The modification of methyltransferase-like (METTL) enzymes plays important roles in various cellular responses by regulating microRNA expression. However, how m6A modification is involved in stress granule (SG) formation in the early stage of acute ischemic stroke by affecting the biogenesis processing of microRNAs remains unclear. Here, we established a middle cerebral artery occlusion (MCAO) model in rats and an oxygen-glucose deprivation/reperfusion (OGD/R) model in primary cortical neurons and PC12 cells to explore the potential mechanism between m6A modification and SG formation. The in vivo results showed that the level of infarction and apoptosis increased while SG formation decreased significantly within the ischemic cortex with improved reperfusion time after 2 h of ischemia. Consistent with the in vivo data, an inverse association between the apoptosis level and SG formation was observed in PC12 cells during the reperfusion period after 6 h of OGD stimulation. Both in vivo and in vitro results showed that the expression of METTL3 protein, m6A and miR-335 was significantly decreased with the reperfusion period. Overexpression of the METTL3 and METTL3 gene-knockdown in PC12 cells were achieved via plasmid transfection and CRISPR-Cas9 technology, respectively. Overexpression or knockdown of METTL3 in oxygen-glucose deprivation of PC12 cells resulted in functional maturation of miR-335, SG formation and apoptosis levels. In addition, we found that miR-335 enhanced SG formation through degradation of the mRNA of the eukaryotic translation termination factor (Erf1). In conclusion, we found that METTL3-mediated m6A methylation increases the maturation of miR-335, which promotes SG formation and reduces the apoptosis level of injury neurons and cells, and provides a potential therapeutic strategy for AIS.
Objectives Wedelolactone exhibits regulatory effects on some inflammatory diseases. However, the anti‐inflammatory mechanism of wedelolactone has not been entirely unravelled. Therefore, the present study focuses on investigating the mechanism of wedelolactone on NLRP3 inflammasome in macrophages and its influence on MSU‐induced inflammation. Materials and Methods BMDM, J774A.1 and PMA‐differentiated THP‐1 macrophages were primed with LPS and then stimulated with ATP or nigericin or MSU crystal in the presence or absence of wedelolactone. The cell lysates and supernatants were collected to detect NLRP3 inflammasome components such as NLRP3, ASC and caspase 1, as well as pyroptosis and IL‐1β production. In addition, the anti‐inflammatory effects of wedelolactone on MSU‐induced peritonitis and arthritis mice were also evaluated. Results We found that wedelolactone broadly inhibited NLRP3 inflammasome activation and pyroptosis and IL‐1β secretion. Wedelolactone also block ASC oligomerization and speck formation. The inhibitory effects of wedelolactone were abrogated by PKA inhibitor H89, which also attenuated wedelolactone‐enhanced Ser/Thr phosphorylation of NLRP3 at PKA‐specific sites. Importantly, wedelolactone could abate MSU‐induced IL‐1β production and neutrophils migration into peritoneal cavity, and reduced caspase 1 (p20) and IL‐1β expression in the joint tissue of MSU‐induced arthritis. Conclusion Our results indicate that wedelolactone promotes the Ser/Thr phosphorylation of NLRP3 to inhibit inflammasome activation and pyroptosis partly through potentiating PKA signalling, thus identifying its potential use for treating MSU‐induced peritonitis and gouty arthritis.
Under harmful environmental conditions, stress granules (SGs), macromolecular aggregates that are associated with cell survival and death, are produced in the eukaryotic cytoplasm. However, whether and how microRNAs (miRNAs/miRs) modulate SG formation induced by acute ischemic stroke has not been investigated. In the present study, a rat model of middle cerebral artery occlusion (MCAO) was utilized and miRNA array profiling and reverse transcription-quantitative polymerase chain reaction were performed. The results revealed that miR-335 was downregulated during acute ischemic stroke, which was concomitant with reduced SG formation, enhanced apoptosis levels and increased Rho associated protein kinase 2 (ROCK2) expression. In the MCAO rat and serum-free cell models, miR-335 treatment upregulated SG formation, alleviated the ischemia-induced infarction, and decreased ROCK2 protein expression and apoptosis levels. By contrast, when compared with miR-335 treatment, the inhibition of miR-335 resulted in reduced SG formation and higher ROCK2 expression and apoptosis levels. Target prediction analysis and luciferase 3′-untranslated region reporter assay identified ROCK2 as the direct target of miR-335. Furthermore, ROCK2 silencing enhanced SG formation and attenuated the level of apoptosis in the serum-free cell model. In addition, ROCK2 silencing markedly inhibited the effect of miR-335 on SG formation and apoptosis levels. Unexpectedly, the phosphorylation of T-cell intracellular antigen-1 was significantly inhibited by miR-335 in the MCAO rat model, which provides a reasonable explanation for the promotional effect of miR-335 on SG formation by specifically targeting ROCK2. In conclusion, these results demonstrate that miR-335 promotes SG formation and inhibits apoptosis by reducing ROCK2 expression in acute ischemic stroke, which provides a possible therapeutic target for brain injury.
Parkinson's disease (PD) is a neurodegenerative disease characterized by the selective loss of dopaminergic neurons in the substantia nigra (SN). In a previous study, the authors demonstrated that ferritin heavy chain 1 (FTH1) inhibited ferroptosis in a model of 6-hydroxydopamine (6-OHDA)-induced PD. However, whether and how microRNAs (miRNAs/miRs) modulate FTH1 in PD ferroptosis is not yet well understood. In the present study, in vivo and in vitro models of PD induced by 6-OHDA were established. The results in vivo and in vitro revealed that the levels of the ferroptosis marker protein, glutathione peroxidase 4 (GPX4), and the PD marker protein, tyrosine hydroxylase (TH), were decreased in the model group, associated with a decreased FTH1 expression and the upregulation of miR-335. In both the in vivo and in vitro models, miR-335 mimic led to a lower FTH1 expression, exacerbated ferroptosis and an enhanced PD pathology. The luciferase 3′-untranslated region reporter results identified FTH1 as the direct target of miR-335. The silencing of FTH1 in 6-OHDA-stimulated cells enhanced the effects of miR-335 on ferroptosis and promoted PD pathology. Mechanistically, miR-335 enhanced ferroptosis through the degradation of FTH1 to increase iron release, lipid peroxidation and reactive oxygen species (ROS) accumulation, and to decrease mitochondrial membrane potential (MMP). On the whole, the findings of the present study reveal that miR-335 promotes ferroptosis by targeting FTH1 in in vitro and in vivo models of PD, providing a potential therapeutic target for the treatment of PD.
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