The METTL3/m6A Reader Protein YTHDF1 Regulates Endothelial Cell Pyroptosis by Enhancing NLRP3 Expression to Affect Soft Tissue Injury

Background: Pyroptosis is inflammation-associated programmed cell death triggered by activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, which plays a crucial role in acute soft tissue injury (ASTI). This study aimed to explore whether methyltransferase-like 3 (METTL3) can regulate NLRP3 expression through N6-methyladenosine (m6A) modification to mediate endothelial cell pyroptosis and thus affect soft tissue injury.

Methods: An experimental ASTI rat model was created by inducing muscle injury through striking the rat muscle. In vitro, an ASTI cell model was established using human umbilical vein endothelial cells (HUVECs) stimulated with lipopolysaccharide (LPS) and ATP. The severity of ASTI in rats was evaluated using H&E staining. To assess protein levels, Western blot and Immunohistochemistry (IHC) analyses were performed, focusing on METTL3, pyroptosis-associated proteins, and m6A reader proteins. Immunofluorescence (IF) assay was conducted to examine the expression of NLRP3 and CD31. The levels of inflammatory cytokines were measured using an ELISA assay, while flow cytometry was used to detect levels of ROS and cellular pyroptosis. The m6A levels in cells were analyzed by RNA m6A colorimetry. The interactions between METTL3 and NLRP3, and YTHDF1 and NLRP3 were analyzed using RIP and RNA pull-down assays, respectively.

Results: METTL3 and YTHDF1 were significantly upregulated in ASTI rats and LPS-ATP-induced HUVECs. Knockdown of METTL3 ameliorated ASTI and inhibited cellular pyroptosis. Knockdown of METTL3 reduced the levels of total m6A and NLRP3 m6A in HUVECs and suppressed NLRP3 expression. Meanwhile, knockdown of YTHDF1 decreased NLRP3 protein expression without affecting NLRP3 mRNA levels. In addition, overexpression of NLRP3 was able to reverse the effect of METTL3 on LPS-ATP-induced endothelial cell pyroptosis.

Conclusion: The METTL3/m6A reader protein YTHDF1 regulates endothelial cell pyroptosis by enhancing NLRP3 expression to affect soft tissue injury.