Eight-week-old male C57BL/6J mice were obtained from the laboratory of Jinan University’s animal center and used in this study. All animals were fed in cages and bred in a temperature-controlled room maintained at 22 ± 1°C on a 12-h light-dark cycle with standard food and water. All animal experimental procedures complied with Institutional Animal Care and Use Committee of Jinan University.

According to previously reported surgical methods (19), a mouse model of AAA or sham was induced by perivascular application of PPE (Sigma-Aldrich; 4 U/mg protein) or saline. Briefly, after induction of anesthesia with 3% isoflurane, the lower three-fourths of the infrarenal abdominal aorta was exposed and isolated from the vena cava. 30uL elastase was applied to exposed aortic adventitia and infiltrated for 30 minutes. Then, the abdominal cavity, especially the adventitia, needed to be rinsed with saline. Finally, the abdomen was closed routinely with 6-0 silk sutures. After 14 days, all mice were sacrificed by carbon dioxide inhalation, while the aortas were isolated for pathological examination.

To investigate the effect of ATL-I on AAA, a total of 18 mice were divided by random number table into sham group, AAA group, and ATL-I group with 6 samples in each group. After modeling, mice in ATL-I groups received the ATL-I (20mg/kg/day, 73069-13-3, MCE) by gavage daily for the next 14 days, and mice in AAA group were given the same amount of sterile PBS (20–23). The molecular structure of ATL-I was shown in Figure 1A .

To investigate whether ATL-I affects AAA through AMP-activated protein kinase (AMPK) signaling pathway, 24 mice were randomly divided into four groups of six mice in each group. The treatment of the sham group, AAA group and ATL-I group was the same as the groups mentioned above. In the ATL-I+CC group, mice were simultaneously given compound C (CC, 300mg/kg/day, 866405-64-3, MCE) and ATL-I (20mg/kg/day).

The diameter of the subrenal abdominal aorta was measured by B-ultrasound imaging (Vevo 2100, Visual Sonic Inc.) before and 14 days after modeling. After anesthesia with 1.5% isoflurane, the aorta of mouse was detected during systole by a blinded investigator. AAA was defined as the maximum diameter of the subrenal aorta that dilates more than 50% of its baseline (The diameter of the abdominal aorta examined before modeling) (24).

Aortic samples isolated from the mice were fixed with 4% paraformaldehyde for 24h and embedded in paraffin. Serial sections (5μm each) were used for HE staining, elastic fiber staining (G1596, Solarbio) and immunofluorescence staining. The severity of elastic fiber degradation was evaluated on a standard described in a previous study: score 1, <25% degradation; score 2, 25% to 50% degradation; score 3, 50% to 75% degradation; score 4, >75% degradation (25).

Paraffin slides were incubated in xylene and alcohol of different gradients for deparaffin and rehydration. The slides were placed in 10 mM sodium citrate buffer (pH 6, BL619A, Biosharp) at 85°C for 30 minutes and cooled at room temperature for 20 minutes. Then, the slides were incubated in 5% horse serum for 30 minutes at room temperature. Next, the slides were incubated at 4°C overnight with the following primary antibodies: CD68 (1:200, ab125212; Abcam), CD80 (1:200, ab254579, Abcam), CD206 (1:200, AF2535, R&D Systems), α-SMA (1:500, ab5694, Abcam). The following day, the slides were incubated with the corresponding secondary antibodies and DAPI at room temperature for 30 minutes. Immunofluorescence images were obtained using confocal laser scanning microscopy (Leica) and analyzed using Image-J software.

Serum was obtained by centrifugation after blood collection through inferior vena cava. The content of IL-1β, IL-6, IL-10, alanine aminotransferase (ALT), aspertate aminotransferase (AST), creatine, and blood urea nitrogen (BUN) was analyzed using a commercially available ELISA kit (ml098416; ml098430; mIC50274-1; SP30122; CSB-E12649m; LE-M0613; RF8291; MLbio; Spbio; Cusabio; Isite; Ruifan) according to the manufacturer’s protocol. The results were calculated using the standard curve method and expressed as pg/ml.

Total RNA was extracted from the subrenal abdominal aorta using Trizol Reagent (155960-18, Ambion). Then, 1ug of total RNA was subjected to reverse transcription based on the manufacturer’s instructions. The mRNA expression of IL-1β, IL-6 and IL-10 in aorta was analyzed through PCR system (BIO-RAD). The primer sequences are shown in Table 1 .

Total protein was extracted with RIPA lysate. Protein concentration was quantified by bicinchoninic acid (BCA) protein assay kit (P0010, Beyotime). The protein was separated by 10% SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and incubated in 5% BSA for 1 hour at room temperature. Then, the membranes were incubated overnight with primary antibodies AMPK (1:1000, 2365, CST), p-AMPK (1:1000, 2535, CST), MMP-9 (1:1000, ab283575, Abcam), MMP-2 (1:1000, ab86607, Abcam), β-actin (1:1000, ab8226, Abcam) at 4°C. The membranes were incubated with secondary antibodies for 1 hour at room temperature the next day. Finally, the protein bands were visualized with the ECL system (BIO-RAD).

All data were presented as the mean ± SD and executed using GraphPad Prism 8.0 software. Statistical differences between groups were performed by one-way ANOVA. P<0.05 was considered as statistically significant.

