T-BHP Induced Oxidative Stress Injury And Pyroptosis In Endothelial Cells: Roles Of NF-?B1/miR-144-3p/FOXP1 Axis

Background Coronary atherosclerotic heart disease refers to the heart disease caused by atherosclerosis of the coronary arteries,which leads to the stenosis or occlusion of the vessel wall,resulting in myocardial hypoxia or necrosis,referred to as coronary heart disease(CHD).Endothelial dysfunction due to endothelial injury and/or death is considered to be the initiating link of CHD.Factors such as hyperglycemia,hyperlipidemia,nicotine,and hypertension are common causes of endothelial cell death.Oxidative stress caused by overproduction of reactive oxygen species(ROS)may be one of their common mechanisms.ROS in endothelial cells mainly comes from mitochondria,NADPH oxidase(NOX)and Xanthine oxidase(XO).ROS are considered to be a bridge for the activation of the NLRP3 inflammasome by stimuli such as hyperlipidemia,hyperglycemia,nicotine,and hypertension.The NLRP3 inflammasome is composed of the intracellular recognition receptor NLRP3,apoptosis-associated speck-like protein(ASC)and effector protein caspase-1.Activation of the NLRP3 inflammasome is essentially caspase-1 autoactivation.Activated caspase-1 can mediate the maturation of interleukin-1?(IL-1?)and interleukin-18(IL-18).In addition,activated caspase-1 can promote the maturation of the porin Gasdermin D(GSDMD),leading to the formation of membrane pores.The formation of membrane pores can not only cause cell expansion or even rupture,but also promote the release of IL-1? and IL-18,maintaining and amplifying the inflammatory response.Therefore,NLRP3 inflammasome activation is one of the important mechanisms of ROS-mediated endothelial cell swelling accompanied by inflammation.Forkhead box protein P(FOXP)is a large multidomain transcriptional regulator that binds to DNA through a forkhead or "winged helix" domain located near its carboxy terminus.In addition,Foxp family members also include C2H2 zinc fingers and leucine zipper motifs that promote self and homodimerization.Foxp1 is one of the four Foxp subfamily members and has a variety of biological functions,such as involvement in tumor progression,lung and esophagus development,maintenance of naive T cell quiescence,follicular helper T cell differentiation and participation in B cell development,etc..Notably,Foxp1 as a transcriptional repressor directly regulates NLRP3 inflammasome components,including NLRP3,Caspase-1,and IL-1?,identified as gatekeepers of vascular inflammation.Therefore,Foxp1 may be a potential target for the treatment of endothelial dysfunction.Micro RNA(miRNA)is an evolutionarily highly conserved non-coding RNA of about 19-22 nt in length,which can regulate gene expression post-transcriptionally.miRNAs degrade m RNA or inhibit its translation in the RNA-induced silencing complex(RISC)by binding to the 3'-untranslated region(3'-UTR)of m RNA.Accumulating evidence suggests that miRNAs play a key role in the development of cardiovascular disease,suggesting that miRNAs can be considered as potential drug targets for the treatment of cardiovascular disease.It has been reported that oxidative stress stimulates the production of several miRNAs,which are defined as oxidative stress-responsive miRNAs.miR-144 is a classic oxidative stress-responsive miRNA.Notably,several studies have shown that miR-144 plays an important role in endothelial cell injury.Therefore,miR-144 may be a potential target for the treatment of endothelial dysfunction.Objective In this study,tert-butyl hydroperoxide(t-BHP)was used to induce oxidative stress in human umbilical vein endothelium(HUVEC)to determine whether t-BHP could activate the NLRP3 inflammasome,leading to EC pyroptosis.In the HUVEC oxidative stress model constructed by t-BHP,the regulation mechanism of NF-?B1/miR-144-3p/FOXP1 axis on cell pyroptosis was deeply studied.Methods 1.HUVECs were treated with 0,25 ?M,50 ?M,and 100 ?M t-BHP for 1h,respectively.Endothelial