Alginate oligosaccharide indirectly affects toll-like receptor signaling via the inhibition of microRNA-29b in aneurysm patients after endovascular aortic repair

Abstract

Endovascular aortic repair (EVAR) is often followed by aneurysm recurrence. Alginate oligosaccharide (AOS) has potential antitumor properties as a natural product while the related mechanisms remain unclear. Toll-like receptor (TLR) signaling is associated with inflammatory activity of aneurysm and may be affected by miR-29b. Thus, inhibitory function of AOS on aneurysms was explored by measuring the important molecules in TLR4 signaling. After EVAR, a total of 248 aortic aneurysm patients were recruited and randomly assigned into two groups: AOS group (AG, oral administration 10-mg AOS daily) and control group (CG, placebo daily). The size of residual aneurysms, aneurysm recurrence, and side effects were investigated. Aneurysm recurrence was determined by Kaplan-Meier analysis. After 2 years, eight and two patients died in the CG and AG, respectively. The sizes of residual aneurysms were significantly larger in the CG than in the AG (P<0.05). The incidence of aneurysm recurrence was also significantly higher in the CG than in the AG (P<0.05). AOS treatment reduced the levels of miR-29b, TLR4, mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NF-kappa B), interleukin 1 (IL-1) beta, and interleukin 6 (IL-6). Overexpression and silence of miR-29b increased and reduced the level of TLR4, phospho-p65 NF-kappa B, phospho-p38 MAPK, IL-1 beta, and IL-6. Spearman's rank correlation analysis shows that the level of miR-29b is positively related to the levels of TLR4, NF-kappa B, IL-1 beta, and IL-6 (P<0.05). Thus, AOS represses aneurysm recurrence by indirectly affecting TLR signaling via miR-29b.

Keywords: alginate oligosaccharide; anti-inflammatory agent; aortic aneurysms; microRNA-29b; minimally invasive endovascular repair; mitogen-activated protein kinase; outcome assessment; toll-like receptor signaling pathway.

Figure 1

Electrospray ionization mass spectrometry analysis of the DP of digested AOS from alginate sodium with produced mass spectra as [M + K]⁺. Notes: Mass spectra were visualized following the separation of DP3 ([M + K]⁺ =651 Da); mass spectra were visualized following the separation of DP4 ([M + K]⁺ =849 Da); mass spectra were visualized following the separation of DP5 ([M + K]⁺ =1,047 Da); and mass spectra were visualized following the separation of DP6 ([M + K]⁺ =1,245 Da). The mass spectrometer was set over the range m/z 500–1,500. Abbreviations: AOS, alginate oligosaccharide; DP, degree of polymerization.

Figure 2

Present study flowchart. Notes: CG, the patients received 10 mg placebo daily in a control group. AG, the patients received 10 mg AOS daily in the AOS group. Abbreviations: AA, aortic aneurysm; CG, control group; AOS, alginate oligosaccharide; AG, AOS group; TLR4, toll-like receptor 4; NF-kappa B, nuclear factor kappa B.

Figure 3

Scatter dot plot of the AA size variances in different groups. Notes: CG-B, before EVAR and placebo treatment. AG-B, before EVAR and AOS treatment. CG-A, after EVAR and 2-year placebo treatment. AG-A, after EVAR and 2-year AOS treatment. AG, AA patients received 10 mg AOS daily and CG, AA patients received 10 mg placebo daily. AA size was measured by ultrasound scanning. Maximum transverse and anterior–posterior external diameters were measured in infrarenal aorta, which is perpendicular to aortic axis. There was a statistical difference if P<0.05. Abbreviations: AA, aortic aneurysm; AG, AOS group; AOS, alginate oligosaccharide; CG, control group; EVAR, endovascular aortic repair.

Figure 4

Relative levels of miR-29b in different groups. Notes: CG-B, before EVAR and placebo treatment. AG-B, before EVAR and AOS treatment. CG-A, after EVAR and 2-year placebo treatment. AG-A, after EVAR and 2-year AOS treatment. N=124, 124, 116, and 122 cases in CG-B, AG-B, CG-A, and AG-A groups, respectively. AG, AA patients received 10 mg AOS daily and CG, AA patients received 10 mg placebo daily. There was a statistical difference if P<0.05. Abbreviations: AA, aortic aneurysm; AG, AOS group; AOS, alginate oligosaccharide; CG, control group; EVAR, endovascular aortic repair.

Figure 5

The effects of AOS on the levels of TLR4, NF-kappa B, IL-1 beta, and IL-6 in AA patients. Notes: (A) The effects of AOS on the levels of TLR4. (B) The effects of AOS on the levels of NF-kappa B. (C) The effects of AOS on the levels of IL-1 beta. (D) The effects of AOS on the levels of IL-6. N=124, 124, 116, and 122 cases in CG-B, AG-B, CG-A, and AG-A groups, respectively. AG, AA patients received 10 mg AOS daily and CG, AA patients received 10 mg placebo daily. CG-B, the patients before taking placebo. CG-A, the patients after taking placebo for two years. AG-B, the patients before taking AOS. AG-A, the patients after taking AOS for two years. There was a statistical difference if P<0.05. Abbreviations: AA, aortic aneurysm; AG, AOS group; AOS, alginate oligosaccharide; CG, control group; IL-6, interleukin 6; TLR, toll-like receptor; NF-kappa B, nuclear factor kappa B.

