| Atherosclerosis(AS)is the most common disease of the cardiovascular system,which has become the number one killer of human death.Therefore,the research on AS pathogenesis has attracted much attention.With the rapid development of life sciences,extensive expression of inflammation-related factors such as C-reactive protein and interleukin-6(IL-6)have been detected in various stages of AS development in recent years,which shows that the immune inflammatory response plays an important role in the occurrence,development and outcome of AS.IL-6 is an inflammatory factor of AS,promotes the development of plaques,and participates in the formation of AS,which has become one of the important markers of AS plaques.After IL-6 binds to its receptor,it could activate the janus kinases/signal transducer and activator of transcriptions(JAK-STAT)signal pathway,which is closely related to the immune inflammatory response,thus affecting the interaction between inflammatory factors,enhancing the cascade of inflammatory responses,and then becoming an important key pathological link in AS.Numerous studies have found that activation of the IL-6/JAK2/STAT3 signaling pathway may cause a variety of pathological manifestations and promote the occurrence and development of AS,such as dysfunction of vascular endothelial cells,migration and proliferation of vascular smooth muscle cells,proliferation,differentiation and migration of inflammatory cells and over-expression of pro-inflammatory cytokines.Therefore,inhibiting the IL-6/JAK2/STAT3 signaling pathway activated may effectively alleviate the pathological symptoms of AS and provide an important experimental basis for the development of anti-AS drugs.Urotensin(U? ?)was currently known as the most potent vasoconstrictor substance,which formed a U?/UT system with G protein coupled receptor 14(GPR14)and played a vital role in promoting AS.Urantide was a U? receptor antagonist derived from U?,which can effectively inhibit the U?/UT system and devitalize its biological function.Earlier studies found that binding U? to its receptor GPR14 may activate the IL-6/JAK2/STAT3 signaling pathway and participate in the progress of AS through inflammatory mechanisms;while urantide can block the U?/UT system and thus inhibit the activation of this pathway,and alleviate the progress of AS,however,the specific mechanism of action was unclear.However,whether urantide antagonizes the U?/UT system will affect the IL-6/JAK2/STAT3 signaling pathway has not been studied.Therefore,in this paper,rats with AS were used as the research object to study the regulation and mechanism of urantide on IL-6/JAK2/STAT3 signaling pathway,and to explore the relationship between urantide and the upstream and downstream proteins of this signaling pathway and inflammatory mediators activated by this signaling pathway.This study aims to clarify that urantide's anti-AS target is related to inhibition of the IL-6/JAK2/STAT3 signaling pathway protein cascade,and to develop it as a new anti-AS targeted therapeutic drug against the abnormal activation of the IL-6/JAK2/STAT3 signaling pathway.Objective: To investigate the effect and mechanism of urantide on the IL-6/JAK2/ STAT3 signaling pathway in the thoracic aorta of rats with AS,so as to provide new therapeutic targets for the prevention and treatment of AS.Methods: 1.We established the AS rat model by a high-fat diet and intraperitoneal injection of vitamin D3(VD3),then randomly divided the rats into two groups: the normal control group and the model group;after the model group was successfully prepared,rats with AS were divided into five groups: the AS model group,the simvastatin group,and the 3 days' using of urantide group,7 days' using of urantide group and 14 days' using of urantide group.There are 30 rats in each group.2.The automatic biochemical analyzer detects the levels of total cholesterol (TC),triglyceride(TG),high density lipoprotein(HDL),low density lipoprotein(LDL)and calcium ion(Ca2+)in rat serum of the normal control group and the model group,and calculates AS index;3.Hematoxylin and eosin staining(HE)staining was used to observe the morphological differences of the rat's thoracic aorta in these groups.4.The immunofluorescence staining was used to detect IL-6,p-JAK2 and p-STAT3 protein expression and their localization in the thoracic aorta of each group.5.Real-time fluorescence quantitative PCR(RT-q PCR)was used to detect the m RNA expression levels of IL-6,JAK2 and STAT3 in the thoracic aorta of each group.6.Western blotting was used to detect the expression levels of U?,GPR14,IL-6,JAK2,p-JAK2,STAT3,and p-STAT3 proteins in the thoracic aorta of each group of rats.7.SPSS 20.0 software was used to statistically analyze the experimental data,and it was expressed as mean ± standard deviation(Mean ± SD).The independent sample t test was used for comparison between the two groups;one-way ANOVA was used for comparison among multiple groups,and LSD-t test was used for pairwise comparison.The difference was statistically significant at P<0.05.Results: 1.The model preparation results showed that: compared with the normal control group,the serum levels of TC,TG,LDL and Ca2+ in the model group were significantly increased(P<0.01),the HDL level was significantly reduced(P<0.01),and the AS index was significantly increased(P<0.01);meanwhile,in the model group,the intima of the thoracic aorta was raised,the arrangement of medial smooth muscle cells was disordered,the elastic fiber layer was broken,and there were typical AS pathological changes such as foam cells,calcified tissues and fiber caps,and central necrotic substances.2.The results of HE staining showed that,in the AS model group,more prominent AS lesions occurred in the thoracic aorta of the rat,including intima rupture,calcified tissue,a large amount of central necrotic substance in the atherosclerotic plaque and cholesterol crystal gap;compared with the AS model group,the thoracic aorta lesions of the urantide experimental group were significantly reduced and were time-dependent.3.The results of immunofluorescence staining showed that the IL-6,p-JAK2 and p-STAT3 proteins were expressed in a small amount in the thoracic aorta of the normal control group.Compared with the normal control group,IL-6,p-JAK2 and p-STAT3 was abundantly expressed and the fluorescence intensity was increased in the AS model group.IL-6,p-JAK2 and p-STAT3 are expressed in atheromatous plaque and surrounding tissues,where the fluorescence intensity was obviously stronger than other parts.Compared with the AS model group,the fluorescence intensity of IL-6,p-JAK2 and p-STAT3 in the thoracic aorta of urantide experimental groups gradually weakened.4.RT-q PCR results showed that compared with the normal control group,the m RNA expression levels of IL-6,JAK2 and STAT3 in the thoracic aorta of the AS model group were increased(P<0.01);the groups treated with the urantide,compared with the AS model group,their relative expression levels of IL-6,JAK2,and STAT3 genes showed a significant downward trend(P <0.01).5.Western blotting results showed that compared with the normal control group,U?,GPR14,IL-6,p-JAK2,JAK2,p-STAT3 and STAT3 proteins were significantly increased in AS rat thoracic aorta(P<0.01);The groups treated with the urantide,compared with the AS model group,their expression of U?,GPR14,IL-6,p-JAK2,JAK2,p-STAT3,and STAT3 was significantly reduced(P<0.01).The proteins of UII,GPR14 and IL-6 had a significant downward trend,and the expression of p-JAK2,JAK2,p-STAT3,and STAT3 protein was better at the seventh day after giving urantide.Conclusion The peptide compound urantide may effectively reduce U ?and GPR14 protein expression in the thoracic aorta of rats with AS,and down-regulate the expression of genes and proteins related to the IL-6/JAK2/STAT3 signaling pathway.Therefore,this study concluded that by antagonizing the U?/UT system,urantide may inhibit the activation of the IL-6/JAK2/ STAT3 signaling pathway thereby improving the pathological changes of rats with AS. |
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