Mechanisms Of Interleukin Enhancer Binding Factor 3 In Regulating Plaque Stability In Atherosclerosis

1 BackgroundThe prevalence of cardiovascular disease in China is continuing to rise.The"China Cardiovascular Diseases Report 2017" estimates that the current number of cardiovascular diseases is 290 million.In 2015,cardiovascular disease mortality still ranked first,higher than cancer and other diseases.Atherosclerosis(AS)is the most important pathological basis of cardiovascular disease and the primary pathology underlying cardiovascular disease development is atherosclerosis.Atherosclerosi's morbidity and mortality are increasing day by day.The formation and development of atherosclerotic plaques mainly include abnormal accumulation of inflammatory cells,formation of foam cells,proliferation of smooth muscle cells,increased synthesis of extracellular matrix,and concomitant arterial vascular remodeling.Recent studies have shown that atherosclerosis is a chronic inflammatory response condition of the blood vessel wall.There are abundant inflammatory cells,activated immune cells,and cytokines in the atherosclerotic plaque.There is increasing evidence that inflammatory factors and their associated signaling pathways are closely related to the progression of atherosclerosis,but the exact molecular mechanism remains unclear.ILF3 protein is a kind of double-stranded RNA(dsRNA)-binding protein.As one of the members of the activated T lymphocyte nuclear factor(NFAT)transcription factor family and is involved in the control of early inflammatory responses in activated lymphocytes.ILF3 participates in a series of cellular processes including DNA metabolism,transcription,translation,RNA stabilization,and microRNA biosynthesis.Previous studies of ILF3 have focused on tumors.In recent years,studies have found that ILF3 may be associated with diseases of the cardiovascular system,such as regulation of bloodlipid,thrombosis,myocardial infarction,and post-traumatic stress disorder-mediated heart disease.Whether ILF3 is related to the formation and development of atherosclerosis or not has not been reported at home and abroad.This article intends to detect the expression of ILF3 in different atherosclerotic plaques and to determine whether ILF3 is related to the development of atherosclerosis.2 Objectives(1)Expression and distribution detection of ILF3 in normal heart,liver,spleen,lung,kidney in mice.(2)To clarify the expression of ILF3 in vulnerable plaques of atherosclerosis in mice,rabbits and humans,respectively.(3)Detection of ILF3 expression level in mouse peripheral blood.3 Method3.1 Establishment of Animal Atherosclerotic ModelPart one:Mouse carotid atherosclerosis modelEight-week-old male ApoE-/-mice(n=75)were randomly divided into the following groups:A:Control(n=15):Give a normal diet for 12 weeks;B:Cannula(n=15):The surgery was performed with the silastic tube placed around the left common carotid artery and given a high-fat diet from the same day to 12 week;C:Statin+Cannula(n=15):The surgery was performed with the silastic tube placed around the left common carotid artery and given a high-fat diet from the same day to 12 week;from the eighth week,simvastatin(9.4mg/kg/d)was given to the mice tntil the 12 week;D:Cannula+Triggering(n=15):The surgery was performed with the silastic tube placed around the left common carotid artery and given a high-fat diet from the same day to 12 week,before the end of the experience,jointly triggered experiments was performed to stimulate the plaque to rupture;E:Statin+Cannula+Triggering(n=15):The surgery was performed with the silastic tube placed around the left common carotid artery and given a high-fat diet from the same day to 12 week;from the eighth week,simvastatin(9.4mg/kg/d)was given to the mice tntil the 12 week.,before the end of the experience,jointly triggered experiments was performed to stimulate the plaque to rupture.Part two:Rabbit abdominal aorta atherosclerosis modelthree-month-old male purebred New Zealand white rabbits(n=50)were randomly divided into the following groups:A:Control(n=10):Give a normal diet for 12 weeks;B:Balloon-injury(n=10):Abdominal aorta balloon injury surgery was performed and given a high-fat diet from the same day to 12 week;C:Statin+Balloon-injury(n=10):Abdominal aorta balloon injury surgery was performed and given a high-fat