseases. However, the effects of PSO with allergic rhinitis (AR) are yet to be reported. In the present study, an in vitro AR model was generated by inducing JME/CF15 human nasal epithelial cells with IL­13, after which MTT was used to assess the cytotoxicity of PSO. The expression levels of inflammatory cytokines (granulocyte­macrophage colony­stimulating factor and Eotaxin) were determined by ELISA. Furthermore, the expression of inflammatory IL­6 and ­8, as well as mucin 5AC, was assessed by reverse transcription­quantitative PCR and western blotting, and cellular reactive oxygen species were detected using a 2',7'­dichlorodihydrofluorescein diacetate fluorescent probe. Western blotting was also used to detect the expression and phosphorylation of c­Fos and c­Jun in the activator protein 1 (AP­1) pathway, as well as the expression of cystatin­SN (CST1). PSO inhibited the inflammatory response and mucus production in IL­13­induced JME/CF15 cells. Furthermore, the levels of c­Fos and c­Jun phosphorylation in the AP­1 pathway were decreased in IL­13­induced JME/CF15 cells following PSO treatment. The expression of pathway proteins was activated by the addition of PMA, an AP­1 pathway activator, which concurrently reversed the inhibitory effects of PSO on the inflammatory response and mucus formation. The addition of an AP­1 inhibitor (SP600125) further inhibited pathway activity, and IL­13­induced inflammation and mucus formation was restored. In conclusion, PSO regulates the expression of CST1 by inhibiting the AP­1 pathway, thus suppressing the IL­13­induced inflammatory response and mucus production in nasal mucosal epithelial cells.