The Role Of TET2/SDHB In The Low Shear Stress Induced Pyroptosis Of HUVECs And Its Mechanism

[Background] Inflammation of Vascular endothelial cells(VECs)induced by low shear stress plays a key role in the initiation and progression of atherosclerosis(As),but its underlying regulatory mechanism is still unclear.Recent studies have shown that pyroptosis,an inflammatory programmed form of cell death,which is closely related to the occurrence and development of As.Our previous studies have shown that Tet methylcytidine dioxygenase 2(TET2)is a shear stress sensitive gene,low shear stress down-regulates the expression of TET2,and TET2 shRNA up-regulates the expression of Succinic acid dehydrogenase complex B(SDHB)in vascular endothelial cells.Here,we will explore the role and mechanism of TET2/SDHB in low shear stress-induced VEC pyroptosis.[Objective] To explore the regulation mechanism of VEC pyroptosis induced by low shear stress.[Methods] 1.The expression levels of pyroptosis proteins NLRP3,GSDMD,Caspase-1,IL-18,IL-1?,TET2 and SDHB in human umbilical vein endothelial cells(HUVECs)under low shear stress and physiological shear stress were observed using a parallel plate flow chamber system.2.Four HUVECs cell lines were constructed by lentivirus transfection technology,including HUVECs cell lines transfected with low TET2 expression,HUVECs cell lines with high TET2 expression,HUVECs cell lines with high SDHB expression and HUVECs with low SDHB expression.3.Western blot was used to detect the effects of TET2 shRNA and SDHB overexpression on the expression of pyroptosis-related proteins NLRP3,GSDMD,Caspase-1,IL-18 and IL-1?.4.The protein expression levels of SDHB in TET2 shRNA or TET2 overexpression HUVECs was detected by western blot,and the activity of SDH was detected by succinic acid dehydrogenase kit.5.Western blot was used to detect the expressions of NLRP3,GSDMD,Caspase-1,IL-18 and IL-1? proteins in TET2 overexpression or SDHB shRNA-treated HUVECs under low shear stress.6.The effects of SDHB overexpression on mitochondrial morphology and function of HUVECs were detected by transmission electron microscopy,Flow cytometry,ATP assay kit and reactive oxygen species(ROS)fluorescence probe.7.ROS fluorescence probes were used to detect the effect of ROS scavenging agent N-acetylcystine(NAC)on ROS levels in SDHB overexpression HUVECs,and Western blot was used to detect the expression levels of pyroptosis-related proteins NLRP3,GSDMD,Caspase-1,IL-18 and IL-1? in SDHB overexpression HUVECs.8.The effect of Histone deacetylase2(HDAC2)inhibitor(MS-275)on the expression of HDAC2 protein in VECs was detected by Western blot.Western blot was used to detect the influence of MS-275 on the expression of HUVECs SDHB overexpressing TET2,the ROS fluorescence probe was used to detect the level of mitochondrial ROS,and the western blot was used to detect the influence of pyroptosis-related proteins NLRP3,GSDMD,Caspase-1,IL-18 and IL-1? in VECs.[Results] Low-shear stress up-regulated the expression of NLRP3,GSDMD,Caspase-1,IL-18,IL-1? and SDHB,and down-regulated the expression of TET2.The results of fluorescence microscope and Western blot indicated that HUVECs with low expression of TET2,high expression of TET2,low expression of SDHB and high expression of SDHB were successfully constructed.Western blot results showed that the expression levels of NLRP3,GSDMD,Caspase-1,IL-18 and IL-1? of HUVECs pyroptosis related proteins were up-regulated in TET2 shRNA group.Moreover,the low-shear stress-induced HUVECs pyroptosis was significantly inhibited by TET2 overexpression.TET2 shRNA upregulated SDHB expression and increased SDH activity.TET2 overexpression down-regulated SDHB expression and decreased SDH activity.the expression of pyroptosis-related proteins NLRP3,GSDMD,Caspase-1,IL-18 and IL-1? was up-regulated by SDHB overexpression.The expression of low-shear stress-induced HUVECs pyroptosis was inhibited by SDHB shRNA.Transmission electron microscopy showed that mitochondria in SDHB overexpression group characterized with decreased number,the increased volume,the shortened mitochondrial ridge,and the increased focal vacuoles.Flow cytometry showed that most cells were distributed in Q3 region and the proportion of Q2/Q3 was decreased,which showed that the mitochondrial membrane potential of HUVECs in SDHB overexpression group was abnormal.The ROS fluorescence probe test results showed that the ROS level of SDHB overexpression group was significantly increased than that of control group.ROS production was the ROS scavenger NAC inhibited the ROS increase induced by SDHB overexpression.Western blot results showed the expression levels of pyroptosis-related proteins NLRP3,GSDMD,Caspase-1,IL-18 and IL-1? were significantly down-regulated were decreased by NAC in SDHB overexpression group.MS-275 significantly inhibited the expression of HDAC2 in vascular endothelial cells,but overexpression of TET2 had no significant effect on the expression of HDAC2 protein.Compared with the overexpressed TET2 group,MS-275 upregulated the expression levels of HUVECs SDHB and pyroptosisrelated proteins NLRP3,GSDMD,Caspase-1,IL-18 and IL-1?.[Conclusion] Low shear stress down-regulated the expression of TET2,activated the SDHB/ROS pathway,and caused the pyroptosis of VECs.