Inhibitory Effect And Mechanism Of Urantide On MAPK Signaling Pathway In Atherosclerotic Rats

Atherosclerosis?AS?plays an important role in cardiovascular diseases as the pathological basis of many diseases.Research on the pathogenesis and prevention of AS has been a hot topic in medical research.As early as 1999,some scholars proposed the"inflammation theory"of AS,pointing out that the development process of AS always accompanied the inflammatory reaction.Pathological changes such as lipid deposition,endothelial cell damage,and smooth muscle cell proliferation in AS are involved in the involvement of various inflammatory factors and cells.The interaction and interaction of inflammatory factors promote the development of AS.The production and action of inflammatory factors in AS is in the fine inflammatory regulation network of the body.It affects the expression and activity of inflammation-related downstream effector proteins by acting on the signal transduction pathway to affect the genes that play a key regulatory role,thereby participating in AS formation.Therefore,in-depth study of the AS inflammatory signaling pathway or its key signal transduction molecules is of great significance for the intervention of AS disease from the perspective of inflammation.Mitogen-activated protein kinases?MAPKs?are present in all eukaryotic cell regulation processes and are the main signaling pathways regulating inflammatory factors and mediator activation.Once activated,MAPK signal transduction pathways achieve diversified coordination and comprehensive response to cell stimulation,and control the synthesis and release of inflammatory factors,which are closely related to the occurrence and development of AS.Inhibition of activation of this pathway is a good strategy for treating AS.Urotensin ??U??is the most potent vasoconstrictor active substance currently known to bind to its receptor G protein-coupled receptor 14?GPR14?to form the U?/UT system.Promote the development of AS.Urantide is derived from the human U? fragment and is considered to be the most potent antagonist of U?-specific receptors.Our previous study found that U? binds to its receptor GPR14,which activates the MAPK signaling pathway and participates in the development of AS through inflammatory mechanisms.Urantide inhibits the activation of this pathway and blocks the promotion of U? by AS,but the specific mechanism still not clear.In this study,the MAPK signal pathway was used as the entry point.Serological,morphological,immunohistochemical,qRT-PCR,Western blot and other experimental techniques were used to study the effect of Urantide on the MAPK signaling pathway in the thoracic aorta of AS rats.The mechanism of action of the signaling pathway provides a new direction for the clinical treatment of AS.Objective:To investigate the inhibitory effect of Urantide on MAPK signaling pathway in AS rats,and to elucidate the relationship between Urantide anti-AS target and protein cascade of MAPK signaling pathway,and provide new ideas for Urantide treatment of AS.Methods:1.Each group of rats was fed a high-fat diet and combined with intraperitoneal injection of vitamin D₃?VD₃?for 3 days to establish an AS rat model.180 Wistar rats were randomly divided into normal control group?NC?,model group?AS?,simvastatin group,Urantide 3 d,7 d,14 d group.Body weight was weighed before the experiment,before the drug,and at the end of the experiment.2.Automatic biochemical analyzer was used to detect serum calcium(Ca²⁺),triglyceride?TG?,total cholesterol?TC?and low density lipoprotein?LDL?and high in each group.High density lipoprotein?HDL?level.3.HE staining was used to observe the pathological changes of thoracic aorta in each group.4.Immunohistochemical staining technique was used to observe the effect of Urantide on the expression of p38,JNK and ERK in the thoracic aorta of each group.5.Immunofluorescence staining technique was used to observe the expression of pp38,pJNK and pERK in the thoracic aorta of each group.6.Real-time quantitative PCR?RT-qPCR?was used to detect the effect of Urantide on p38,JNK and ERK mRNA expression in thoracic aorta of each group.7.Western blot analysis was used to detect the effect of Urantide on the expression of p38,pp38,JNK,pJNK,ERK and pERK proteins in rat thoracic aorta.8.Statistical analysis was performed using SPSS 19.0 software and the data were expressed as mean±standard deviation?x±s?.One-way analysis of variance was used to perform statistical tests on differences between groups.The least significant difference method?LSD-t test?was used to test the comparison between the two groups.P<0.05 indicates that the difference was statistically significant.Results:1.The body weight of the rats in the AS group was significantly lower than that in the normal control group?P<0.01?.Rats in each administration group had different degrees of weight gain after administration?P<0.05 or P<0.01?.2.Serological results showed that the levels of Ca²⁺,TC,TG and LDL in the serum of the AS group were significantly higher than those in the normal control group?P<0.01?,and the HDL level was significantly lower?P<0.01?.Compared with the AS group,serum levels of Ca²⁺,TG,TC and LDL were decreased in the Urantide group?P<0.05 or P<0.01?,and HDL levels were increased?P<0.05 or P<0.01?.3.HE staining results showed that typical AS lesions occurred in the thoracic aorta of rats in the AS group,while the degree of thoracic aorta AS lesions gradually decreased in the Urantide administration group with the prolonged administration time.4.Immunohistochemical staining showed that the positive staining of p38,JNK and ERK in the thoracic aorta of AS group was significantly increased compared with the normal control group?P<0.01?.With the prolongation of administration time,Urantide gradually decreased the expression of p38,JNK and ERK in the thoracic aorta of each group compared with the AS group?P<0.01?.5.Immunofluorescence staining showed that pp38,pJNK and pERK were slightly expressed in the thoracic aorta of normal control rats.Compared with the normal control group,the positive staining of pp38,pJNK and pERK in the AS group was significantly increased in the rat thoracic aorta?P<0.01?.Urantide significantly reduced the expression of pp38,pJNK and pERK in rat thoracic aorta,reaching or close to the level of positive drug group?P<0.01?.6.Real-time quantitative PCR results showed that the expressions of p38,JNK and ERK genes in the thoracic aorta of the AS group were significantly higher than those in the normal control group?P<0.01?.Compared with the AS group,the gene expression of p38,JNK and ERK decreased in the thoracic aorta of each administration group with the prolongation of the administration time?P<0.01?.7.The results of immunoblotting showed that the expressions of p38,pp38 and JNK,pJNK,ERK and pERK in the thoracic aorta of the AS group were significantly higher than those in the normal control group?P<0.01?.The protein expressions of p38,pp38,JNK,pJNK,ERK and pERK in the thoracic aorta of the rats in each administration group were significantly lower than those in the AS group?P<0.05 or P<0.01?.Conclusion:1.Urantide can reduce the levels of Ca²⁺,TG,TC and LDL in serum of AS rats,and increase the level of HDL.With the prolongation of administration time,the degree of AS in the thoracic aorta is gradually reduced,and the symptoms of AS in rats are relieved.2.Urantide down-regulates the expression levels of p38,JNK,ERK mRNA and protein phosphorylation in thoracic aorta,and has anti-inflammatory effects on the thoracic aorta of AS rats.