| BackgroundAbdominal aortic aneurysm is one of the important causes of adult death,and about 150,000-200,000 people dies of abdominal aortic aneurysm every year in the world.At present,the incidence of AAA in China is increasing year by year.Through ultrasonic examination,the incidence of AAA in men over 65 years old reaches 9%.Abdominal aortic aneurysm(AAA)is a kind of local or extensive permanent expansion caused by the pressure of blood flow impact caused by the destruction of the middle structure of artery,which often leads to high morbidity and mortality due to the appearance of aortic dissection and aneurysm rupture.Because the specific molecular mechanism of pathological features of AAA is not clear,the clinical treatment of patients is still limited to surgical repair.Lack of medication means that patients with small AAA or those who are not suitable for surgical repair have no active treatment options,so it is urgent to explore effective drugs to prevent their progression.The formation of AAA is a multi-factor process,which mainly involves the infiltration of inflammatory cells,the degradation of matrix metalloproteinases and the apoptosis and phenotype transformation of smooth muscle cells.In AAA animal model,inflammatory cells such as macrophages,neutrophils,mast cells and CD4+T cells infiltrated before abdominal aorta dilated.Studies have shown that immune inflammatory cell infiltration is one of the important pathogenesis of AAA,and it runs through the whole process of AAA.Macrophages,as effector cells of innate immune system,play an important role in inflammatory response.They can devour bacteria and necrotic substances and secrete pro-inflammatory factors,such as Interleukin(IL),Interferon(IFN),tumor necrosis factor(TNF),chemokines,etc.We also confirmed this view by collecting abdominal aorta tissues of AAA patients and using immunohistochemical staining.In addition,previous studies have found that cyclosporine and steroid anti-inflammatory drugs can reduce the size of AAA in animal models;Macrolide and nonsteroidal anti-inflammatory drugs can reduce the growth rate of AAA in humans.To sum up,we believe that in-depth study of the inflammatory response mechanism of AAA may provide a basis for drug treatment of AAA.ILF3,also known as interleukin enhancer binding factor 3,contains two main protein isomers:NF90/NF110,which is a double-stranded RNA(dsRNA)binding protein and mainly participates in different physiological activities such as mRNA transportation,translation,transcription and signal transduction.In the past,ILF3 has been identified as a new factor affecting dyslipidemia and stroke subtypes.Xie et al.showed that ILF3 can mediate the upregulation of BMP2 and down-regulation of STAT1,thus promoting the calcification of atherosclerotic plaque.The study of temporal lobe epilepsy found that ILF3-AS1 promoted the expression of inflammatory cytokines and MMPs by targeting miR-212.In addition,it was found that overexpression of ILF3 could inhibit the up-regulation of miR-215-5p gene,thus inhibiting the inflammatory injury of septic H9C2.These studies suggest that ILF3 plays an important role in the occurrence and development of inflammatory diseases and cardiovascular diseases.But the role of ILF3 in AAA has not been discussed.To further clarify the relationship between ILF3 and AAA,we extracted primary mouse peritoneal macrophages,and established AAA model by Ang Ⅱ infusion and Cacl2 infiltration,so as to explore the specific effect of ILF3 on AAA and its molecular mechanism.Objectives1.To clarify the expression changes of ILF3 in the diseased tissue of abdominal aortic aneurysm,and explore the relationship between ILF3 expression changes and macrophages in the diseased area.2.To clarify the mechanism of ILF3 in macrophages in the development of abdominal aortic aneurysm.3.To explore the molecular mechanism that ILF3 regulates Keap1-Nrf2 signaling pathway in macrophages and affects the local inflammatory response.Method1.To clarify the expression changes of ILF3 in the diseased tissue of abdominal aortic aneurysm,and explore the relationship between ILF3 expression changes and macrophages in the diseased area.1.1 Collecting abdominal aortic tissues of patients with abdominal aortic aneurysm and healthy people,and selecting Apoe-/-mice of 8 weeks old,embedding Ang II pump or Nacl pump subcutaneously,and taking materials to keep abdominal aortic tissues of mice after 28 days.1.1.1 RT-PCR and Western Blot were used to detect the expression of ILF3 at RNA level and protein level in the diseased area of abdominal aorta in control group and AAA disease group.1.1.2 The co-localization of ILF3 and macrophage marker CD68 in control group and model group was detected by immunofluorescence staining.1.2 The primary peritoneal macrophages of ILF3M-WT mice were extracted.After being stimulated by Ang II,the changes of ILF3 level in the cells at specific concentration and time were detected by Western Blot.2.To clarify the mechanism of ILF3 in macrophages in the development of abdominal aortic aneurysm.2.1 In vivo