diet from the same day to 12 week,from the eighth week,simvastatin(9.4mg/kg/d)was given to the mice tntil the 12 week;D:Balloon-injury+triggering(n=10):Abdominal aorta balloon injury surgery was performed and given a high-fat diet from the same day to 12 week,before the end of the experience,two drug-triggered injections was performed at 24h and 48h before the end of the experiment to stimulate plaque rupture;E:Statin+Balloon injury+triggering(n=10):Abdominal aorta balloon injury surgery was performed and given a high-fat diet from the same day to 12 week,from the eighth week,simvastatin(9.4mg/kg/d)was given to the mice tntil the 12 week,two drug-triggered injections was performed at 24h and 48h before the end of the experiment to stimulate plaque rupture.3.2 Body weight and serum index measurementAll animals were weighed before sacrifice and blood was collected from the apex cordis.Serum total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL),high-density lipoprotein(HDL)were measured.3.3 Histopathological and immunohistochemical detectionCannulated carotid arteries of the mice and the injuried abdominal aorta of the rabbits were individually stained by oil red lipids to detect lipid deposition.Sirius red staining was used to detect collagen.Immunohistochemical staining of CD68 and a-SMA was performed to detect macrophages and smooth muscles.The vulnerability index of the plaque was calculated,and the vulnerability index =(macrophage + lipid)positive area%/(smooth muscle cell + collagen)positive area%.Immunohistochemical staining of ILF3,IL-6 and TNF-a was used to detect the expression of inflammatory factors.Immunohistochemistry was used to detect the expression of ILF3 in the heart,liver,spleen,lung,kidney of normal mice.3.4 Immunofluorescencethe expression of ILF3 in macrophages and smooth muscle cells in human aortic plaque was detected by double primary antibodies which including those against CD68 and ILF3,a-SMA and ILF3.3.5 ELISA assay of serum ILF3 in miceELISA assay was used to detecte the concentration level of ILF3 in different models.3.6 Statistical analysisAll statistics were analyzed using the SPSS 19.0 software package.All experiments were repeated at least 3 times.The measurement data is expressed as mean ± standard deviation,and the count data is expressed as a numerical value and a percentage.An independent sample t test was used for comparison between the two groups.P<0.05 was considered to be statistically significant.4 Results4.1 The general conditions of animalsEach group of mice and New Zealand white rabbits were weighed before sacrifice,and serum concentration of TC,TG,LDL-C,and HDL-C were measured.The results showed that compared with the normal diet group,body weight increased,The concentration of TC,TG,LDL-C and HDL-C increased(P<0.05).There were no statistical differences among each model groups.By the end of the experiment,a total of 1 mouse died and 6 New Zealand white rabbits died.Eventually.74 mice completed the experiment and 44 rabbits completed the experiment.4.2 Plaque vulnerability of each groupsCompared with the control group,the lipid content,collagen content,macrophage and smooth muscle cells in the plaque of the cannula/balloon injury group increased.Compared with the untreated group,statin treatment can reduce the lipid content in the plaque,increased collagen content,decreased the number of macrophages,increased the number of smooth muscle cells,reduced plaque vulnerability(P<0.05);compared with the unruptured group,the rupture group showed increased lipid cotnet and reduced collagen content,increased macrophages and reduced smooth muscle cells,and the plaque vulnerability is increased(P<0.05);in the cannula/balloon injury group,2/15 mice developed plaque rupture,no rabbit plaque rupture,In the cannula/balloon strain plus trigger group,plaque rupture occurred in 7/14 mice,and plaque rupture occurred in 4/8 rabbits,indicating that drug triggering increased plaque rupture;cannula/balloon strain plus statin treatment Plaque rupture occurred in 1/15 mice,plaque rupture occurred in rabbits,plaque rupture occurred in 5/15 mice in cannula/balloon injury plus statin plus trigger group,and 2/9 rabbits developed plaque rupture,furtherlly indicates that statin treatment can increase plaque stability.4.3 Expression of inflammatory factors in plaqueCompared with