Study On The Mechanism Of P.g-LPS Regulating GSDMD Through TET2 To Mediate The Pyroptosis Of Human Umbilical Vein Endothelial Cells

Objective:Periodontitis causes systematic long-term,low-grade chronic inflammation and is a risk factor for atherosclerosis.At present,the direct molecular mechanism by which periodontal pathogens promote atherosclerosis has not been elucidated.Vascular endothelial cell injury and dysfunction are the initiation and key events in the pathological process of atherosclerosis.Pathogen infection can trigger an inflammatory and immune cascade by activating GSDMD(gasdermin D),the key executive protein of pyroptosis,leading to inflammatory injury of endothelial cells.In recent years,Tet methylcytosine dioxygenase 2(TET2)plays a key role in regulating inflammation and immune homeostasis.However,the role of TET2 gene in regulating pyroptosis remains unclear.Therefore,the aim of this study is to investigate the role of TET2 in Porphyromonas gingivalis-Lipopolysaccharide(P.g-LPS)-induced pyroptosis in vascular endothelial cells and the molecular mechanism of TET2 in GSDMD cleavage.This study provides a basic theoretical basis for periodontitis to aggravate cardiovascular diseases,and provides a potential therapeutic target for the prevention and treatment of vascular inflammatory immune diseases.Methods:Different concentrations of Porphyromonas gingivalis-Lipopolysaccharide(P.gLPS)(0,0.1,0.5,1,5μg/m L)treated Human Umbilical Vein Endothelial Cells(HUVECs)for different time(0,12,24,48h).The morphological changes of the cells were observed under a microscope.Cell Counting Kit-8(CCK-8)assay was used to detect the effects of P.g-LPS on the proliferation of HUVECs.Flow cytometry were used to detect the effects of P.g-LPS on the cell cycle and apoptosis of HUVECs.Western Blot was used to detect the protein expression of gasdermin D(GSDMD),the key execuator of pyroptosis,to determine the optimal concentration and time of P.gLPS inducing HUVECs pyroptosis model.Western Blot was used to evaluate the effect of P.g-LPS on TET2 protein expression.HUVECs were transfected with Green fluorescent protein(GFP)-tagged TET2 shRNA lentivirus and TET2 overexpression plasmid DNA for 48-72 hours.Then,Western Blot was used to detect the expression of TET2 in HUVECs to construct TET2 knockdown and TET2 overexpression HUVECs models.Subsequently,the experiments were divided into 4 groups: Control group(Control);LPS group;Experimental groups(TET2 shRNA group,TET2 overexpression group);Composite experimental groups(LPS+ TET2 shRNA group,LPS+ TET2 overexpression group).Western Blot was used to detect the expression of the nonclassical inflammasome pathway markers Caspase-4(Cysteinyl aspartate specific proteinase-4,Caspase-4),Caspase-5 and classical inflammasome pathway markers NLRP3(NOD-like receptors 3,NLRP3),Caspase-1,GSDMD,GSDMD-N and IL-1β(Interleukin-1β,IL-1β)and IL-18 in each group.Results:1.The results of CCK8 and flow cytometry showed that compared with the control group,most of the cells treated with 1μg/m L P.g-LPS for 48 h were swollen and round under the microscope.At the same time,P.g-LPS induced HUVECs growth arrest at G1 phases and G2/M phases(P < 0.05).It also significantly inhibited the proliferation of HUVECs(P < 0.0001),but did not promote the HUVECs apoptosis(P > 0.05).Western blot results also showed that 1μg/m L P.g-LPS treatment for 48 h significantly increased the expression of GSDMD,a key executive protein of pyroptosis(P < 0.0001).Therefore,treatment with 1μg/m L P.g-LPS for 48 h was the optimal condition for constructing P.g-LPS-induced HUVECs pyroptosis model.2.Western blot results showed that with the increase of P.g-LPS concentration and the extension of treatment time,the expression of TET2 protein in HUVECs gradually decreased.Compared with the control group,1μg/ml P.g-LPS treatment for48 h significantly down-regulated the expression of TET2 protein(P < 0.0001)3.The results of fluorescence microscopy and Western blot showed that compared with the negative control group,the number of green fluorescent cells in HUVECs transfected with LV-TET2 shRNA was more than 80%,and the expression of TET2 was decreased by 60%(P<0.001),while the number of green fluorescent cells in HUVECs transfected with TET2 overexpression plasmid DNA was more than 80%.The expression of TET2 was increased by 70%(P<0.001).These results indicated that the TET2 knockdown and TET2 overexpression HUVECs models were successfully constructed.4.Compared with the control group,LPS group and TET2 shRNA group,The LPS+TET2 shRNA compound group significantly increased the protein expression of Caspase-4,Caspase-5,NLRP3,Caspase-1,GSDMD,GSDMD-N,IL-1β,and IL-18 in HUVECs(P <0.001).On the contrary,TET2 overexpression down-regulated the protein expression of Caspase-4,Caspase-5,NLRP3,Caspase-1,GSDMD,GSDMD-N,IL-1β,and IL-18(P < 0.05).However,compared with the LPS group,the LPS + TET2 overexpression compound group had significantly down-regulated the expression of proteins related to pyroptosis(P <0.05).In conclusion,TET2 overexpression can inhibit the pyroptosis of HUVECs and partially reduce the expression of proteins related to pyroptosis up-regulated by P.g-LPS in HUVECs.Conclusions:P.g-LPS synergistically induces pyroptosis and inflammatory injury by downregulating TET2 and activating NLRP3/Caspase-1 canonical inflammasome and Caspase-4/5 non-canonical inflammasome pathways to promote GSDMD cleavage and the expression of inflammatory factors IL-1β and IL-18 in HUVECs.TET2 overexpression partially reverse the effect of P.g-LPS on promoting GSDMD-mediated pyroptosis in HUVECs.The role of TET2 in inflammatory injury of vascular endothelial cells induced by periodontal infection was clarified,which provided a new scientific basis and potential clinical prevention and treatment strategy for the causal relationship between periodontitis and cardiovascular diseases.