cell survival was assessed by CCK-8 assay,and LDH activity in cell supernatant was measured to determine cell injury.The expression levels of NLRP3,Pro-caspase1,ASC,C-caspase-1,Pro-IL-1?,C-IL-1? and N-GSDMD were also measured using Western bolt.The protein immunoprecipitation was applied to clarify the NLRP3 inflammasome at the optimal concentration.2.HUVECs were treated with 50 ?M t-BHP for 0,0.5h,1h and 1.5h,respectively.Endothelial cell survival was assessed by CCK-8 assay,and cell damage was determined by detecting LDH activity in cell supernatants.The expression levels of NLRP3,Pro-caspase1,ASC,C-caspase-1,Pro-IL-1?,C-IL-1? and N-GSDMD were also measured using Western bolt.3.Knockdown of NLRP3 expression in the HUVEC oxidative stress model increased HUVEC cell survival and decreased LDH activity.Knockdown of NLRP3 expression in HUVEC oxidative stress model decreased the expression levels of pyroptosis-related proteins C-caspase-1,C-IL-1? and N-GSDMD.4.To clarify whether knockdown of ASC can alleviate t-BHP-induced oxidative stress injury in HUVEC.The transfection efficiency of si-ASC was detected by RT-q PCR and Western Blot techniques.The survival rate of endothelial cells was assessed by the CCK-8 detection kit,and the endothelial cell damage was assessed by the LDH activity detection kit.At the same time,Western bolt technology was used to detect the expression levels of pyroptosis marker proteins C-caspase-1,C-IL-1? and N-GSDMD in cells.5.To investigate whether the caspase-1 inhibitor VX-765 can alleviate t-BHP-induced oxidative stress injury in HUVEC.The survival rate of endothelial cells was assessed by the CCK-8 detection kit,and the endothelial cell damage was assessed by the LDH activity detection kit.At the same time,Western bolt technology was used to detect the expression levels of pyroptosis marker proteins C-caspase-1,C-IL-1? and N-GSDMD in cells.6.Different concentrations of t-BHP were used to act on HUVEC to build a model of HUVEC oxidative stress injury.RT-q PCR was used to detect the change trend of pri-miR-144 and miR-144-3p.In addition,50 ?M t-BHP was used to act on HUVEC at different time points to construct an oxidative stress injury model of HUVEC,and RT-q PCR was used to detect the change trend of pri-miR-144 and miR-144-3p.7.To study the regulation mechanism of NF-?B1 on MIR-144 transcription initiation.The HUVEC oxidative stress injury model was constructed by t-BHP and the expression of NF-?B1 was knocked down,and the expressions of pri-miR-144-3p and miR-144-3p were detected by RT-q PCR.Chromatin immunoprecipitation(Ch IP)was used to demonstrate whether NF-?B1 could bind to the transcription initiation region of MIR-144.The dual-luciferase reporter gene assay was used to detect whether NF-?B1 could activate the transcription of MIR-144.8.HUVECs were transfected with miR-144-3p mimics to determine whether overexpression of miR-144-3p could induce NLRP3 inflammasome activation in HUVECs.The transfection efficiency was detected by RT-q PCR.The survival rate of endothelial cells was assessed by the CCK-8 detection kit,and the endothelial cell damage was assessed by the LDH activity detection kit.At the same time,Western bolt technology was used to detect the expression levels of pyroptosis marker proteins NLRP3,Pro-caspase1,ASC,C-caspase-1,Pro-IL-1?,C-IL-1? and N-GSDMD in cells.9.HUVECs were transfected with miR-144-3p inhibitor to determine whether knockdown of miR-144-3p expression could alleviate t-BHP-induced NLRP3 inflammasome activation and oxidative stress injury in HUVECs.The transfection efficiency was detected by RT-q PCR.The survival rate of endothelial cells was assessed by the CCK-8 detection kit,and the endothelial cell damage was assessed by the LDH activity detection kit.At the same time,Western bolt technology was used to detect the expression levels of pyroptosis marker proteins NLRP3,Pro-caspase1,ASC,C-caspase-1,Pro-IL-1?,C-IL-1? and N-GSDMD in cells.10.To