the control group,the expression of ILF3,IL-6,and TNF-a in the plaque of the cannula/balloon injury group increased;compared with the cannula/balloon injury group,statin treatment can reduce the expression of ILF3,IL-6,TNF-a in plaque(P<0.05);Compared with the cannula/balloon injury group,the expression of ILF3,IL-6,and TNF-a in the cannula/balloon injury plus trigger group increased(P<0.05);Compared with the balloon-injured plus statin group,the expression of ILF3,IL-6,and TNF-a increased in the cannula/balloon injury plus statin plus trigger group(P<0.05).4.4 Expression of ILF3 in mouse normal tissuesThe immunohistochemical staining of ILF3 in normal mice hearts,livers,spleens,lungs,and renal revealed that ILF3 is expressed in myocardium,liver,kidney.3.5 ELISA assay of serum ILF3 in miceCompared with the control group,serum ILF3 concentration in the cannula group increased significantly(P<0.05);compared with the cannula group,statin treatment can reduce the serum ILF3 concentration(P<0.05);the serum ILF3 concentration in the joint trigger group was increased,but Not reach statistical significance.4.6 Expression of ILF3 in human's plaquesImmunofluorescence staining of human's aortic plaques revealed that ILF3 was abundantly expressed in the plaque.immunofluorescence double-label staining of ILF3 and CD68 suggested that ILF3 is mainly expressed in macrophages;immunofluorescence double-label staining of ILF3 and ?-SMA suggested that ILF3 expression in smooth muscle cells limited.5 Conclusion:(1)ILF3 is normally expressed in the heart,liver,and kidney tissues of mice.The expression of ILF3 is increased in plaques and further increased in vulnerable plaques.(2)ILF3 is elevated in peripheral blood of atherosclerotic mice,and statin can reduce ILF3 concentration in peripheral blood.(3)ILF3 is mainly expressed in macrophages in the atherosclerosis plaque.1 BackgroundAtherosclerosis,as the main pathological process of coronary artery disease,has received much attention in recent years.In the early stages of the development of atherosclerosis,oxidized low-density lipoprotein(ox-ldl)causes mononuclear-derived macrophages to aggregate into the blood vessel wall,These macrophages excessively phagocytose cholesterol,leading to the formation of foam cells and promoting the inflammatory response of the arterial wall,and this condition induces a variety of fatal pathological outcomes,such as intra-plaque hemorrhage,plaque rupture,calcification and so on.Inflammation is a key factor in the development of atherosclerosis.lipoprotein metabolism and inflammatory regulation in atherosclerotic plaques have become the focus of research on atherosclerosis.According to the histomorphological features of atherosclerotic plaque,plaques could be divided into stable plaques and vulnerable plaques.At the end of the 20th century,"vulnerable plaques" were used to describe plaques which are prone to rupture.It is the most common cause of acute thrombosis and is considered to be the culprit blood vessel leading to acute coronary syndrome(ACS).The characteristics of vulnerable plaque include:1 volume is relatively large 2 thin fiber cap(smooth muscle and collagen fiber less)3 large lipid nucleus(? 40%of plaque volume)4 a large number of inflammatory cell infiltration(mainly single Nuclear-macrophage)5 positive remodeling 6 increased angiogenesis in plaques.The vulnerable plaque infiltrates a large number of immune inflammatory cells,producing pro-inflammatory factors and hydrolases,which will aggravate the plaque inflammatory response and aggravate the rupture of atherosclerotic plaque.ILF3 is a double-stranded RNA connexin.Previous studies have shown that ILF3 protein is involved in cell cycle regulation and may play a role in RNA transcription,cleavage,editing,RNA export,and subcellular localization,involving cell development.Multiple cellular functions such as cell cycle and viral infection.It is particularly noteworthy that studies in recent years have shown that ILF3 may be involved in cardiovascular system diseases such as blood lipid regulation,thrombosis,myocardial infarction,post-traumatic stress disorder-mediated heart disease,and these studies are concentrated at the genetic level.Whether the level of protein function is unknown or not,whether ILF3 plays an important role in the vulnerability of atherosclerotic plaque has not been explored.2 Objectives(1)illuminate the effect of ILF3 on the atherosclerosic palque vulnerable and its regulatory mechanism in vivo experiments.