miR-9-5p alleviates the development of abdominal aortic aneurysm by regulating the differentiation of CD4+IL-10+T cells via targeting the crosstalk between Nrf2 and NF-κB signaling pathways

Abstract

Background: Abdominal aortic aneurysm (AAA), a gradual segmental dilatation of the abdominal aorta, is associated with a high mortality rate. The pathophysiological molecular mechanisms underlying AAA remain unclear. In recent years, changes in miRNA levels have been reported to be involved in the development and treatment of AAA. This study aimed to investigate the potential targets and underlying mechanisms of miR-9-5p in attenuating AAA progression by modulating the inflammatory response.

Materials and methods: Biochemical kits were used to measure the levels of inflammatory factors, antioxidant enzyme activity, and serum oxidative stress in normal and AAA model mice. miR-9-5p overexpression was achieved by transfecting miR-9-5p mimics into CD4⁺ T cells and administering an miR-9-5p agomir to the mice. The effect of miR-9-5p overexpression was evaluated by detecting the expression level of miR-9-5p in CD4⁺ T cells through qRT-PCR. The NF-κB/Nrf2 pathway levels were assessed using immunofluorescence, western blotting, and quantitative PCR. miR-9-5p expression was modulated by transfecting either miR-9-5p mimics or inhibitors, and the impact on CD4⁺IL-10⁺ T-cell differentiation was analyzed using flow cytometry.

Results: Compared with that in the control group, miR-9-5p expression in CD4⁺ T cells from the peripheral blood of AAA model mice was decreased by 28%. In vivo, miR-9-5p intervention reduced AAA formation in model mice and markedly decreased serum oxidative stress damage and inflammatory factor levels. Furthermore, miR-9-5p intervention significantly increased miR-9-5p levels in CD4⁺ T cells both in vitro and in vivo, increased the proportion of CD4⁺IL-10⁺ T cells, suppressed NF-κB expression, and upregulated Nrf2 and its downstream antioxidant genes. Conversely, these therapeutic effects were abolished when an miR-9-5p inhibitor was administered.

Conclusions: By controlling the interaction between the Nrf2 and NF-κB signaling pathways, miR-9-5p mediates the differentiation of CD4⁺IL-10⁺ T cells and alleviates the development of AAA.

Keywords: Abdominal aortic aneurysm; CD4+ T cells; Nrf2; miR-9-5p; oxidative stress.

Figure 1

miR-9-5p protects against Ang II-induced AAA formation. (A) The scheme of the experimental design. (B) Survival rates after Ang II infusion-induced AAA. (C) Incidence of AAA formation. (D) Maximal abdominal aortic diameter. (E) Representative photomicrographs of H&E-stained renal sections (bar = 50 μm). Data represent the mean scores ± SEM of at least three independent experiments, ^**p < 0.01, Con (Control) group vs. AAA group; ^##p < 0.01, AAA+Ago group vs. AAA+Ago NC group.

Figure 2

miR-9-5p attenuates disorder of redox metabolism and inflammatory response in AAA lesion mice. Levels of serum TOS (A) and TAC (B). Levels of serum cytokines IL-6 (C), IFN-γ (D), TNF-α (E), and IL-10 (F). Data represent the mean scores ± SEM of at least three independent experiments, ^*p < 0.05, ^**p < 0.01, Con group vs. AAA group; ^##p < 0.01, ^#p < 0.05, AAA+Ago group vs. AAA+Ago NC group. TAC, total antioxidant capacity; TOS, total oxidative stress; IL-6, interleukin-6; IFN-γ, interferon-gamma; IL-10, Interleukin-10; TNF-α, tumor necrosis factor alpha.

Figure 3

miR-9-5p regulates the differentiation of CD4⁺IL-10⁺ T cells in AAA lesion mice. (A) Gating strategy: forward scatter (FSC) and side scatter (SSC) gating were used to discriminate viable cells from cell debris. Within the lymphocyte gate, CD3 was used as a T cell maker. (B) Representative dot plots of CD4⁺IL-10⁺ T cells. (C) The percentage of CD4⁺IL-10⁺ T cells. (D) The expression of miR-9-5p. (E) Pearson’s correlation analysis between the levels of miR-9-5p and the percentage of CD4⁺IL-10⁺ T cells. Data represent the mean scores ± SEM of at least three independent experiments, ^*p < 0.05, ^**p < 0.01, Con group vs. AAA group; ^##p < 0.01, ^#p < 0.05, AAA+Ago group vs. AAA+Ago NC group.

Figure 4

miR-9-5p regulates the expressions of NF-κB-Nrf2 pathway of CD4⁺ T cells in AAA lesion mice. (A) The protein expressions of the NF-κB-Nrf2 pathway and its downstream target genes were evaluated by western blot. (B) The protein level of nuclear NF-κB. (C) The protein level of nuclear Nrf2. (D) The protein level of HO-1. (E) The protein level of NQO1. (F) Nuclear translocation of Nrf2 and NF-κB were determined by immunofluorescent staining (bar = 10 μm). Data represent the mean scores ± SEM of at least three independent experiments, ^*p < 0.05, ^**p < 0.01, Con group vs. AAA group; ^##p < 0.01, ^#p < 0.05, AAA+Ago group vs. AAA+Ago NC group. NF-κB, nuclear factor kappaB; Nrf2, NF-E2-related factor 2; NQO1, NAD(P)H quinone oxidoreductase 1; HO-1, heme oxygenase-1.

Figure 5

miR-9-5p Directly Targets the NF-κB. (A) Target miRNAs intersection analysis for NF-κB. (B) Analysis of intersections among AAA-related miRNAs and NF-κB target miRNAs. (C) Potential binding sites between miR-9-5p and NF-κB 3′UTR predicted by TargetScan. (D) Dual-luciferase assays showing repression of wild-type NF-κB -3′UTR following transfection of miR-9-5p mimics or negative control. (E) The levels of miR-9-5p and NF-κB mRNA in 293T cells after miR-9-5p overexpression and inhibition. Data represent the mean scores ± SEM of at least three independent experiments, ^*p < 0.05, ^**p < 0.01, Con group vs. AAA group; ^##p < 0.01, ^#p < 0.05, AAA+Ago group vs. AAA+Ago NC group. NF-κB, nuclear factor kappaB.

Figure 6

miR-9-5p promotes the differentiation of CD4⁺IL-10⁺T cells by targeting the expressions of NF-κB-Nrf2 pathway in vitro. (A) The expression of miR-9-5p. (B) Representative dot plots of CD4⁺IL-10⁺ T cells. (C) The percentage of CD4⁺IL-10⁺ T cells. (D-H) The protein expressions of the NF-κB-Nrf2 pathway and its downstream target genes. (I) Nuclear translocation of Nrf2 and NF-κB were determined by immunofluorescent staining (bar = 10 μm). All data are expressed as the mean ± SD, ^*p < 0.05, ^**p < 0.01, Con group vs. AAA group; ^##p < 0.01, ^#p < 0.05, AAA+Ago group vs. AAA+Ago NC group. NF-κB, nuclear factor kappaB; Nrf2, NF-E2-related factor 2.