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

Pyroptosis is a kind of programmed cell death characterized by pro-inflammatory,which can promote atherosclerosis（As）.In our previous study,we found that Trimethylamine N-oxide（TMAO）,a metabolite of intestinal flora,promotes the pyroptosis of vascular endothelial cells（VECs）by inducing reactive oxygen species（ROS）.However,the mechanism of ROS induction is not clear.ROS is mainly produced by mitochondria,and mitochondrial electron transport chain is the main place where mitochondrial reactive oxygen species（mtROS）are produced.Cytochrome b（CYTB）is encoded by mitochondrial DNA（mtDNA）and is a component of mitochondrial electron transport chain complex III.We speculate that TMAO may induce mtROS to promote VECs pyroptosis by regulating the expression level of CYTB.This study showed that TMAO can down-regulate the expression of CYTB in human umbilical vein endothelial cells（HUVECs）,cause HUVECs pyroptosis and Mitochondrial dysfunction（MDF）,Overexpression of CYTB antagonizes the effect of TMAO.Further,silencing CYTB promotes mtROS production,and mtROS scavenger MitoTEMPO can inhibit HUVECs pyroptosis caused by CYTB silencing.In addition,TET methylcytosine dioxygenase 2（TET2）has demethylation activity.It was found that the expression of CYTB was positively regulated by TET2.Overexpression of TET2 can increase the level of CYTB protein,but decrease it when TET2 is interrupted.And and TMAO inhibited the expression of TET2 and promoted the methylation level of CYTB gene promoter.In summary,TMAO induces HUVECs pyroptosis through the TET2–CYTB–mtROS pathway.[Objective]To investigate the role and mechanism of CYTB in TMAO-induced pyroptosis in HUVECs.[Methods]1.The effect of TMAO on pyroptosis of cells was detected.Cultivate HUVECs,after incubation with 600μmol/L TMAO for 24 hours,the expression of pyroptosis related protein and the level of pro-inflammatory cytokines were detected,the percentage of PI positive staining was detected by Hoechst 33342/PI staining,and the rupture of VECs membrane was detected by scanning electron microscope.2.The effect of TMAO on mitochondrial function was detected.The protein expression of Nuclear Respiratory Factor 1（NRF1）,nuclear respiratory factor 2（NRF2）and Peroxisome proliferator-activated receptor-γcoactivator-1α（PGC-1α）,and the expression of NADH dehydrogenase subunit 2（ND2）at gene level were detected.The function of mitochondria was detected by ROS probe（MitoSOX）and Flou-4AM.The morphology of mitochondria was observed by transmission electron microscope.3.The effects of CYTB on pyroptosis and mitochondrial function were detected.We transfected siRNA CYTB and overexpressed lentivirus CYTB respectively,knocked down or overexpressed CYTB,detected the transfection efficiency by Western blot and RT-qPCR,and detected the expression of pyroptosis proteins in endothelial cells after transfection by Western blot.4.Detect the regulatory effect of TET2 on CYTB expression.The CpG island of CYTB gene promoter was analyzed by bioinformatics,and the promoter region of CYTB gene was methylated by TBS analysis.Lentiviral vectors with low and high expression of TET2 were constructed and transfected into VECs,Western blot to detect the changes of TET2-CYTB pathway and the effect of TMAO on this pathway.[Results]1.After TMAO treatment,the expression of NLRP3,GSDMD,GSDMD-N,Pro-caspase-1,Caspase-1 HUVECs pyroptosis related proteins detected by Western blot and proinflammatory interleukin-1β（IL-1β）by ELISA increased significantly,and the percentage of PI positive staining increased after TMAO treatment.Scanning electron microscope showed that VECs membrane ruptured and membrane pores appeared on the cell membrane.2.The results of RT-qPCR and Western blot showed that TMAO could reduce the level of mitochondrial gene level in HUVECs.MitoSOX detection and Flou-4AM results showed that mtROS in HUVECs increased and Ca²⁺influx decreased under TMAO conditions,and transmission electron microscopy showed abnormal morphological changes of mitochondria in HUVECs,such as crest shortening and vesicle formation.3.Western Blot and RT-qPCR showed that silencing CYTB or overexpressing CYTB was effective.The expression levels of pyroptosis marker protein and IL-1βwere increased after CYTBsiRNA.But it could be inhibited by mtROS scavenger（MitoTEMPO）.After pretreatment with MitoTEMPO,the mRNA expression of IL-1β,intracellular calcium and the percentage of PI positive cells in HUVECs transfected with CYTBsiRNA were decreased,while the mRNA expression of CYTB and ND2 were increased.Western blot detection showed that overexpression of CYTB could inhibit the expression of pyroptosis-related proteins and the level of IL-1βwhich promoted by TMAO.The percentage of PI positive cells in HUVECs,calcium level,ND2 at mRNA level and mtROS level could be inhibited by overexpressed CYTB.4.Western blot and RT-qPCR found that the expression of CYTB and TET2 in HUVECs decreased under TMAO condition.Western blot and RT-qPCR detected the expression level of CYTB in the Overexpression/interrupted TET2,and found that the expression of CYTB decreased when TET2 interrupted,and the expression of CYTB increased when TET2 overexpression.TBS Methylation sequencing analysis showed that the total average methylation level of 15 CpG gene promoter regions in the TMAO group was significantly higher than that in the control group,and the methylation level in the promoter region of CpG site 6（14804 site）in the TMAO group was significantly higher than that in the control group,indicating that the mitochondrial gene CYTB was regulated by methylation.[Conclusion]1.TMAO induced HUVECs pyroptosis,but was inhibited by overexpression of CYTB and TET2.2.TMAO down-regulated the expression of CYTB and TET2,induced mtROS,TET2 to positively regulate the expression of CYTB.3.Silencing of CYTB gene promoted HUVECs mitochondrial dysfunction and pyroptosis,but was inhibited by mtROS scavenger MitoTEMPO.4.TMAO promoted CYTB gene promoter methylation.