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Abstract Vascular endothelial injury initiates atherosclerosis (AS) progression. N-Acetylneuraminic acid (Neu5Ac) metabolic disorder was found to intensify endothelial mitochondrial damage. And GLS2-associated glutaminolysis disorder contributed to mitochondrial dysfunction. However, mechanisms underlying Neu5Ac-associated mitochondrial dysfunction as well as its association with GLS2 remains unclear. In this study, we constructed GLS2−/−ApoE−/− mice by using HBLV-GLS2 shRNA injection. And methods like immunofluorescence, western blotting, transmission electron microscopy were applied to detect profiles of endothelial injury and AS progression both in vivo and in vitro. We demonstrated that Neu5Ac accumulation increased GLS2 expression and promoted glutaminolysis disorder, which further induced endothelial mitochondrial dysfunction via a pyroptosis-dependent pathway in vivo and in vitro. Mechanically, Neu5Ac interacted with SIRT3 and led to FOXO3a deacetylation and phosphorylation, further facilitated c-Myc antagonism and ultimately increased GLS2 levels. Inhibition of GLS2 could improve mitochondrial function and mitigate pyroptosis process. In addition, blocking Neu5Ac production using neuraminidases (NEUs) inhibitor could rescue endothelial damage and alleviate AS development in ApoE−/− mice. These findings proposed that Neu5Ac induced GLS2-mediated glutaminolysis disorder and then promoted mitochondrial dysfunction in a pyroptosis-dependent pathway. Targeting GLS2 or inhibiting Neu5Ac production could prevent AS progression.

Tetramethylpyrazine (TMP), a Chinese traditional herbal extraction widely used in treating cardiovascular diseases, could attenuate vascular endothelial injuries, but the underlying mechanism remains incomprehensive. Vascular glycocalyx coating on the endothelium would be damaged and caused endothelial dysfunction in the inflammatory microenvironment, which was the initial factor of morbidity of many vascular diseases, such as atherosclerosis (AS). Here, we thoroughly investigated the molecular mechanism of TMP on vascular endothelial glycocalyx in the LPS-induced inflammatory model both in vitro and in vivo. Results showed that pretreatment with TMP significantly inhibited glycocalyx degradation and monocytes adhesion to the endothelial process. Moreover, TMP pretreatment inhibited the expression of HPSE1 (a major degrading enzyme of endothelial glycocalyx), Toll-like receptor 4 (TLR4), and the translocation of nuclear factor kappa B p65 (NF-κB p65). We were utilized withTLR4 siRNA, NF-κB inhibitor, and HPSE1 overexpression analysis confirmed TMP’s protection on endothelial glycocalyx injury, which further contributed to the monocyte-endothelial adhesion process. It was indicated that TMP might suppress glycocalyx degradation through TLR4/NF-κB/HPSE1 signaling pathway. Taken together, our results enriched the occurrence molecular mechanism of glycocalyx shedding and molecular regulation mechanism of TMP in protecting integrity of the glycocalyx structure during inflammation. As TMP is currently used in clinical applications, it may be considered a novel strategy against atherosclerosis through its ability to protect endothelial glycocalyx.