Biochemical and biophysical research communications · 2025
High glucose-induced alternative splicing of MEF2D in macrophages promotes vascular chronic inflammation in type 2 diabetes mellitus by mediating M1 macrophage polarization.
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He Yuqi, Luo Jie, Xie Haitao
Аннотация
OBJECTIVES: To investigate the effects of a high-glucose environment in type 2 diabetes mellitus (T2DM) on Myocyte enhancer factor 2d (MEF2D) selective splicing and its impact on the disease process and mechanism.
METHODS: Human monocyte (THP-1) cells were induced into macrophages with phorbol 12-myristate 13-acetate (PMA), and treated with high glucose for 24 h. PCR confirmed MEF2D splicing products. MEF2D or MEF2D-AS overexpression vectors were transfected into macrophages, and ELISA detected inflammatory factors; flow cytometry analyzed MI/M2 phenotypes; and levels of LC3, PI3K, and LAMP2 were measured. Autophagic flux detection; co-immunoprecipitation detected MEF2D and KCNMA1 interaction; WB and RT-qPCR assessed KCNMA1 expression. Macrophages co-cultured with endothelial cells were analyzed by ELISA for vascular inflammation factors MMP-9, Cys-C, and hsCRP.
RESULTS: High glucose-induced alternative splicing of MEF2D at 86-132 aa. MEF2D-AS group showed higher inflammatory factors, increased M1 phenotype, lower autophagy gene expression, and higher vascular inflammation factors compared to MEF2D group. Autophagy activator Rapamycin or KCNMA1 overexpression reversed these effects. MEF2D targeted KCNMA1, and MEF2D-AS overexpression led to decreased KCNMA1, increased inflammatory factors, M1 polarization, autophagy inhibition, and higher vascular inflammation factors.
CONCLUSION: High glucose induces MEF2D alternative splicing in macrophages, inhibiting autophagy and promoting M1 polarization via KCNMA1 down-regulation, thus promoting chronic inflammation in T2DM vessels.
METHODS: Human monocyte (THP-1) cells were induced into macrophages with phorbol 12-myristate 13-acetate (PMA), and treated with high glucose for 24 h. PCR confirmed MEF2D splicing products. MEF2D or MEF2D-AS overexpression vectors were transfected into macrophages, and ELISA detected inflammatory factors; flow cytometry analyzed MI/M2 phenotypes; and levels of LC3, PI3K, and LAMP2 were measured. Autophagic flux detection; co-immunoprecipitation detected MEF2D and KCNMA1 interaction; WB and RT-qPCR assessed KCNMA1 expression. Macrophages co-cultured with endothelial cells were analyzed by ELISA for vascular inflammation factors MMP-9, Cys-C, and hsCRP.
RESULTS: High glucose-induced alternative splicing of MEF2D at 86-132 aa. MEF2D-AS group showed higher inflammatory factors, increased M1 phenotype, lower autophagy gene expression, and higher vascular inflammation factors compared to MEF2D group. Autophagy activator Rapamycin or KCNMA1 overexpression reversed these effects. MEF2D targeted KCNMA1, and MEF2D-AS overexpression led to decreased KCNMA1, increased inflammatory factors, M1 polarization, autophagy inhibition, and higher vascular inflammation factors.
CONCLUSION: High glucose induces MEF2D alternative splicing in macrophages, inhibiting autophagy and promoting M1 polarization via KCNMA1 down-regulation, thus promoting chronic inflammation in T2DM vessels.