Antagonizing The Activity Of RORγt With Digoxin Attenuates The Progression Of Experimental Abdominal Aortic Aneurysm

Part Ⅰ Protein expression of IL-17A, ROR y t in human abdominal aortic aneurysm lesionsObjective To investigate the protein expression of IL-17A, RORyt in the aortic wall specimens.Methods The aortic wall specimens from abdominal aortic aneurysm patients and patients who underwent aortic surgery with non-aneurysm were collected and divided into two groups:Abdominal Aortic Aneurysm group (AAA group, n=10) and Control group (n=10). Histopathologic analysis and Western blot test were utilized for study. The elastica van Gieson (EVG) staining and immunohistochemical (a-SM actin, CD31, CD4, and Mac2) staining were performed for histopathologic analysis. And the protein levels of IL-17A and RORyt were also analyzed via Western blot analysis.Results The elastin fragmentation, SMC destruction, and neovascularization within the adventitia and inflammatory cell (such as CD4+T cells and Mac2+macrophages) infiltration in the aortic sections were noted in abdominal aortic aneurysm lesions via hiistopathologic analysis. Moreover, compared with the control specimens, the protein level of IL-17A and RORyt were conspicuously increased in aortic tissues from AAA patients.Conclusions The role of IL-17in AAA is still controversial, but our research indicates that the protein expression of IL-17A and RORyt are obviously increased in aortic aneurysm tissues, and inflammatory cells infiltrate in the aortic sections. The IL-17A may promote the progression of abdominal aortic aneurysm. Part Ⅱ Digoxin confers protection against the experimental abdominal aortic aneurysm induced by Angiotensin Ⅱ in ApoE-/-miceObjective To investigate the effect and mechanism of digoxin treatment in Angiotensin Ⅱ (Ang Ⅱ) induced experimental abdominal aortic aneurysm in ApoE-/-mice.Methods A mouse experimental abdominal aortic aneurysm model was established by subcutaneous Ang Ⅱ infusion in ApoE-/-mice (Ang Ⅱ/APOE model). all ApoE-/-mice (16weeks age, male, n=80) were randomly divided into the following4groups: Sham group, saline infusion with0.5%DMSO treatment (intraperitoneal injection [iP], n=20); Control group, Ang Ⅱ infusion with0.5%DMSO treatment (iP, n=20); Low-dose group, Ang Ⅱ infusion with low-dose digoxin treatment (Sigma-Aldrich,20jig/day per mouse)(iP, n=20); and High-dose group, Ang Ⅱ infusion with high-dose digoxin (40μg/day per mouse)(iP, n=20). The aortic diameter was measured via ultrasound imaging at30MHz using a high-resolution Vevo2100Microimaging System, and the suprarenal aorta was chosen as the measuring point in the Ang II/APOE model on days0,7,14,21, and28thereafter. To investigate the effect of digoxin on Ang II-induced hypertension, the Softron BP-2010Series, a noninvasive tail-cuff system, was utilized for the measurement of Systolic Blood Pressure (SBP). Twenty-eight days after Ang Ⅱ infusion, the mice were anesthetized and the abdominal aortic specimens were harvested. The hematoxylin and eosin(H&E) elastica van Gieson (EVG) staining, immunostaining (α-SM actin, CD31, CD4, and Mac2) and immunofluorescent staining were performed for histopathologic analysis. Flow cytometry was utilized to analyze the proportion of lymphocytes in the spleen. The protein levels of IL-17A, RORyt, MCP-1, IFN-y, RANTES and MMP-2, MMP-9were determined by Western blot analysis.Results (1) Compared with the Control group, the AAA incidence in the High-dose group was significantly reduced from70%(Control group) to35%, the survival ratio was notably increased in the High-dose group.(2) Digoxin treatment had no effect on Ang II-induced hypertension.(3) High-dose treatment preserves the structure of the aortic wall in the Ang II/APOE model.(4) High-dose treatment attenuates local inflammatory cell infiltration in the Ang II/APOE Model.(5) High-dose treatment suppresses the protein expression of pro-inflammatory mediators and MMPs in the Ang II/APOE model.(6) High-dose treatment changes the Th17cells, Tregs percentage in the splenocytes of APOE-/-mice.Conclusions Treatment with digoxin, especially high dose treatment inhibits the Th17/IL-17A-related inflammatory responses, preserves the structure of the aortic wall, reduces local inflammatory cell infiltration and inflammatory factors expression, attenuates the progression of abdominal aortic aneurysm. Part Ⅲ Digoxin Treatment confers protection against the experimental abdominal aortic aneurysm induced by porcine pancreatic elastase in C57BL/6J miceObjective To investigate the effect and mechanism of digoxin treatment in porcine pancreatic elastase (PPE) induced experimental abdominal aortic aneurysm in C57BL/6J mice.Methods To fully investigate the effect of digoxin, a selective antagonist of the activity of RORyt, another mouse experimental abdominal aortic aneurysm model was established by intra-aortic PPE perfusion in C57BL/6J mice (PPE/C57model). All C57BL/6J mice (10weeks age, male, n=42) were randomly divided into the following3groups:Sham group, saline perfused with0.5%DMSO treatment (iP, n=12); Control group, PPE perfused with0.5%DMSO (iP, n=15); and Digoxin group, PPE perfused with digoxin (40μg/day per mouse)(iP, n=15). The aortic diameter was measured via ultrasound imaging at30MHz using a high-resolution Vevo2100Microimaging System, and the infra-renal abdominal aorta was chosen as the measuring point in the PPE/C57model on days0,7and14thereafter. Fourteen days after PPE perfusion, the mice were anesthetized and the abdominal aortic specimens were harvested. The elastica van Gieson (EVG) staining and immunostaining (a-SM actin, CD31, CD4, and Mac2) were performed for histopathologic analysis. The protein levels of IL-17A, MCP-1, IFN-y and MMP-2were determined by Western blot analysis.Results (1) Compared with the Control group, the AAA incidence in the Digoxin group was significantly reduced (33.3%vs71.4%, P<0.05).(2) Digoxin treatment preserves the structure of the aortic wall in the PPE/C57model.(3) Digoxin treatment attenuates local inflammatory cell infiltration in the PPE/C57model.(4) Digoxin treatment suppresses the protein expression of pro-inflammatory mediators and MMP-2in the PPE/C57model.Conclusions Treatment with digoxin inhibits the Th17/IL-17A-related inflammatory responses, preserves the structure of the aortic wall, reduces local inflammatory cell infiltration and inflammatory factors expression, attenuates the progression of abdominal aortic aneurysm in a model-independent manner.