clarify whether miR-144-3p regulates Foxp1.After overexpression of miR-144-3p in HUVEC,RT-q PCR and Western Blot were used to detect the expression of Foxp1.After knockdown of miR-144-3p in HUVEC oxidative stress model,RT-q PCR and Western Blot were used to detect the expression of Foxp1.In addition,the dual-luciferase reporter gene assay was used to confirm that miR-144-3p binds to Foxp1 m RNA 3'-UTR.11.n the HUVEC oxidative stress model,miR-144-3p inhibitor and si-Foxp1 were simultaneously transfected to investigate whether miR-144-3p mediates the activation of NLRP3 inflammasome and oxidative stress injury through Foxp1.The survival rate of endothelial cells was assessed by the CCK-8 detection kit,and the endothelial cell damage was assessed by the LDH activity detection kit.At the same time,Western bolt technology was used to detect the expression levels of pyroptosis marker proteins NLRP3,Pro-caspase1,C-caspase-1,Pro-IL-1?,C-IL-1? and N-GSDMD in cells.12.Foxp1 was overexpressed in the HUVEC oxidative stress model to determine whether Foxp1 could alleviate t-BHP-induced NLRP3 inflammasome activation and oxidative stress injury in HUVEC.The survival rate of endothelial cells was assessed by the CCK-8 detection kit,and the endothelial cell damage was assessed by the LDH activity detection kit.At the same time,Western bolt technology was used to detect the expression levels of pyroptosis marker proteins NLRP3,Pro-caspase1,C-caspase-1,Pro-IL-1?,C-IL-1? and N-GSDMD in cells.Results 1.With the increase of t-BHP concentration,the survival rate of HUVEC cells gradually decreased,and the LDH activity gradually increased.By Western blot detection,it was found that with the increase of t-BHP concentration,the expression levels of pyroptosis-related proteins gradually increased.When the concentration of t-BHP was 50 ?M,the expression of pyroptosis-related proteins was the highest.At the same time,CO-IP assay found that the enrichment of NLRP inflammatory complex was significantly increased when the concentration of t-BHP was 50 ?M.2.HUVECs were treated with 50 ?M t-BHP at different time points.With the increase of t-BHP induction time,the survival rate of HUVEC cells gradually decreased,and the LDH activity gradually increased.Western blot detection showed that with the increase of t-BHP induction time,the expression levels of pyroptosis-related proteins gradually increased.3.Knockdown of NLRP3 expression in the HUVEC oxidative stress model increased HUVEC cell survival and decreased LDH activity.Knockdown of NLRP3 expression in HUVEC oxidative stress model decreased the expression levels of pyroptosis-related proteins C-caspase-1,C-IL-1? and N-GSDMD.4.Knockdown of ASC expression in the HUVEC oxidative stress model increased HUVEC cell survival and decreased LDH activity.Knocking down the expression of ASC in the HUVEC oxidative stress model decreased the expression levels of pyroptosis-related proteins C-caspase-1,C-IL-1? and N-GSDMD.5.The addition of the caspase-1 inhibitor VX-765 to the HUVEC oxidative stress model increased HUVEC cell viability and decreased LDH activity.The addition of the caspase-1 inhibitor VX-765 to the HUVEC oxidative stress model reduced the expression levels of pyroptosis-related proteins C-caspase-1,C-IL-1? and N-GSDMD.6.In the HUVEC oxidative stress model constructed by t-BHP,the expressions of pri-miR-144 and miR-144-3p were increased in a dose-dependent and time-dependent manner.7.In the HUVEC oxidative stress model constructed by t-BHP,knockdown of NF-?B1 expression significantly decreased the expression of pri-miR-144 and miR-144-3p.The results of Ch IP experiments showed that the enrichment of NF-?B1 in the promoter region of MIR-144 was significantly increased.The dual-luciferase reporter gene assay showed that NF-?B1 could activate the transcription of MIR-144.8.Overexpression of miR-144-3p in HUVEC resulted in decreased HUVEC cell viability and increased LDH activity.Western blot detection