| Atherosclerosis（AS）is a disease with pathological characteristics that chronic inflammatory injury caused by disorder of lipid metabolic balance in the body,involving various pathophysiological processes such as lipid absorption infiltration,endothelial inflammatory injury-response,adaptive immune response and activation of mononuclear macrophage system.At first,the high risk areas of AS patients were mainly in western countries such as Europe and the United States,and now it has become an important disease concerned by the whole world,which has seriously affected people’s life and health.At present,the AS incidence rate in our country is increasing year by year and the age tends to be young.Therefore,it is particularly important to deeply study the pathogenesis of AS in order to effectively prevent the occurrence and development of the disease.In recent years,with the rapid development of science and technology,a variety of theories have been proposed in the academic community to explain the pathogenesis of AS（such AS:immune response theory,lipid infiltration theory,inflammation theory,mononuclear macrophage theory,etc.）,but there is still no theory that can fully explain the pathophysiological mechanism of AS.At present,most scholars believe that AS is an inflammatory disease with extensive involvement of a large number of inflammatory factors in each stage of AS progression.The disorder of lipid metabolism is the main basis of AS.The lipids in blood infiltrate into vascular endothelial cells through chemical modification such as oxidation,dissolution and aggregation,thus causing sustained inflammatory injury,resulting in the chemotaxis of mononuclear macrophages and recruitment to the injured site of endothelial cells to phagocytose lipids and form foam cells.And finally led to the occurrence of AS.The UⅡ/UT system formed by UrotensinⅡ（UII）and G protein coupled receptor 14（GPR14）has the functions of vasoconstrictive activity,vasodilation,influence on substance metabolism,collagen synthesis,etc.It is widely distributed among tissues and organs of various animal species including humans（such as vascular endothelium,myocardium,liver,kidney,spleen,lung,etc.）.This system is involved in a variety of AS-related pathophysiological processes（Such as macrophage proinflammatory factor expression,vascular calcification,endothelial function impairment）by regulating the expression level of active substances in the body,and plays a major role in the progression of cardiovascular diseases such AS.Urantide,a peptide compound,is a receptor inhibitor derived from human UⅡ,which can inhibit the activation state of UⅡ/UT system and prevent its biological function.Previous studies have found that UⅡcan activate MAPK and IL-6/JAK2/STAT3 signaling pathways when combined with GPR14,and Urantide can effectively inhibit the activation of these two pathways and slow down the progression of AS.The Wnt/β-catenin signaling pathway has been strictly controlled during evolution,and is involved in regulating many physiological functions of cells,such as cell proliferation and migration,determining cell fate,and cell self-renewal.Normally,this signaling pathway is closed,but it can be activated when stimulated by external signals.Wnt-secreted proteins can combine with specific receptors to form a complex,which transmits signals step by step downstream to activate the key moleculeβ-catenin and participate in transcriptional regulation in the nucleus,resulting in corresponding biological effects.Abnormal activation of Wnt/β-catenin pathway can be seen in many diseases such AS malignant tumor,tissue fibrosis,neurodegenerative diseases,and so on.Targeted treatment of Wnt/β-catenin pathway has become an emerging therapeutic pathway.Due to the wide range of the signaling pathway,the relationship between AS and the signaling circuit has also become a hot topic of attention.The Wnt/β-catenin signaling pathway is involved in various pathophysiological stages of AS,including vascular endothelial dysfunction,overexpression of pro-inflammatory factors,and macrophage activation,thereby regulating the disease process.Therefore,inhibition of the activation state of this pathway in the course of AS disease may effectively slow down the evolution and progression of AS,providing a new direction for the development of anti-AS drugs.Can Urantide control AS by regulating Wnt/β-catenin pathway after antagonizing UⅡ/UT system?In order to further study this key issue,Apo E-/-AS mice were taken AS the research object and Wnt/β-catenin signaling pathway was taken as the entry point to explore the prevention and control effect of Urantide on AS and its influence on the expression of key signal molecules in the Wnt/β-catenin signaling cascade pathway.The aim is to elucidate whether its target is related to the Wnt/β-catenin pathway,and to provide reliable experimental data for the development of novel clinical anti-AS targeted drugs targeting the Wnt/β-catenin signaling pathway.Objective:In this study,The Wnt/β-catenin signaling pathway was taken AS the entry point to explore whether the peptide compound Urantide plays a role in preventing and treating AS by regulating the Wnt/β-catenin signaling pathway in the thoracic aorta of Apo E-/-AS mice after antagonizing the UII/UT system,and to clarify its specific mechanism of action.To provide effective experimental data for the development of novel targeted drugs for the clinical prevention and treatment of AS targeting the Wnt/β-catenin signaling pathway.Methods:1.Apo E-/-AS mice model was constructed by high-fat diet,and C57BL/6J mice were used as normal control group.After the successful construction of the pathological model,mice were divided into the following groups according to the principle of randomization:AS model group,positive drug group（simvastatin）,three