[The protective effect and mechanism of inosine on AngⅡ-induced aortic aneurysms in mice]

Objective: To investigate the protective effects of inosine on angiotensin Ⅱ (AngⅡ)-induced aortic aneurysm in mice and its underlying mechanisms. Methods: This study was conducted in three phases. In the first phase, 19 male C57BL/6 mice aged 6 to 8 weeks were randomly divided into a control group (n=9, without any intervention) and a model group (n=10, with a mini-osmotic pump implanted subcutaneously in the back to continuously infuse Ang Ⅱ at a dose of 2 000 ng·kg⁻¹·min⁻¹ for 14 days to establish the aortic aneurysm model). At 14 days after modeling, non-targeted metabolomics technology was employed to detect the plasma metabolite profiles in both groups to screen for differential metabolites. Serum samples were collected from patients diagnosed with aortic aneurysm (aortic aneurysm group) and healthy controls (healthy control group) at the First Affiliated Hospital of Xinjiang Medical University from October 2023 to May 2024, and serum inosine levels were measured using enzyme-linked immunosorbent assay (ELISA). In the second phase, another 40 male mice of the same strain and age were randomly divided into four groups: control group 2 (no intervention), control intervention group (daily intraperitoneal injection of inosine at 400 mg/kg for 14 consecutive days), model group 2 (same as the first phase), and model intervention group (Ang Ⅱ infusion with concurrent daily intraperitoneal injection of inosine at 400 mg/kg for 14 consecutive days), and there were 10 mice per group. At 14 days after modeling, ultrasound was used to measure the maximum diameters of the aortic arch and abdominal aorta. HE, EVG and Masson staining were performed to observe the distribution of elastin and collagen fibers as well as inflammatory cell infiltration in aortic tissues. ELISA was used to detect serum levels of inflammatory cytokines including interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α). Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was employed to measure the mRNA expression levels of IL-6, IL-1β, TNF-α, and α-smooth muscle actin (α-SMA) in aortic tissues. Immunofluorescence staining was used to detect α-SMA protein expression in aortic tissues, and immunohistochemical staining was performed to detect the protein expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 in aortic tissues. Molecular docking analysis was conducted to explore the binding mode between inosine and STING protein. Western blot was used to detect the protein expression levels of phosphorylated STING (P-STING) and STING in aortic tissues. In the third phase, in vitro experiments were carried out using human aortic smooth muscle cells (HASMC). The cells were cultured to passages 4 to 5, which were treated with AngⅡ at 1 μmol/L (AngⅡ group) and AngⅡ combined with inosine at 100 μmol/L (AngⅡ+inosine group). STING-specific siRNA was transfected into HASMCs to construct a STING knockdown model, the knockdown cells were divided into two groups for intervention: one group was treated with 1 μmol/L AngⅡ alone (siSTING+AngⅡ group), and the other group was treated with 1 μmol/L AngⅡ combined with 100 μmol/L inosine (siSTING+AngⅡ+inosine group), and qRT-PCR was used to detect the mRNA levels of IL-6, IL-1β, and TNF-α. Results: Metabolomic analysis revealed that metabolic pathway was significantly enriched in the model group, and the purine metabolite inosine levels were higher than that in the control group (P<0.05); ELISA results showed that serum inosine levels of patients in aortic aneurysm group were higher than those in healthy control group (P<0.000 1). Ultrasound results demonstrated that the maximum diameters of the aortic arch and abdominal aorta in model 2 group were significantly greater than those in control 2 group (both P<0.000 1). Compared with the model group 2, the model intervention group had markedly smaller aortic diameters (all P<0.05). HE, EVG and Masson staining results indicated that compared with control group 2, model group 2 mice exhibited significantly increased inflammatory cell infiltration in the aortic wall, higher elastin degradation scores, and greater collagen volume fractions, whereas the model intervention group showed significantly milder pathological changes than model group 2 (all P<0.05). Immunofluorescence and qRT-PCR results showed that the protein and mRNA expression levels of α-SMA in aortic tissues were higher in the model intervention group than in model group 2 (all P<0.05). Immunohistochemistry results revealed that the protein expression levels of MMP-2 and MMP-9 in aortic tissues were lower in the model intervention group than in model group 2 (all P<0.05). ELISA showed that serum IL-6 levels were lower in the model intervention group than in model group 2 (P<0.000 1); qRT-PCR of the model intervention group aortic tissues revealed that mRNA levels of IL-6, IL-1β, and TNF-α were lower in the model intervention group (all P<0.05). The molecular docking analysis indicated a binding energy of -6.2 kcal/mol between inosine and STING. Western blot showed that the protein expression levels of P-STING and STING in aortic tissues of the model intervention group were lower than those in model group 2 (all P<0.001). In vitro experiments, qRT-PCR results showed that the mRNA levels of IL-6 and IL-1β in the AngⅡ+inosine group were lower than those in the AngⅡ group (both P<0.001); the mRNA levels of IL-6 and IL-1β in the siSTING+AngⅡ+inosine group were higher than those in the AngⅡ+inosine group (both P<0.05). Conclusions: Plasma inosine levels are significantly elevated in mice with AngⅡ-induced aortic aneurysm and in patients with aortic aneurysm. Inosine supplementation may exert a protective effect on aortic aneurysm by inhibiting STING signaling pathway activation, reducing inflammatory responses, and downregulating MMP expression, thereby alleviating structural damage to the aortic wall.