Inhibition Effects of Nippostrongylus brasiliensis and Its Derivatives against Atherosclerosis in ApoE-/- Mice through Anti-Inflammatory Response

Abstract

Atherosclerosis (AS) is a dominant and growing cause of death and disability worldwide that involves inflammation from its inception to the emergence of complications. Studies have demonstrated that intervention with helminth infections or derived products could modulate the host immune response and effectively prevent or mitigate the onset and progression of inflammation-related diseases. Therefore, to understand the molecular mechanisms underlying the development of atherosclerosis, we intervened in ApoE^-/- mice maintained on a high-fat diet with Nippostrongylus brasiliensis (N. brasiliensis) infection and immunized with its derived products. We found that N. brasiliensis infection and its derived proteins had suitable protective effects both in the initial and progressive stages of atherosclerosis, effectively reducing aortic arch plaque areas and liver lipid contents and downregulating serum LDL levels, which may be associated with the significant upregulation of serum anti-inflammatory cytokines (IL-10 and IL-4) and the down-regulation of proinflammatory cytokines (TNF-α and IFN-γ) in the serum. In conclusion, these data highlighted the effective regulatory role of N. brasiliensis and its derived proteins in the development and progression of atherosclerosis. This could provide a promising new avenue for the prevention and treatment of atherosclerosis.

Keywords: Nippostrongylus brasiliensis; apolipoprotein-E-deficient mouse; atherosclerosis; hookworm; intervention; protective effect.

Figure 1

N. brasiliensis infection could prevent atherosclerosis. (A): Schematic diagram of the experimental design through N. brasiliensis infection intervention in atherosclerosis. (B): Body weight changes in mice in the model (n = 6) and N. brasiliensis infection groups (n = 6) during the initial stages of atherosclerosis; (C,D): Representative images of Oil Red O Staining of liver and quantitative data relating to lipid percentage in mice from the model group (n = 6) and N. brasiliensis infection group (n = 6); (E,F): Representative images of Oil Red O Staining of the aortic roots of mice in the model (n = 6) and N. brasiliensis-infected groups (n = 6) and quantitative data relating to relative plaque area. (G–I): Serum cytokine levels in the model group (n = 6) and N. brasiliensis-infected group (n = 6). The “n” indicates the number of biological replicates. p < 0.05 considered significant; ns = not significant, ^* p < 0.05; ^** p < 0.01; ^**** p < 0.0001 as determined by Student’s t-test comparisons of two groups or one-way ANOVA followed by Dunnett multiple comparisons test to compare more than two groups. Error bars represent SEMs.

Figure 2

N. brasiliensis-derived proteins could prevent atherosclerosis. (A): Schematic diagram of the experimental design of N. brasiliensis-derived protein intervention in atherogenesis. (B): Body weight changes in vehicle (n = 6)-, L3-protein (n = 6)-, and L5-protein (n = 6)-treated mice during the initial stages of atherosclerosis; (C,D): Representative images of Oil Red O Staining and quantitative data of lipid percentage in the liver of vehicle (n = 6)-, L3-protein (n = 6)-, and L5-protein (n = 6)-treated mice; (E,F): Representative images of Oil Red O Staining and quantitative data on relative plaque area in aortic roots of vehicle (n = 6)-, L3-protein (n = 6)-, and L5-protein (n = 6)-treated mice; (G–J): Serum cytokine levels in mice treated with vehicle (n = 6), L3-protein (n = 6), and L5-protein (n = 6). The “n” indicates the number of biological replicates. p < 0.05 considered significant; ns = not significant, ^* p < 0.05; ^** p < 0.01; ^**** p < 0.0001 as determined by Student’s t-test comparisons of two groups or one-way ANOVA followed by Dunnett multiple comparisons test to compare more than two groups. Error bars represent SEMs.

Figure 3

N. brasiliensis infection slowed the progression of atherosclerosis. (A): Schematic diagram of the experimental design of N. brasiliensis infection to intervene in the progression of atherosclerosis. (B,C): Representative images of Oil Red O Staining of liver and quantitative data of lipid percentage in model (n = 5) and N. brasiliensis-infected (n = 5–7) mice; (D,E): Representative images of Oil Red O Staining of aortic roots and quantitative data of relative plaque area in model (n = 5) and N. brasiliensis-infected (n = 5–7) mice. (F–I): Serum levels of total cholesterol, triglycerides, LDL, and HDL in the model group (n = 5) and N. brasiliensis-infected group (n = 5–7) mice; (J–N): Serum levels of cytokines in the model group (n = 5) and N. brasiliensis-infected group (n = 6–7). The “n” indicates the number of biological replicates. p < 0.05 considered significant; ns = not significant, ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001; ^**** p < 0.0001 as determined by Student’s t-test comparisons of two groups or one-way ANOVA followed by Dunnett multiple comparisons test to compare more than two groups. Error bars represent SEMs.

Figure 4

N. brasiliensis-derived proteins slowed the progression of atherosclerosis. (A): Schematic diagram of the experimental design of N. brasiliensis-derived protein intervention in atherosclerosis progression. (B,C): Representative images of Oil Red O Staining of liver and quantitative data of lipid percentage in vehicle (n = 4)-, L3-protein (n = 6)-, and L5-protein (n = 6)-treated mice; (D,E): Representative images of Oil Red O Staining of aortic roots and quantitative data of relative plaque area in vehicle (n = 6)-, L3-protein (n = 6)-, and L5-protein (n = 6)-treated mice; (F–I): Total cholesterol, triglyceride, LDL and HDL levels in serum of vehicle(n = 6)-, L3-protein(n = 6)-, and L5-protein(n = 6)-treated mice. (J–M): Cytokine levels in the serum of vehicle (n = 6)-, L3 protein (n = 6)-, and L5 protein (n = 6)-treated mice. The “n” indicates the number of biological replicates. p < 0.05 considered significant; ns = not significant, ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001; ^**** p < 0.000 1 as determined by Student’s t-test comparisons of two groups or one-way ANOVA followed by Dunnett multiple comparisons test to compare more than two groups. Error bars represent SEMs.