Role Of Serum Amyloid A In The Pathogenesis Of Aortic Dissection And Targeting Theraphy

Objective:The aim of this study was to investigate the value of serum amyloid A(SAA)in the diagnosis and prognostic evaluation of acute aortic dissection(AAD)and the molecular mechanism of its mediated NF-κB signaling pathway in the pathogenesis of AAD.To investigate the mechanism of SAA in the pathogenesis of AAD and its intervention from multiple levels of gene,protein and molecular mechanism.To investigate the association between single nucleotide polymorphisms(SNPs)in SAA2 gene and the incidence of AAD in Xinjiang Han population at the genetic level.To determine the efficacy and feasibility of SAA as a potential biomarker for the diagnosis and prognostic evaluation of AAD at the clinical level by detecting its expression in serum and aortic tissue of patients with AAD.At the level of animals and cells,SAA was systematically explored to induce TGF-β synthesis by activating NF-κB signaling pathway,and then activating TGF-β/Smad signaling pathway to promote the phenotypic conversion of VSMCs,while aggravating extracellular matrix degradation and inflammatory response,and damaging aortic structure leading to the occurrence of AAD.However,inhibiting NF-κB activity could effectively reduce the incidence of dissection and reduce aortic wall injury,providing new ideas and basis for the prevention and treatment of AAD.1)To investigate the association between SAA2 gene polymorphism and AAD in Xinjiang Han population;2)To explore the effectiveness and feasibility of SAA as a marker for the diagnosis and prognostic evaluation of AAD;3)To investigate the mechanism of SAA in aortic wall injury by activating NF-κB signaling pathway.Methods:Part I: 182 patients with AAD and 101 healthy controls were selected.The gene polymorphism of SAA2 was genotyped and haplotype constructed by Taqman genotyping technique.The association between SAA2 gene polymorphism and AAD in Xinjiang Han population was analyzed.Part II: A case-control study was conducted to investigate the expression of SAA in serum of patients with AAD and to analyze the efficacy and feasibility of SAA as a diagnostic and prognostic marker of AAD.Part III: AAD model was established in male C57BL/6J mice aged 3 weeks.Aortic dilatation was detected by ultrasound in small animals,and aortic wall destruction was determined by HE staining and EVG staining.Serum inflammatory factor expression was measured by ELISA in AAD mice,and the expression of NF-κB signaling pathway proteins and downstream target proteins was detected by Western blot.Part IV: Human aortic VSMCs were selected and the injury model of VSMCs was established by exogenous stimulation with SAA.CCK8,TUNEL and scratch assay were used to detect the migration,proliferation and apoptosis of VSMCs.ELISA was used to detect the expression of inflammatory factors in the supernatant,and Western blot was used to detect the expression of NF-κB signaling pathway proteins and downstream target proteins in VSMCs.Results:Part I: 1)In this study,we described the frequency and difference of SAA2 gene polymorphism in AAD in Xinjiang Han population,and also found that the mutant genotype A-and--at rs58546509 of SAA2 gene may be risk factors for AAD;2)rs56373142 of SAA2 gene may not be associated with the occurrence of AAD in Xinjiang Han population.Part II: 1)SAA was significantly increased in the serum of AAD patients compared with healthy controls(P<0.001),and further subgroup analysis showed that SAA expression was higher in AAD patients with chest pain duration<24 hours as well as death,with no difference between genders;2)SAA,systolic blood pressure(SBP),and white blood cell(WBC)were identified as independent risk factors for AAD by multivariate Logistic regression analysis.The diagnostic efficacy of the above parameters was determined by plotting ROC curves and nomograms were constructed for quantitative prediction of the occurrence of AAD using SAA,SBP,and WBC.The predictive model was assessed using the Hosmer-Lemeshow goodness-of-fit test and calibration curves.ROC curve analysis was used to verify the predictive efficacy of the prediction model for AAD patients,and the AUC value of this model could reach 0.994 and the sensitivity and specificity of this model reached more than 90%,which had good diagnostic value for AAD.3)According to the optimal cut-off value of SAA,AAD patients were divided into high-risk group and low-risk group,and the patients were followed up for an average of20.3 months and Kaplan-Meier survival analysis was used to analyze the survival of high-risk group and low-risk group,and the results showed that the incidence of adverse events in the high-risk group was significantly higher than that in the low-risk group;4)Western blot confirmed that SAA was significantly highly expressed in the aortic tissue of AAD patients compared with