To explore whether ATL-I could restrain AAA progression, we first detected the diameter of the infrarenal abdominal aorta on the 14^th day after modeling. As revealed by Figure 1B , the aorta diameter in the AAA group was significantly increased, which was reversed after ATL-I treatment. At the same time, ultrasound data showed that the infrarenal aorta was dilated with a maximum diameter of 1.23 ± 0.21 mm in the AAA group. However, the baseline of its diameter was 0.57 ± 0.02 mm, and the maximum infrarenal aortic diameter was only slightly increased to 0.91 ± 0.02 mm in the ATL-I group ( Figures 1C, D ). Ultimately, ATL-I decreased the incidence of AAA ( Figure 1E ). Additionally, we observed that ATL-I couldn’t change the levels of ALT, AST, creatine and BUN, indicating that it had no obviously toxic side effects on liver and renal function in vivo ( Supplementary Figures S1A–D ). HE staining results displayed that the adventitia was thicker, accompanied with recruitment of vast inflammatory cells in AAA, and the terrible condition was improved following ATL-I treatment ( Figures 1F, G ). Taken together, ATL-I administration inhibited the progression of AAA.

Inflammation has been considered as a central player in the progression of AAA. There were a large number of macrophages (CD68+) in the adventitia of AAA ( Figures 2A, D ). To examine whether the inhibitory effects of ATL-I on AAA progression were associated with changes in M1 or M2 macrophages, we subjected abdominal aortic tissue for immunostaining using CD80 to stain M1 macrophages and CD206 to identify M2 macrophages. As shown in Figures 2B, E the significantly increased CD8 staining was observed in the adventitial region of the aneurysm, which was markedly reduced following ATL-I administration. Nevertheless, ATL-I treatment significantly augment aggregation of M2 macrophages in adventitia ( Figures 2C, F ). Subsequently, we compared the differences in the levels of inflammatory cytokines among three groups using ELISA and qPCR experiments. As illustrated in Figures 2G–J , the expression of proinflammatory cytokines, such as IL-1β and IL-6, was elevated in the aneurysm and circulating system of mice with AAA, which was restrained after ATL-I treatment. By comparison, decreased anti-inflammatory cytokine IL-10 expression in AAA were reversed by ATL-I ( Figures 2K, L ). As such, our results demonstrated the anti-inflammatory activity of ATL-I on AAA.

Macrophages and VSMCs generate MMP-2 and MMP-9, therefore triggering steady destruction of extracellular matrix (ECM). We next examined α-SMA expression and elastin alteration. As shown in Figures 3A, B , a large amount of VSMCs was lost and remaining VSMCs were disordered in aneurysm, whereas the VSMCs disruption was markedly ameliorated after ATL-I supplement. Similarly, ATL-I administration improved the condition of elastin that showed obvious fracture and degradation in AAA tissue ( Figures 3C, D ). We further verified the levels of MMP-2 and MMP-9 using western blotting. MMP-2 and MMP-9 were elevated in the AAA group, which was mitigated after ATL-I therapy ( Figures 3E, F ). Collectively, these results suggested that ATL-I inhibited MMP-2 and MMP-9 expression, thereby attenuating pathological lesions of aorta.

Though there was no difference in AMPK expression in three groups, the expression of phospho- AMPK (p-AMPK) was downregulated in AAA tissues, which was upregulated following ATL-I treatment ( Figure 4A ). Therefore, we speculated that ATL-I might exert its inhibitory effect on AAA by activating AMPK phosphorylation. As expected, the administration of compound C, an inhibitor of AMPK, abolished the salutary effects of ATL-I on AAA. It was found that the diameter of aneurysm and adventitial thickness were apparently increased in the ATL-I+CC group ( Figures 4B–D ). Compound C reduced the phosphorylation level of AMPK, and led to a larger diameter of abdominal aorta and a higher incidence of AAA, compared with ATL-I group ( Figures 4E, F ). Thus, ATL-I treatment retarded AAA formation partially by targeting AMPK.

Subsequently, we examined the levels of macrophages and inflammatory cytokines after the application of compound C. Compared with the ATL-I group, the fluorescence intensity of CD68 was robustly increased in ATL-I+CC group ( Figures 5A, D ). Similarly, compound C reduced the inhibitory effect of ATL-I on M1 macrophages ( Figures 5B, E ). In contrast, increased CD206 expression was observed in the adventitia in the ATL-I group, which was restricted by compound C administration ( Figures 5C, F ). Meanwhile, ATL-I-induced decreased levels of IL-1β and IL-6 in the aorta and circulating system were reraised after the administration of compound C ( Figures 5G–J ). Our data also demonstrated that ATL-I-triggered the activation of IL-10 was significantly decreased following compound C treatment ( Figures 5K, L ). Taken together, our results supported the notion that AMPK was required for ATL-I to exert its anti-inflammatory effects on AAA.

Using immunostaining and elastic fiber staining, we observed that the α-SMA expression was dampened in the ATL-I+CC group, accompanied by the aggravated deterioration of elastic fibers, which was in line with pathological injury results of AAA as described above ( Figures 6A–D ). Furthermore, we noticed that the supplement of compound C impaired the inhibitory effects of ATL-I on MMP-2 and MMP-9 expression, as displayed in Figures 6E, F . Collectively, these findings suggested that ATL-I mitigated the pathological lesion of AAA by activating AMPK signaling pathway.