(2)illuminate the effect of ILF3 on the inflammatory factor secreted bymacrophages and lipid phagocytosis and study the relevant mechanism.3 Method3.1 Construction of Ientiviral vectorsThree pairs of shRNA plasmids of ILF3 gene were designed,and the interference efficiency was detected by RT-PCR and Western blotting.A shRNA sequence with the highest interference efficiency was cloned into the lentiviral vector containing the green fluorescent protein(GFP)reporter gene.The target interference sequence(Sh-ILF3)of ILF3 is:5'-GCCAATGGACTGAAGTCATGT-3',and the negative control sequence(Sh-NC)is:5'-TTCTCCGAACGTGTCACGT-3'.3.2 Construction of macrophage-specific knockout miceMacrophage conditional overexpression of Rosa26-ILF3 mice were purchased from Beijing viewsolid biological technology co,ltd.3.3 Establishment of mice atherosclerotic modelEight-week-old male ApoE-/-mice(n=45)underwent the cannula surgery of left common carotidartery to establish the atherosclerosic plaque vunerable model.After eight weeks' high-fat diet,different lentiviral injections were given via the tail vein,according to different transfection,mice were randomly divided into the following groups:(1)NS group(n=15),without intervention(2)Empty vector group(NC group,n=15),transfected with empty vector carrying only GFP(3)ShRNA-ILF3 intervention group(LV-ILF3(-)group,n= 15),transfected with ShRNA-ILF3 lentivirusAfter transfection,the high-fat diet was continued until 12 weeks.The mice were killed by euthanasia,the tissues were taken,some were stored in liquid nitrogen,and some were fixed in 4%paraformaldehyde for at least 24 hours.3.4 Body weight and blood lipid index testThe weight of the mice were measured before the end of the experiment.The apical blood of the mice were taken and the serum total cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL),high density lipoprotein(HDL)and Blood sugar levels were measured.3.5 aorta oil red O detectionThe aorta was removed from 4%paraformaldehyde.After washing with running water,the perivascular tissue was dissected under microscope.The aorta was dissected along the long axis for oil red O staining.After staining,the blood vessels were fixed on the white plate and photographed.3.6 Histopathology and immunohistochemistryThe left carotid artery was taken out from 4%paraformaldehyde,washing with running water,and embedded in OTC,and frozen sections were taken.The sections were staining with oil red O to identify the lipid deposition,Sirius red to visualize the collagen,CD68 and ?-SMA immunohistochemical staining to identify the macrophages and the amooth muscle cells(SMC).The plaque vulnerability index was calculated,and the vulnerability index =(macrophage + lipid)positive area%/(smooth muscle cells + collagen)positive area%.Immunohistochemistry was used to detect the expression of IL-6 and TNF-? in plaques.3.7 Real-time quantitative RT-PCR detectionFresh carotid tissue was taken,RNA was extracted,and real-time quantitative RT-PCR was used to detect the expression of IL-6 and TNF-a at the level of mRNA.3.8 Extraction and culture of mouse peritoneal macrophagesSix-week-old Rosa26-ILF3 mice and 6-8-week-old C57/B6 mice were injected with 6%starch intraperitoneally.After 72 hours,the induced primary peritoneal macrophages were extracted and the peritoneal macrophages were identified by immunofluorescence staining of CD68.3.9 Cell grouping and stimulationExperiment I:Defining the role of ox-LDL in inducing ILF3 production in mouse peritoneal macrophages.C57/B6 mouse peritoneal macrophages were stimulated with different concentrations of ox-LDL(Oug/ml,25ug/ml,50ug/ml,75ug/ml,100ug/ml)for 24h,and the expression of ILF3 mRNA and protein was observed;50ug/ml ox-LDL were used to stimulated mouse peritoneal macrophages at different times(Oh,4h,8h,12h,24h,48h),and the expression of ILF3 mRNA and protein levels was boserved.Experiment 2:To investigate the role of ILF3 in ox-LDL-induced macrophage foaming,inflammatory factor secretion and related signaling pathways.