CABG patients;5)The expression of MMP9 and TGF-βwas also significantly increased in the serum of patients with AAD,which indicated that extracellular matrix degradation and TGF-β/Smad signaling pathway activation were involved in the development of AAD.Part III: 1)AAD mouse model was successfully established in this study,and the modeling rate was up to 90%.When PDTC was administered to inhibit NF-κB signaling pathway,the modeling rate of AAD decreased to40%;2)Echocardiography showed that the ascending aorta and abdominal aorta of AAD mice were significantly dilated,and the aortic systolic and diastolic diameter and ejection velocity were significantly increased in the AAD group,which decreased when PDTC was intervened;3)HE and EVG staining showed the aortic wall tear and elastic fiber loss,and it was found that the aortic structure was destroyed,the cells were lost,and the elastic fiber layer was significantly torn in the AAD group,and the aortic destruction was alleviated when PDTC inhibitors were administered;4)The transcription level of SAA in liver and the expression level of SAA in serum of AAD mice were significantly higher than those of WT mice,suggesting that when AAD occurs,SAA is mainly synthesized by liver transcription and secreted into the circulation to participate in subsequent reactions;5)Cytoplasmic protein IκBα was significantly decreased and P65 and P50 nuclear entry was significantly increased in the AAD group compared with the WT group,suggesting that NF-κB signaling pathway was activated in AAD mice to allow IκBα degradation and P65 and P50 nuclear entry,while no significant IκBα degradation and P65 and P50 nuclear entry were observed in the PDTC group;6)The expression of TGF-β1,Smad2/3,and the synthetic protein OPN was significantly increased and the expression of the contractile proteins α-SMA and SM22α was significantly decreased in the aortas of AAD mice compared with WT.This suggests that NF-κB activation can further activate the TGF-β/Smad signaling pathway to promote the abnormal phenotypic conversion of mediated VSMCs,showing the opposite trend when PDTC inhibitors were administered.In addition,we found that MMP9 expression was significantly elevated in the aortas of AAD mice compared with WT and PDTC groups,indicating increased degradation of the extracellular matrix;7)ELISA detected the expression of IFN-α,IL-1β,IL-6,and TNF-α,and the results showed that all four inflammatory factors were significantly highly expressed in the AAD group compared with the WT group,and the expression levels of inflammatory factors were decreased when PDTC was administered to inhibit NF-κB activation compared with the AAD group.Part IV: 1)This study showed that after exogenous stimulation of VSMCs with SAA,the proliferation and migration ability of cells decreased significantly and the number of apoptotic cells increased with the increase of stimulation time and concentration;2)VSMCs were stimulated with SAA at a concentration of 10μg/ml,and the translocation activation of NF-κB was measured at different stimulation time points(0min,5min,15 min,30min,60 min and 24h),respectively.The results showed that cytoplasmic protein IκBα gradually decreased and P65 and P50 nuclear entry significantly increased with the extension of stimulation time;3)After exogenous stimulation of VSMCs with different concentrations of SAA,the expression of TGF-β1,Smad2/3 and synthetic protein OPN was significantly increased,and the expression of contractile protein α-SMA and SM22α was significantly decreased with increasing stimulation time and concentration.The expression showed the opposite trend when PDTC inhibitors and TGF-β inhibitors were administered;4)After exogenous stimulation of VSMCs with different concentrations of SAA,the expression of MMP in cells and inflammatory factors in supernatants increased significantly with increasing stimulation time and concentration,and the expression of the above proteins showed the opposite trend when PDTC inhibitors and TGF-β inhibitors were administered.Conclusion:1)This study found that rs58546509 polymorphism of SAA2 gene was a risk factor for AAD in Xinjiang Han population;2)At the clinical level,SAA is not only significantly elevated in the tissues and serum of patients with AAD,but also has good value in the diagnosis and prognostic evaluation of AAD or can be used as a potential biomarker for early diagnosis and prognostic evaluation of AAD;3)In this study,we confirmed at the cellular and animal levels that SAA induces TGF-β synthesis by activating NF-κB,which in turn activates the TGF-β/Smad signaling pathway and promotes phenotypic switching in VSMCs.At the same time,activated NF-κB induced increased secretion of MMP9 and related inflammatory factors,and injury of aortic structure led to the development of AAD,while inhibition of NF-κB activity effectively reduced the incidence of dissection.