Effect Of NLRP3 On TNFα -induced LDL Trancytosis Across Endothelial Cells And Targeted Molecular Intervention

Objective Atherosclerosis(AS)is a chronic inflammatory disease,but its pathogenesis has not yet fully elucidated.In many theories,the“response to retention hypothesisis” is widely concerned.The theory emphasizes the deposition of apolipoprotein B100 containing lipoproteins,such as low-density lipoprotein(LDL)in the vascular wall is the initial step of atherogenisis.Our previous studies have shown that TNFα can promote LDL trancytosis across the endothelium and promote AS.NLRP3 inflammasome is a large molecule protein complex that regulates IL-1β production,which is closely related to the initiation and development of AS.However,whether or not NLRP3 play a role in TNFα-induced LDL trancytosis is not clear.“Inflamed”endothelial cells play an important role in the pathogenesis of AS.It’s an attractive therapeutic target.The aim of this study was to investigate the role of NLRP3 inflammasome in TNFα-induced LDL trancytosis.We further developed a liposome si RNA delivery system targeting “Inflamed” endothelial cells and evaluted the use of the targeted si RNA delivery system to deliver NLRP3 si RNA to the inflamed endothelial cells,the specificity of delivery and the effect on TNFα-induced NLRP3 inflammasome activationMethods(1)TNFα was added into human umbilical vein endothelial cells(HUVEC)transfected with NLRP3 si RNA.The expression of NLRP3,IL-1β and caspase-1 was detected by Western Blot,and the LDL trancytosis was evaluated by an in-vitro trancytosis model.(2)Liposomes were prepared by membrane hydration and VCAM-1 binding peptides were ligated to prepare peptides targeting cationic liposomes(PCL)as si RNA vectors.The Key parameters of liposome were detected by DLS.The serum stability of si RNA-PCL was detected by agarose gel electrophoresis and immunofluorescence.The targeted delivery of si RNA-PCL in vitro was determined by immunofluorescence.The toxicity of si RNA-PCL was assessed using CCK8.In order to evaluated the efficacy of the si RNA-PCL in vitro,HUVECs were treated with si RNA-PCL.The expression of NLRP3 was detected by Western Blot.Result(1)TNFα could activate the NLRP3 inflammasome in endothelial cells and increase the production of IL-1β and caspase-1.After silencing NLRP3,TNFα-induced expression of IL-1β and caspase-1 was inhibited and TNFα-induced LDL trancytosis across HUVECs was also blocked.(2)We optimized the si RNA-PCL preparation process by detecting the si RNA-PCL particle size,Zeta potential and dispersion coefficient in different composition ratio.We found that the si RNA-PCL system was the most stable when the molar ratio of DOTAP: DOPC was 40: 35,the molar ratio of MAL-DSPE-PEG2000: peptides was 3: 1 and the mass ratio of liposome to si RNA was 100: 1.Si RNA-PCL was more stable than naked si RNA and could effectively enter LPS pre-treated endothelial cells in medium containing 50% serum.The toxicity of si RNA-PCL was much lower than Lipo2000.Si RNA-PCL could effectively target inflamed endothelial cells and inhibit the expression of NLRP3 protein.Conclusion TNFα could activate NLRP3 inflammasome.NLRP3 inflammasome involved in TNFα-induced LDL trancytosis.Si RNA-PCL could effectively target inflamed endothelial cells and inhibit the expression of NLRP3.The VCAM1-targeted PCL carrying si RNA may serve as an effective si RNA delivery vehicle to perform targeted antiatherogenic therapy.