(1)C57/B6 mouse peritoneal macrophages were extracted and Sh-ILF3 lentiviral vector transfected and then cells were stimulated with ox-LDL and divided into the following three groups:NC group;NC +ox-LDL stimulation group;SI+ ox-LDL stimulation group;(2)Extraction of mouse peritoneal macrophages,divided into:C57/B6 group(WT group)and Rosa26-ILF3 group(ILF3 group).experiment 3:To investigate the role of ILF3 in lps-induced macrophage inflammatory response:C57/B6 mouse peritoneal macrophages were extracted and transfected with Sh-ILF3 lentiviral vector and then stimulated with LPS.The cells were divided into the following three groups:NC group;NC+LPS stimulation group;SI+LPS stimulation group;3.7 Real-time quantitative RT-PCR detection Macrophages of each group were collected,RNA was extracted,and real-time quantitative RT-PCR detection ILF3?Arg1?iNOS?CD86?IL-6?TNF-??IL-1??IL-10 expression of mRNA.3.8 Western Blot detectionCollect macrophages from each group,extract proteins,and detect the expression of ILF3.ABCAI?ABCGI?SRA1?LOX1?ACAT1?IL-6?IL-10?NF-?B?NF-?B(p65)?AKT?p-AKT proteins by Western Blot at the protein level.The strength is expressed as the ratio of the target protein to the integrated optical density of the?-actin band.3.9 Immunofluorescence staining After the cells were collected,the expression of il3in the cells was detected by immunofluorescence staining.3.10 oil red O stainingAfter stimulation of each group,oil O staining was performed to detect the lipid eposition in the cells.3.11 Statistical analysisAll statistics were analyzed using the SPSS 19.0 software package.All experiments were repeated at least 3 times.The measurement data is expressed as mean ± standard deviation,and the count data is expressed as a numerical value and a percentage.An independent sample t test was used for comparison between the two groups.P<0.05 was considered to be statistically significant.4 Results4.1 The general conditions of animalsThe weight of each group of mice was weighed before sacrifice.The concentrations of serum TC,TG,LDL-C and HDL-C were measured.The results showed that there was no difference between groups,indicating that ILF3 virus transfection did not affect the lipid metabolisim of mice.4.2 Expression of ILF3 in plaque after lentivirus transfectionThe results of immunohistochemistry showed that there was no statistical difference between the ILF3 level in the sh-nc group and the control group.The expression of ILF3 in the sh-ILF3 group was significantly lower than that in the sh-nc group(P<0.05).The RT-PCR results were consistent with the immunohistochemistry results.This indicates that the ILF3 virus can effectively interfere with the expression of ILF3 in mouse plaques.4.2 Expression level of ILF3 in plaque after lentivirus transfectionThe results of immunohistochemistry showed that there was no statistical difference between the ILF3 level in the sh-nc group and the control group.The expression of ILF3 in the sh-ILF3 group was significantly lower than that in the sh-nc group(P<0.05).The RT-PCR results were consistent with the immunohistochemistry results.This indicates that the ILF3 virus can effectively interfere with the expression of ILF3 in mouse plaques.4.3 The effect of ILF3 on the composition and vulnerability of atherosclerotic plaqueCompared with the control group,sh-ILF3 can reduce the content of lipids and macrophages in plaque,increase the content of smooth muscle and collagen,and reduce plaque vulnerability(P<0.05).4.4 Effect of ILF3 on the expression level of inflammatory cytokines in AS plaque results of Immunohistochemistry and RT-PCR showed that the levels of IL-6 and TNF-a in sh-ILF3 group were significantly lower(P<0.05).4.5 Extraction of mouse peritoneal macrophagesThe mouse peritoneal macrophages were extracted successfully,and the immunofluorescent staining of macrophage marker CD68 was performed to identify the purity of the extracted cells.