Urantide treatment groups（20mg/Kg,30mg/Kg,40mg/Kg）,a total of 5 groups,each group 12.Control group and AS model group were given equal volume normal saline by caudal vein injection for two weeks.The positive group was given simvastatin 5 mg/kg intragastric administration for 14 days.The treatment group was given different concentrations of Urantide（20 mg/kg,30 mg/kg,40 mg/kg）by caudal vein injection for 14 consecutive days.The plaque formation in the thoracic aorta of mice was observed by ultrasound imaging technology.2.The levels of triglyceride（TG）,total cholesterol（TC）,low density lipoprotein（LDL）and high density lipoprotein（HDL）in serum of mice in normal control group and model group were detected by automatic biochemical analyzer,and the AS index was calculated.3.Hematoxynlin and eosin staining（H&E staining）was used to observe the morphological changes of thoracic aorta tissues of Apo E-/-mice in each group.4.The protein expression and localization of UII and GPR14 in the thoracic aorta of Apo E-/-mice in each group were detected by immunohistochemical staining（IHC staining）.5.The protein expression and localization of Wnt3a andβ-catenin in the thoracic aorta of Apo E-/-mice in each group were detected by immunofluorescence staining（IF staining）.6.The m RNA expression levels of UII,GPR14,Wnt3a andβ-catenin in the thoracic aorta of Apo E-/-mice in each group were detected by Real-time fluorescence quantitative PCR（RT-q PCR）.7.The protein expression levels of UⅡ,GPR14,Wnt3a,β-catenin,GSK-3β,p-GSK-3β-S9,CK-1,APC and Axin-2 in the thoracic aorta of Apo E-/-mice were detected by Western blotting（WB）.8.The experimental data were expressed as mean±standard deviation（Mean±SD）,and SPSS 26.0 software was used for statistical analysis of the data.One-way analysis of variance（ANOVA）was used for comparison between multiple groups,LSD-t test was used for comparison within groups,and independent sample t test was used for comparison between the two groups.P<0.05 indicated that the difference was statistically significant.Results:1.The preparation of Apo E-/-mice AS model showed that the levels of serum triglyceride（TG）,total cholesterol（TC）and low density lipoprotein（LDL）in AS model group were significantly higher than those in normal control group（P<0.01）,the level of high density lipoprotein（HDL）was significantly lower than that in control group（P<0.01）,and the AS index was significantly increased.In the AS model,the thoracic aorta intima was raised,the arrangement of smooth muscle cells in the media was disordered,and there were some typical AS pathological changes,such as subintimal foam cells,pathological calcification,cholesterol crystals fissure and fibrous cap.2.Doppler ultrasonography showed that the blood vessels in the normal control group were smooth and the cavities were normal.In the model group,atherosclerotic plaques were formed in the thoracic aortic arch,with rough vascular wall,protrusion and lumen stenosis.Urantide treatment group showed no obvious protrusion,no stenosis of lumen.3.Hematoxynlin and eosin staining（H&E staining）results showed that rupture and destruction of thoracic aorta intima,accumulation of necrotic materials and calcified tissues in plaques were observed in the AS model group under light microscope.The pathological changes of thoracic aorta in Urantide administration groups were effectively alleviated.4.The results of immunohistochemical staining（IHC staining）showed that a small amount of UII and GPR14 brown and yellow positive particles were expressed in the thoracic aorta of the normal control group.The expressions of UII and GPR14 proteins in the thoracic aorta of mice in AS model group were significantly increased,mainly in atherosclerotic plaques,foam cells and surrounding tissues.However,the expression levels of UII and GPR14 in Apo E-/-mice decreased after Urantide administration.5.The results of immunofluorescence staining（IF staining）showed that Wnt3a andβ-catenin,key proteins of the Wnt/β-catenin signaling cascade pathway,were slightly expressed in the thoracic aorta of mice in the normal control group.The expressions of Wnt3a andβ-catenin proteins in the thoracic aorta of mice in AS model group were increased,which were mainly found in atherosclerotic plaques and surrounding tissues of mice.After Urantide administration,the expression levels of Wnt3a andβ-catenin in the thoracic aorta were decreased.6.The results of Real-time fluorescence quantitative PCR（RT-q PCR）showed that compared with the normal control group,the m RNA expressions of UⅡ,GPR14,Wnt3a andβ-catenin in thoracic aorta tissues of mice in AS model group were up-regulated（P<0.01）.compared with the AS model group,the m RNA expression levels of UⅡ,GPR14,Wnt3a andβ-catenin in Urantide groups were significantly down-regulated（P<0.01）.7.The results of Western blotting（WB）showed that:Compared with the normal control group,the protein levels of UⅡ,GPR14,Wnt3a,β-catenin and p-GSK-3β-S9 in thoracic aorta tissue of mice in AS model group were significantly increased（P<0.01）,while the protein levels of GSK-3β,CK-1,APC and Axin-2 were significantly decreased.Compared with AS model group,the protein expressions of UⅡ,GPR14,Wnt3a,β-catenin and p-GSK-3β-S9 in Urantide groups were down-regulated（P<0.05）,and the protein levels of GSK-3β,CK-1,APC and Axin-2 were increased（P<0.05）.Conclusions:The peptide Urantide can effectively reduce the gene and protein expression levels of UII and GPR14 in the thoracic aorta of Apo E-/-AS mice.Regulate the expression level of related proteins in the Wnt/β-catenin signaling pathway.These results indicate that Urantide regulates the Wnt/β-catenin signaling pathway by antagonizing the UⅡ/UT system,alleviates the degree of pathological changes in Apo E-/-AS mice,and achieves the purpose of preventing and treating AS. |
PDF Full Text Request