| [Background]Inflammation of vascular endothelial cells（VECs）induced by low shear stress（LSS）plays an important role in the occurrence and development of atherosclerosis（As）,However,its regulatory mechanism is still unclear.Recent studies have shown that pyroptosis,an inflammatory and programmed cell death,is benefited to the progression of As.Our previous research showed that Tet methylcytidine dioxygenase2（TET2）is a shear stress sensitive gene and down-regulated under low shear stress.Succinate is an important metabolite of the tricarboxylic acid cycle（TCA）.It can combine with its specific receptor G protein-coupled receptor 91（GPR91）to conduct signal transduction and affect immunity and inflammation and other biological processes.In this article,we will explore the role and mechanism of TET2/succinate/GPR91 in LSS-induced pyroptosis of VECs.[Objective]To explore the regulation mechanism of VEC pyroptosis induced by low shear stress.[Methods]1.Using a parallel plate flow chamber system,the effects of LSS on the expression of TET2,Nod-like receptors 3（NOD-like receptors 3,NLRP3）,Gasdermin D-N tunel（GSDMD-N）,Caspase-1（Cysteinyl aspartate specific proteinase-1）,Interleukin-18（IL-18）and IL-1βin human umbilical vein endothelial cells（HUVECs）were observed.2.Using high-fat diet apo E-/-mouse atherosclerosis model,the relationship between As lesions and serum succinate content was detected.3.The low TET2 expression HUVEC was constructed with lentiviral transfection,and the transfection efficiency was detected with fluorescence microscope and Western blot.The effect of LSS and TET2sh RNA on GPR91 and succinate content was detected with Western blot;4.Exogenous succinate analogue diethyl succinate（Diethyl succinate,DS）was added to detect the effect of succinate on the expression of GPR91 and pyroptosis-related proteins NLRP3,GSDMD-N,Caspase-1,IL-18 and IL-1β;5.GPR91 inhibitor was used to detect the effect of GPR91 on HUVECs pyroptosis induced by TET2sh RNA and succinate analog DS.6.The effects of TET2sh RNA and succinate analog DS and GPR91 inhibitor on the expression of protein kinase C（PKC）and the phosphorylation of NLRP3 were detected with westeren blot.7.The high-fat diet supplied with succinate apo E-/-mice was used to detect the effect of succinate on As.[Results]Low shear stress up-regulates the expression of pyroptosis-related proteins NLRP3,GSDMD-N,Caspase-1,IL-18,IL-1βand down-regulates the expression of TET2 in HUVECs,indicating that low shear stress down-regulates TET2 and induces pyroptosis of HUVECs.After 12weeks of high-fat diet,significant atherosclerotic lesions were formed in apo E-/-mice,and the serum succinate content was increased,indicating that the accumulation of succinate is closely related to atherosclerotic lesions.Western blot showed that low shear stress and TET2sh RNA promote the accumulation of succinate and up-regulate the expression of GPR91.The succinate analog DS promotes the accumulation of succinate,up-regulates GPR91 and induces pyroptosis of HUVECs.GPR91 inhibitor NF-56-EJ40inhibited HUVECs pyroptosis induced by TET2sh RNA and succinate analog DS.These results indicate that low shear stress promotes HUVECs pyroptosis through TET2/succinate/GPR91.Western blot showed that TET2sh RNA and succinate analog DS up-regulated PKC expression and NLRP3 phosphorylation in HUVECs.After GPR91 inhibitor treatment,the up-regulation of PKC expression and NLRP3 phosphorylation induced by TET2sh RNA and succinate analog DS was inhibited,indicating that low shear stress promotes PKC expression and NLRP3 phosphorylation modification through TET2/succinate/GPR91.In addition,the high-fat diet supplied with succinate apo E-/-mice have higher blood lipid levels and more obvious atherosclerotic lesions than high-fat diet apo E-/-mice,indicating that succinate promotes the formation and progression of As.[Conclusion]Low shear stress down-regulates the expression of TET2,activates the succinate/GPR91/PKC/NLRP3 pathway,which induces HUVEC pyroptosis. |
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