Ceramide Promotes Vascular Endothelial Cell Pyroptosis Through TXNIP-NLRP3-GSDMD Pathway

Background:Vascular endothelial cell injury is a significant pathological feature of many diseases.Pyroptosis of endothelial cells is a new cause of endothelial dysfunction in multiple diseases.Ceramide acts as a potential bioactive mediator of inflammation and increases endothelial vascular permeability in sepsis 、 acute lung injury、 acute myocardial infarction and other diseases.Ceramides are also involved in a variety of biological effects,such as cell apoptosis,proliferation,differentiation,etc.However,it is not yet known whether it can aggravate vascular endothelial injury by inducing cell pyroptosis.This study investigated whether ceramide could induce pyroptosis and its possible underlying mechanism.Methods:1.Human umbilical vein endothelial cells（HUVECs）were exposed to various concentrations of ceramide（0、10、20、30、40、50、60 μmol/L）for 12 h,24 h,48 h.The cell survival rate was measured using the cell counting kit-8 assay.2.After treating human umbilical vein endothelial cells with different concentrations of verapamil（0,10,20,40,60,80,100 μmol/L）for 12 h,the cell viability was determined using CCK-8 kit,so as to screen out the appropriate concentration of verapamil for subsequent experiments.3.Cells were divided into control,control si-RNA and TXNIP si RNA groups,and the effect of si-RNA on TXNIP expression was detected by reverse transcription-polymerase chain reaction（RT-PCR）and western blot（WB）.4.The cells were divided into 0 μmol/L ceramide,5 μmol/L ceramide,10 μmol/L ceramide,20μmol/L ceramide,control,20 μmol/L ceramide,20 μmol/L ceramide + 20μmol/L verapamil,20 μmol/L verapamil group and 20 μmol/L ceramide +TXNIP si RNA.Cells in different groups were then treated for 12 hours.Then the expressions of TXNIP,NLRP3,Caspase-1 and GSDMD genes were detected by reverse transcription-polymerase chain reaction technology,and the expressions of TXNIP,NLRP3,Caspase-1,Cas P1-P20,GSDMD and GSDMD-NT proteins were detected by western bloting.5.The cells were seeded in 6-well plates at appropriate density and the supernatant was collected after drugs treatment.LDH release level and IL-1β and IL-18 level in supernatant were detected,and PI positive cells were detected by flow cytometry and Hoechst33342/ PI double staining kit.6.Cells in different groups were treated accordingly,caspase-1 activity was detected by Caspase-1 activity detection kit,and endothelial cell permeability was detected by FITC method.Results:1.The viability of HUVECs decreased gradually with the increase in ceramide concentration and time.When the concentration of ceramide was 20 μmol/L and treated for 12 hours,the cell viability was about 65% compared with the control group.Therefore,the concentration of ceramide was 20 μmol/L and the treatment time was 12 hours as the best experimental conditions.Similarly,when the concentration of verapamil was 0-20 μmol/L,there was no significant effect on cell viability,so 20μmol/L verapamil was selected as the subsequent pretreatment concentration.2.Si-RNA reduced the expression of TXNIP gene and protein.3.With the increase of the concentration of ceramide treatment,the expressions of TXNIP,NLRP3,Caspase-1 and GSDMD increased（P<0.05）.Meanwhile,the protein expression levels of TXNIP,NLRP3,Caspase-1,Casp1 P20,GSDMD and GSDMD-NT were up-regulated（P<0.05）.When verapamil or si-TXNIP was co-incubated with ceramide,compared with the ceramide group,the gene expression levels of TXNIP,NLRP3,Caspase-1 and GSDMD were down-regulated（P<0.05）.Meanwhile,the protein expression levels of TXNIP,NLRP3,Caspase-1,Casp1 P20,GSDMD and GSDMD-NT decreased（P<0.05）.4.With the increase of ceramide concentration,The release of LDH,IL-1β and IL-18 in the supernatant increased and PI positive staining cells upregulated（P<0.05）.However,when given verapamil or si-TXNIP,the level of LDH,IL-1β and IL-18 in the supernatant decreased（P<0.05）.Flow cytometry and Hoechst33342/ PI staining also appeared a decrease in the number of positive staining cells（P<0.05）.5.After treatment with different concentrations of ceramide,the activity of caspase-1also increased gradually（P<0.05）,and the permeability coefficient percentage of endothelial cells increased gradually（P<0.05）.while,compared with the ceramide-treated group,verapamil or si-TXNIP decreased the activity of caspase-1 and improved the permeability of endothelial cells（P<0.05）.Conclusion:Exogenous administration of ceramide can promote pyroptosis of human umbilical vein vascular endothelial cells and increase endothelial permeability.Meanwhile,TXNIP-NLRP3-GSDMD pathway plays an important role in this process.However,Inhibition of TXNIP expression can reduce cell pyroptosis and improve vascular endothelial permeability.