>99%of cells were macrophages.4.6 Effect of ox-LDL on the expression of ILF3 in mouse peritoneal macrophagesThe murine peritoneal macrophages were stimulated with ox-LDL at Oug/ml,25ug/ml,50ug/ml,75ug/ml,100ug/ml for 24h,it was found that ox-LDL could up-regulate ILF3 mRNA in a concentration-dependent manner.Protein expression.Compared with the normal control group,the ILF3 could be significantly increased after 24h of 25ug/ml ox-LDL stimulation(P<0.01),100ug/ml was the strongest(P<0.01).The murine peritoneal macrophages were stimulated with 50 ug/ml ox-LDL for 0h,4h,8h,12h,24h,48h,and it was found that ox-LDL could up-regulate the expression of ILF3 mRNA and protein in a time-dependent manner.Compared with the normal control group,ILF3 increased after treatment for 4h(P<0.01),peaked at 24h(P<0.01),and continued to 48h.4.7 Detection of transfection efficiency of macrophage Sh-ILF3After ShRNA-NC and ShRNA-ILF transfected into macrophages,the expression levels of ILF3 were detected by real-time quantitative PCR and Western Blot before and after macrophage transfection.4.8 Detection of ILF3 expression in peritoneal macrophages of Rosa26-ILF3 mice The expression levels of ILF3 in peritoneal macrophages of C57/B6 mice and Rosa26-ILF3 mice were detected by real-time quantitative PCR and Western Blot.The results showed that the level of ILF3 in Rosa26-ILF3 mice was significantly higher than that in C57 mice(P<0.01).4.9 ILF3 promotes ox-LDL-induced macrophage foamingC57/B6 mouse peritoneal macrophages were divided into Sh-NC group;ox-LDL stimulation + Sh-NC group;ox-LDL stimulation + Sh-ILF3 group;mRNA and protein expression levels of ABCA1,ABCG1,SRA1,LOX1,ACAT1 were detected in each group.The results showed that after ox-LDL stimulated macrophages,the lipid-transfer related proteins ABCA1,ABCG1 and lipid-transferred proteins LOX1 and ACAT1 were elevated,LOX1 and ACAT1 were statistically significant(P<0.05),after transfection of Sh-ILF3,ABCA1 and ABCG1 were further increased,but ABCG1 did not reach statistical significance,LOX1 and ACAT1 expression decreased(P<0.05).Oil red O staining was performed after stimulation of each group of cells.The results showed that ox-LDL stimulated macrophage and increased lipid deposition.After transfection of Sh-ILF3,lipid deposition was significantly reduced.It is shown that ILF3 has a role in promoting cholesterol deposition.4.10 ILF3 promotes ox-LDL-induced secretion of macrophage inflammatory factorsC57/B6 mouse peritoneal macrophages were divided into Sh-NC group;ox-LDL stimulation + Sh-NC group;ox-LDL stimulation + Sh-ILF3 group;mRNA and protein expression levels of IL-6,INOS,IL-10,ARG1 were detected in each group.The results showed that IL-6,INOS and IL-10 were increased after ox-LDL stimulation of macrophages.After transfection of Sh-ILF3,the expression of pro-inflammatory factors IL-6 and iNOS decreased(P<0.05),while anti-inflammatory factor ARG1 and IL-10 expression was further increased(P<0.05).It is proved that ILF3 has a role in promoting the secretion of inflammatory factors.Peritoneal macrophages were extracted from C57/B6 mice and Rosa26-ILF3 mice.After ox-LDL stimulation for 24 hours,oil red O staining was performed.The results showed that lipid deposition was significantly increased in the ILF3 overexpression group.Above results indicated that ILF3 promoted cholesterol deposition.effect.4.11 ILF3 promotes LPS-induced secretion of macrophage inflammatory factorsC57/B6 mouse peritoneal macrophages were divided into NC group,NC+LPS stimulation group and SI+LPS stimulation group.The mRNA levels of ILF3,Argl,iNOS,CD86,IL-6,TNF-?,IL-1? and IL-10 of each group were detected.The results showed that ILF3,iNOS,CD86,IL-6,TNF-?,IL-1? and IL-10 were increased after LPS stimulation of macrophages(P<0.05).After transfection of Sh-ILF3,proinflammatory cytokines of ILF3,iNOS,CD86,IL-6,TNF-a,IL-1? and IL-6 were decreased,while the expressions of anti-inflammatory factors ARG1 and IL-10 were further increased(P<0.05),which proved that ILF3 promoted the secretion of inflammatory factors.4.12 The role of ILF3 in the signaling pathwayC57/B6 mouse peritoneal macrophages were divided into Sh-NC group;ox-LDL stimulation + Sh-NC group;ox-LDL stimulation + Sh-ILF3 group;expression levels of NF-?B(p-p65)and p-AKT were detected in each group.The results showed that the expression of NF-?B(p-p65)and p-AKT was increased after ox-LDL stimulation of macrophages,and decreased after transfection with Sh-ILF3.This indicates that ILF3 can affect inflammatory factors and cholesterol deposition through the NF-?B signaling pathway and the AKT signaling pathway.5 Conclusion(1)In the APOE-/-mouse plaque vulnerability model,knockout of ILF3 by lentivirus can reduce the expression of inflammatory factors IL-6,TNF-a in plaque,thereby increasing plaque stability.(2)ILF3 could regulate macrophage inflammatory factor secretion and cholesterol deposition,which may be through NF-?B and AKT signaling pathways.