Astragaloside IV Protects against Shear Stress-Induced Glycocalyx Damage and Alleviates Abdominal Aortic Aneurysm by Regulating miR-17-3p/Syndecan-1

Abstract

Background: The present study aimed to analyze the impact of astragaloside IV (AS-IV) on abdominal aortic aneurysm (AAA) and the glycocalyx, elucidating the potential mechanism of AS-IV.

Methods: Rat models of AAA were established using porcine pancreatic elastase. The effects of intraperitoneal AS-IV injection on the morphology, diameter, and glycocalyx of the aorta and the expression of miR-17-3p and Syndecan-1 (SDC1) protein were examined. Differentially expressed miRNAs from peripheral blood samples of healthy individuals, untreated patients with AAA, and treated patients with AAA were identified through sequencing. The relationship between miR-17-3p and SDC1 was validated using a dual-luciferase reporter assay. In vitro, shear stress was induced in human aortic endothelial cells (HAECs) to simulate AAA. Overexpression of miR-17-3p was performed to assess the effects of AS-IV on miR-17-3p and SDC1 expressions, apoptosis, and glycocalyx in HAECs.

Results: AS-IV mitigated aortic damage in AAA rats, reducing the aortic diameter and alleviating glycocalyx damage. In addition, it suppressed the increase in miR-17-3p expression and promoted SDC1 expression in AAA rats. Peripheral blood miR-17-3p levels were significantly higher in patients with AAA than in healthy individuals. miR-17-3p inhibited the SDC1 protein expression in HAECs. In the in vitro AAA environment, miR-17-3p was upregulated and SDC1 was downregulated in HAECs. AS-IV inhibited miR-17-3p expression, promoted SDC1 expression, and mitigated shear stress-induced apoptosis and glycocalyx damage in HAECs. Overexpression of miR-17-3p blocked AS-IV-induced SDC1 expression promotion, glycocalyx protection, and apoptosis suppression in HAECs.

Conclusion: miR-17-3p may damage the glycocalyx of aortic endothelial cells by targeting SDC1. AS-IV may promote SDC1 expression by inhibiting miR-17-3p, thereby protecting the glycocalyx and alleviating AAA.

Figure 1

AS-IV alleviates AAA. Rats were categorized into four groups (n = 10): Sham, AAA, AAA + AS-IV-40, and AAA + AS-IV-40. AAA rat model was constructed using PPE, and the rats in the Sham group received heat-inactivated PPE. AS-IV was administered at doses of 40 mg/kg and 80 mg/kg via gavage. (a,b) Comparison of the aortic inner diameter of rats in each group. (c) The histopathological changes of the abdominal aorta in each group were analyzed using HE staining.  ^∗∗∗P < 0.001 vs. Sham group; ^#P < 0.05, ^##P < 0.01 vs. AAA group; ^{^}P < 0.05 vs. AAA + AI-IV group. AS-IV, astragaloside IV; and AAA, abdominal aortic aneurysm.

Figure 2

AS-IV upregulates SDC1 and protects glycocalyx structure and function in AAA rats. (a,b) Glycocalyx thickness in the abdominal aortic endothelial cells was observed using TEM. (c) Immunohistochemical staining was used to observe SDC1 expression, a key glycocalyx protein, in the abdominal aorta.  ^∗∗∗P < 0.001 vs. Sham group; ^#P < 0.05, ^###P < 0.001 vs. AAA group; ^^P < 0.01 vs. AAA + AS-VI-40 group. AS-IV, astragaloside IV; AAA, abdominal aortic aneurysm; SDC1, syndecan-1; and TEM, transmission electron microscopy.

Figure 3

AS-IV inhibits miR-17-3p expression in endothelial cells of AAA rats. (a) The heat map shows different miRNAs in the peripheral blood of healthy individuals, untreated patients with AAA, and treated patients with AAA. (b) FISH was used to detect miR-17-3p expression in the abdominal aorta. (c) RT-qPCR was used to detect miR-17-3p expression in the abdominal aorta.  ^∗∗∗P < 0.001 vs. Sham group; ^###P < 0.001 vs. AAA group; ^^^P < 0.001 vs. AAA + AS-IV-40 group. AS-IV, astragaloside IV; AAA, abdominal aortic aneurysm; FISH, fluorescence in situ hybridization; and RT-qPCR, real-time quantitative reverse transcription PCR.

Figure 4

miR-17-3p inhibits SDC1 expression in HAECs. (a) The targeted binding sites of miR-17-3p and SDC1 mRNA. (b) The relationship between miR-17-3p and SDC1 was confirmed using the dual-luciferase report assay. (c) miR-17-3p overexpression by transfection. (d, e) Comparison of SDC1 mRNA and protein levels in HAECs in each group. (f) miR-17-3p silencing by transfection. (g, h) Comparison of SDC1 mRNA and protein levels in HAECs in each group.  ^∗∗∗P < 0.001 vs. Mut-SDC1 group or Control group; ^###P < 0.001 vs. miR-17-3p NC group. SDC1, syndecan-1 and HAECs, human aortic endothelial cells.

Figure 5

AS-IV inhibits miR-17-3p expression and promotes SDC1 expression in HAECs. (a) The impact of shear stress on miR-17-3p expression in HAECs. (b–d) The impact of shear stress on SDC1 mRNA and protein expression in HAECs. (e) The impact of different AS-IV concentrations on cell viability of HAECs. (f) The impact of AS-IV on miR-17-3p expression in HAECs. (g–i) The impact of AS-IV on SDC1 mRNA and protein expression in HAECs.  ^∗P < 0.05,  ^∗∗P < 0.01,  ^∗∗∗P < 0.001 vs. 0 dyn/cm² shear stress group or 0 μmol/L AS-IV group. AS-IV, astragaloside IV; AAA, abdominal aortic aneurysm; SDC1, syndecan-1; and HAECs, human aortic endothelial cells.

Figure 6

AS-IV mitigates shear stress-induced damage in HAECs via miR-17-3p inhibition. HAECs were divided into four groups: control, SS (5 dyn/cm² shear stress), SS + AS-IV (100 μmol/L), and SS + AS-IV + miR-17-3p. (a) Comparison of miR-17-3p expressions in each group. (b–d) Comparison of SDC1 mRNA and protein expressions in each group. (e,f) Comparison of cell apoptosis rates, including early and late apoptosis, in each group. Early apoptosis was observed in the B4 quadrant, and late apoptosis was observed in the B2 quadrant. (g) Immunofluorescence staining revealed the CS expression level to assess the glycocalyx damage.  ^∗P < 0.05,  ^∗∗∗P < 0.001 vs. Control group; ^#P < 0.05, ^##P < 0.01 vs. SS group; ^{^^}P < 0.01, ^{^^^}P < 0.001 vs. SS + AS-IV group. AS-IV, astragaloside IV; HAECs, human aortic endothelial cells; SS, shear stress; and CS, chondroitin sulfate.