GALNT6-mediated O-glycosylation on PTGS2 regulates microglia inflammation and ferroptosis following intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a common type of stroke characterized by high mortality and disability rates. Polypeptide N-acetylgalactosaminyl transferase-6 (GALNT6), which can catalyze the O-GalNAc glycosylation, is related to the development of various diseases. Nevertheless, the action of GALNT6 on ICH pathogenesis remains elusive. Hence, this study clarified the role of GALNT6 in ICH pathogenesis and investigated the possible underlying mechanism. The in vivo ICH model was created using collagenase in mice. BV2 microglial cells were subjected to oxygen and glucose deprivation combined with hemin (OGD/H) to create the in vitro ICH model. Neurological dysfunctions were evaluated using the neurological deficit score and the Morris water maze test. CD86, CD206, and Iba1 expression and reactive oxygen species (ROS) production in ICH model mice were measured by immunofluorescence and dihydroethidium staining, respectively. Cell viability was tested utilizing the Cell Counting Kit-8 method. The CD86+CD11b+, CD206 +CD11b+, and lipid ROS levels were assessed using flow cytometry. The secretion levels of inflammation-related factors were measured utilizing the enzyme-linked immunosorbent assay. The binding of GALNT6 to PTGS2 was determined using the co-immunoprecipitation analysis. The O-GalNAc glycosylation of PTGS2 was defined utilizing the lectin pull-down assay. The binding of HIF-1α to the Galnt6 promoter was measured using ChIP-PCR and reporter gene assays. GALNT6 was elevated in ICH mice, and Galnt6 knockdown attenuated spatial memory impairment, inflammation, oxidative stress, and ferroptosis in mice with ICH. Besides, Galnt6 knockdown inhibited OGD/H-induced inflammation and ferroptosis in BV2 microglial cells, while its overexpression induced M1 polarization and inflammation in BV2 microglial cells via ferroptosis. Furthermore, GALNT6 induced O-GalNAc glycosylation of PTGS2, and Galnt6 knockdown inhibited OGD/H-induced M1 polarization, inflammation, and ferroptosis in BV2 microglial cells via PTGS2 inhibition. Moreover, HIF-1α could bind to the Galnt6 promoter and promote Galnt6 transcription. The HIF-1α-GALNT6-PTGS2 axis regulated BV2 microglial polarization, inflammation, and ferroptosis. Together, Galnt6 was transcriptionally regulated by HIF-1α and presented high expression in ICH. GALNT6 induced O-GalNAc glycosylation on PTGS2, and Galnt6 knockdown inhibited inflammation and ferroptosis in ICH via PTGS2 inhibition. Therefore, GALNT6 is a potential target for ICH management.