TMAO Induces Vascular Endothelial Cells Pyroptosis Through TET2-CYTB-ROS Pathway

The study was aimed at identifying that cytochrome b (CYTB) expression regulation by trimethylamine N-oxide (TMAO) can induce mitochondria reactive oxygen species (ROS) and promote vascular endothelial cells (VECs) pyroptosis.

VECs were transfected with TET methylcytosine dioxygenase 2 (TET2)/CYTB overexpression lentivirus, CYTB siRNA, TET2 shRNA, or NC. ROS levels were measured using MitoSOX Red fluorescence, and pyroptosis was evaluated via Hoechst 33342/PI staining. Western blot was used to measure TET2, the NOD-like receptor thermal protein domain associated protein 3 (NLRP3), proteolytic cleavage of gasdermin D (GSDMD), CYTB, and Caspase-1 expression. Interleukin (IL)-1β was quantified by ELISA. The mRNA expression of IL-1β, CYTB, ND2, and TET2 was measured by qRT-PCR. Cellular ultrastructure was examined by electron microscope, and calcium flux was monitored with Fluo-4AM. CYTB methylation was detected using Targeted Bisulfite Sequencing.

This study showed that TMAO can down-regulate the expression of CYTB inVECs, cause VECs pyroptosis and mitochondrial dysfunction (MDF). CYTB overexpression antagonized the effect of TMAO. Further, silencing CYTB promoted mtROS production, and MitoTEMPO, a ROS scavenger, inhibited VECs pyroptosis caused by CYTB silencing. In addition, TET2 had demethylation activity. The expression of CYTB was positively regulated by TET2. TMAO was able to inhibit the expression of TET2 and promote the methylation level of the CYTB gene promoter.

TMAO promotes the methylation level of the CYTB gene promoter and down-regulates the expression of CYTB by inhibiting the expression of TET2. The decreased expression level of CYTB induces ROS, promoting VECs pyroptosis.