showed that the expression levels of pyroptosis-related proteins NLRP3,Pro-caspase1,C-caspase-1,Pro-IL-1?,C-IL-1? and N-GSDMD were increased.9.In the HUVEC oxidative stress model constructed by t-BHP,knocking down the expression of miR-144-3p increased the survival rate of HUVEC cells and decreased the activity of LDH.Western blot detection showed that the expression levels of pyroptosis-related proteins were decreased.10.Overexpression of miR-144-3p in HUVEC decreased Foxp1 m RNA and protein levels.In the HUVEC oxidative stress model constructed by t-BHP,knockdown of miR-144-3p expression increased Foxp1 m RNA and protein levels.The dual-luciferase reporter gene assay showed that miR-144-3p could bind to Foxp1 m RNA 3'-UTR.11.In the HUVEC oxidative stress model constructed by t-BHP,after knocking down the expression of miR-144-3p and Foxp1 at the same time,the survival rate of HUVEC cells decreased and the activity of LDH increased.Western blot detection showed that the expression levels of pyroptosis-related proteins were increased.12.In the HUVEC oxidative stress model constructed by t-BHP,the overexpression of Foxp1 increased the survival rate of HUVEC cells and decreased the activity of LDH.The expression levels of pyroptosis-related proteins were decreased.Conclusions 1.In the HUVEC oxidative stress model constructed by t-BHP,t-BHP induced HUVEC cell damage in a concentration-and time-dependent manner.t-BHP induced NLRP3 inflammasome-mediated pyroptosis in HUVECs in a dose-dependent and time-dependent manner.2.NLRP3 is required for t-BHP to induce NLRP3 inflammasome-mediated pyroptosis in HUVECs.Knockdown of NLRP3 expression can alleviate t-BHP-induced HUVEC cell damage.3.ASC is necessary for t-BHP to induce NLRP3 inflammasome-mediated pyroptosis in HUVECs.Knockdown of ASC expression can alleviate t-BHP-induced HUVEC cell damage.4.Caspase-1 activation is necessary for t-BHP to induce NLRP3 inflammasome-mediated pyroptosis in HUVECs.Inhibition of caspase-1 activity can alleviate t-BHP-induced HUVEC cell damage.5.In the HUVEC oxidative stress model constructed by t-BHP,the expressions of pri-miR-144 and miR-144-3p were increased.Overexpression of miR-144-3p in HUVEC caused HUVEC cell damage and promoted NLRP3 inflammasome-mediated pyroptosis.In the HUVEC oxidative stress model constructed by t-BHP,knocking down the expression of miR-144-3p could alleviate HUVEC cell injury and inhibit NLRP3 inflammasome-mediated pyroptosis.6.In the HUVEC oxidative stress model constructed by t-BHP,NF-?B1 is a transcriptional activator of MIR-144 and promotes the transcription of MIR-144.7.miR-144-3p downregulates Foxp1 expression by targeting Foxp1 m RNA 3'-UTR.8.In the HUVEC oxidative stress model constructed by t-BHP,miR-144-3p mediates HUVEC cell damage and promotes NLRP3 inflammasome-mediated pyroptosis through Foxp1.9.In the HUVEC oxidative stress model constructed by t-BHP,overexpression of Foxp1 can alleviate HUVEC cell injury and inhibit NLRP3 inflammasome-mediated pyroptosis.Innovation and significance This study clarified that t-BHP could activate the NLRP3 inflammasome in endothelial cells,leading to pyroptosis of HUVEC cells.It was found that miR-144-3p plays a regulatory role in NLRP3 inflammasome-mediated pyroptosis in the HUVEC oxidative stress model constructed by t-BHP.It was found that Foxp1 plays a regulatory role in NLRP3 inflammasome-mediated pyroptosis in the HUVEC oxidative stress model constructed by t-BHP.In addition,in the HUVEC oxidative stress model constructed by t-BHP,the role of transcription activator NF-?B1 on the regulation of MIR-144 transcription initiation was further studied.The effect of miR-144-3p on NLRP3-mediated pyroptosis by targeting the downstream gene Foxp1 was studied in depth.This study provides a new research direction for the treatment of coronary atherosclerotic disease in the cardiovascular system,and provides a new theoretical support for the mechanism of action of miR-144-3p.