experimentEight-week-old ILF3M-WT/Apoe-/-(control mice),ILF3M-KO/Apoe-/-(mice with specific ILF3 gene knockout in macrophages)and ILF3M-Tg/Apoe-/-(mice with specific ILF3 gene overexpression in macrophages)were subcutaneously implanted with AngⅡ pump or Nacl pump.After 28 days,the samples were taken and reserved for mice.2.1.1 Observe and count the tumorigenesis rate,mortality rate,tumor weight and tumor diameter of mice.2.1.2 The tumor morphology was observed by HE staining.2.1.3 Verhoeff staining was used to observe the rupture of abdominal aortic elastic plate.2.1.4 Immunohistochemical staining was used to observe the expression of VCAM,ICAM,MCP1,IL6,IL10,TNF-α and other inflammatory factors.2.2 In vitro experiment Primary peritoneal macrophages of ILF3M-WT and ILF3M-KO mice were extracted.2.2.1 Under pathological conditions(Ang Ⅱ stimulation),the changes of inflammatory factors VCAM,ICAM,MCP1,IL6,IL10 and TNF-α expression after ILF3 knockout of macrophages were observed by Western Blot experiment.2.2.2 The expression of matrix metalloproteinases MMPs in supernatant was determined by gelatin zymography.2.2.3 Using protein histochemistry sequencing method,the specific molecular mechanism of ILF3 affecting the progress of abdominal aortic aneurysm was analyzed and verified by KEGG pathway.3.To explore the molecular mechanism that ILF3 regulates Keapl-Nrf2 signaling pathway in macrophages and affects local inflammatory response.In vitro experimentThe primary peritoneal macrophages of ILF3M-WT and ILF3M-Tg mice were extracted,and si-Keap1 and NF-κB inhibitor(JSH-23)were used to intervene the primary peritoneal macrophages of ILF3M-Tg mice.The expression of Keap1,NF-κB pathway and inflammatory factors under different stimuli was observed by Western Blot experiment.Results1.ILF3 increased in abdominal aortic aneurysm disease group.We collected abdominal aorta tissues of healthy people and AAA patients,and abdominal aorta tissues of Nacl group and Ang Ⅱ group of Apoe-/-mice.Immunohistochemical staining,PCR experiment and Western blot experiment confirmed that ILF3 increased in abdominal aortic aneurysm tissue.2.ILF3 is co-located with macrophages obviously in abdominal aortic aneurysm.The abdominal aorta tissues of healthy people and AAA patients,as well as those of Nacl group and Ang Ⅱ group of Apoe-/-mice,were collected.Immunofluorescence staining showed that the expression of ILF3 and macrophages in AAA increased and co-located obviously.3.Ang Ⅱ upregulates the expression of ILF3 in macrophages.The primary peritoneal macrophages of ILF3M-WT mice were extracted and stimulated by Ang Ⅱ at specific concentration and time point.Western Blot experiment showed that the expression of ILF3 in macrophages increased and showed obvious time and concentration dependence with Ang Ⅱ.4.ILF3 knockout of macrophages can reduce the severity of AAA,and over-expression can aggravate the severity of AAA.Two AAA models,Ang Ⅱ pump and Cacl2 infiltration,were selected.HE and VVG staining confirmed that the degree of AAA could be reduced after macrophage ILF3 knockout,but AAA was aggravated after macrophage ILF3 overexpression.5.macrophage ILF3 knockout can reduce the inflammatory reaction in AAA.The primary peritoneal macrophages of ILF3M-WT and ILF3M-KO mice were extracted,stimulated by Ang Ⅱ,and the expression of inflammatory factors was detected by Western Blot.It was found that ILF3 knockout of macrophages could alleviate the inflammatory reaction in AAA.In addition,we collected abdominal aorta tissues of ILF3M-WT/Apoe-/-and ILF3M-KO/Apoe-/-mice,and detected the changes of inflammatory factors in the two groups by immunohistochemical staining,which also showed that macrophage ILF3 knockout could alleviate the inflammatory reaction in AAA.6.Macrophage ILF3 alleviates inflammatory response through Keap1-Nrf2 pathway and NFκB pathway.Protein histochemistry sequencing showed that macrophage ILF3 was related to KeaplNrf2 pathway and NF-κB pathway.Western Blot experiment and immunohistochemical staining showed that ILF3 knockout of macrophage could decrease the expression of Keapl,increase the expression of Nrf2 and inhibit the phosphorylation of NF-κB pathway.In addition,we extracted the primary peritoneal macrophages of ILF3M-WT and ILF3M-Tg mice,and added Ang Ⅱ,si-Keap1 and NF-κB inhibitor(JSH-23)to stimulate them.Western Blot experiment confirmed that ILF3 reduced the inflammatory response through Keap1-Nrf2 pathway and NFκB pathway.ConclusionIn a word,macrophage ILF3 plays an important role in abdominal aortic aneurysm.The results showed that ILF3 knockout of macrophages could control the formation and severity of AAA by inhibiting inflammatory reaction.The loss of macrophage ILF3 can reduce the expression of Keapl protein in vivo,slow down the degradation rate of Nrf2 in vivo,increase the content of Nrf2 protein,and inhibit the activation of NF-κB pathway,so as to effectively alleviate the local inflammatory reaction of the lesion and inhibit the progress of abdominal aortic aneurysm. |
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