Figure 6

The relationship between the level of miR-29b and the levels of TLR4, NF-kappa B, IL-1 beta, and IL-6. Notes: (A) The relation between the level of miR-29b and the level of TLR4. (B) The relation between the level of miR-29b and the level of NF-kappa B. (C) The relation between the level of miR-29b and the level of IL-1 beta. (D) The relation between the level of miR-29b and the level of IL-6. Spearman’s rank correlation test was used to compare the significance of two parameters. There is a positive relationship if the value of rho falls between 0.5 and 1. There was a statistical difference if P<0.05. Abbreviations: IL-6, interleukin 6; NF-kappa B, nuclear factor kappa B; TLR, toll-like receptor.

Figure 7

Effects of AOS on the viability of ECs. Notes: (A) Cell viability of ECs was measured using Trypan blue. (B) Cell viability of ECs was measured using [³H]thymidine uptake. The data were presented as the mean ± standard deviation. N=8 in each group. Abbreviations: AOS, alginate oligosaccharide; ECs, endothelial cells.

Figure 8

The effects of AOS on the growth of ECs. Notes: (A) Real-time analysis of the effects of AOS on the growth of ECs. (B) Trypan blue analysis of the effects of AOS on the viability of ECs. (C) [³H]thymidine incorporation analysis of the effects of AOS on the viability of ECs. N=8. Abbreviations: AOS, alginate oligosaccharide; ECs, endothelial cells.

Figure 9

Relative levels of miR-29b in different groups. Notes: (A) The effects of AOS on the relative levels of miR-29b. (B) The effects of different treatments on relative levels of miR-29b. N=8 for each group. *P<0.05 vs the CG. Abbreviations: AOS, alginate oligosaccharide; CG, control group; SG, miR-29b was silenced by antagomirs; OG, miR-29b was overexpressed.

Figure 10

The effects of AOS on the levels of TLR4, NF-kappa B, IL-1 beta, and IL-6 in ECs. Notes: (A) The effects of AOS on the levels of TLR4. (B) The effects of AOS on the levels of NF-kappa B. (C) The effects of AOS on the levels of IL-1 beta. (D) The effects of AOS on the levels of IL-6. *P<0.05 vs the CG without AOS addition. Abbreviations: AOS, alginate oligosaccharide; CG, control group; ECs, endothelial cells; IL-6, interleukin 6; NF-kappa B, nuclear factor kappa B; TLR, toll-like receptor.

Figure 11

The effects of miR-29b on ECs. Notes: (A) The effects of miR-29b on the levels of TLR4. (B) The effects of miR-29b on the levels of NF-kappa B. (C) The effects of miR-29b on the levels of IL-1 beta. (D) The effects of miR-29b on the levels of IL-6. (E) Trypan blue analysis of the effects of miR-29b on EC viability. (F) [³H]thymidine uptake analysis of the effects of miR-29b on EC viability. *P<0.05 vs the CG. Abbreviations: CG, control group; ECs, endothelial cells; IL-6, interleukin 6; NF-kappa B, nuclear factor kappa B; OG, miR-29b was overexpressed; SG, miR-29b was silenced by antagomirs; TLR, toll-like receptor.

Figure 12

(A–F) Western blot analysis of the effects of miR-29b on the expression of TLR4 signaling molecules. Notes: (A) Western blot analysis for the effects of miR-29b on the expression of p65 NF-kappa B, p38 MAPK, TLR4, IL-1 beta and IL-6. (B) The effects of miR-29b on relative protein level of NF-kappa B. (C) The effects of miR-29b on the expression of phospho-p38 MAPK. (D) The effects of miR-29b on the expression of TLR4. (E) The effects of miR-29b on the expression of IL-1 beta. (F) The effects of miR-29b on the expression of IL-6. *P<0.05 vs the CG. Abbreviations: CG, control group; IL-6, interleukin 6; NF-kappa B, nuclear factor kappa B; OG, miR-29b was overexpressed; SG, miR-29b was silenced by antagomirs; TLR, toll-like receptor.

Figure 13

A cartoon description of AOS from marine algae for the therapy of AA patients. The oligosaccharide has been found to prevent the regrowth of aneurysms by inactivating TLR4, NF-kappa B, IL-1 beta, and IL-6 via the inhibition of miR-29b. Abbreviations: AA, aortic aneurysm; AOS, alginate oligosaccharide; IL-6, interleukin 6; NF-kappa B, nuclear factor kappa B; TLR, toll-like receptor.