Anti-Inflammatory Responses Produced with Nippostrongylus brasiliensis-Derived Uridine via the Mitochondrial ATP-Sensitive Potassium Channel and Its Anti-Atherosclerosis Effect in an Apolipoprotein E Gene Knockout Mouse Model

Abstract

Atherosclerosis (AS) has become the leading cause of cardiovascular disease worldwide. Our previous study had observed that Nippostrongylus brasiliensis (Nb) infection or its derived products could inhibit AS development by inducing an anti-inflammatory response. We performed a metabolic analysis to screen Nb-derived metabolites with anti-inflammation activity and evaluated the AS-prevention effect. We observed that the metabolite uridine had higher expression levels in mice infected with the Nb and ES (excretory-secretory) products and could be selected as a key metabolite. ES and uridine interventions could reduce the pro-inflammatory responses and increase the anti-inflammatory responses in vitro and in vivo. The apolipoprotein E gene knockout (ApoE^-/-) mice were fed with a high-fat diet for the AS modeling. Following the in vivo intervention, ES products or uridine significantly reduced serum and liver lipid levels, alleviated the formation of atherosclerosis, and reduced the pro-inflammatory responses in serum or plaques, while the anti-inflammatory responses showed opposite trends. After blocking with 5-HD (5-hydroxydecanoate sodium) in vitro, the mRNA levels of M2 markers were significantly reduced. When blocked with 5-HD in vivo, the degree of atherosclerosis was worsened, the pro-inflammatory responses were increased compared to the uridine group, while the anti-inflammatory responses decreased accordingly. Uridine, a key metabolite from Nippostrongylus brasiliensis, showed anti-inflammatory and anti-atherosclerotic effects in vitro and in vivo, which depend on the activation of the mitochondrial ATP-sensitive potassium channel.

Keywords: Nippostrongylus brasiliensis; anti-atherosclerosis; mitochondrial ATP-sensitive potassium channel; uridine.

Figure 1

Screening of anti-inflammatory components derived from Nb through UHPLC-MS. (A) Heatmaps of different metabolites between the ES-cultured and control group. (B) Heatmaps of different metabolites treated by intestinal contents of mice infected with Nb. (C,D) Relative expression levels of uridine including ES and Nb infected groups compared with controls. * p < 0.05.

Figure 2

Anti-inflammatory activities observation for the screened uridine via LPS model and BMDM model. (A–C) Serum levels of TNF-α/IL-1β/IL-6 in LPS model obtained using ELISA analysis, respectively (n ≥ 6); (D–G) Serum mRNA relative expressions of TNF-α/IL-6/TGF-β/IL-10 in LPS model obtained using RT-qPCR, respectively (n ≥ 6). (H,I) Western blot analysis for phosphorylation of NF-κB in LPS model and statistical histogram of gel electrophoresis bands (n ≥ 6). Original images can be found in Supplementary information S2. (J,K) Serum mRNA relative expressions of ARG1 in BMDM model obtained using RT-qPCR (n ≥ 6). * p < 0.05 and ** p < 0.01.

Figure 3

Anti-atherosclerosis effects observation following different interventions in AS mouse model. (A) Schematic diagram of experimental design of ES and uridine administration in AS model. (B,F) Body weight changes during the experimental period and comparison on 12th week between groups (n ≥ 6). (C,G) HE staining of mouse liver under 400× magnification and statistics on percentage of fatty areas between groups (n ≥ 5). (D,H) Oil red staining of whole arterial and statistics of lesion areas between groups (n ≥ 5). (E,I) HE staining of plaques within the aortic arch under 100× magnification and statistics of plaque area between groups (n ≥ 5). * p < 0.05 and ** p < 0.01.

Figure 4

Observation of blood lipid levels and inflammatory related factor expression levels in AS mouse model following different interventions. (A–C) Serum levels of total cholesterol/LDL/HDL (n ≥ 6). (D,E) Serum levels of TNF-α and IL-10 in AS model by ELISA (n ≥ 6). (F–H) TNF-α/IL-6/ARG1 mRNA relative expressions levels in aortic arch by RT-qPCR (n ≥ 6). * p < 0.05 and ** p < 0.01.

Figure 5

Role of mitoKATP channel for the uridine’s anti-inflammatory effects in BMDM model through blocked by 5-HD. (A–C) ARG1/CD206/YM1 mRNA relative expressions levels in BMDM model blocked by 5-HD through RT-qPCR (n ≥ 6). (D) Serum levels of IL-10 in BMDM model blocked by 5-HD through ELISA (n ≥ 6). * p < 0.05.

Figure 6

Anti-atherosclerosis effects observation for uridine in AS mouse model following channel blockage by 5-HD. (A) Schematic diagram of experimental design of uridine and 5-HD blockage in AS model. (B,F) Body weight changes during the experimental period and comparison on 12th week between groups (n ≥ 6). (C,G) HE staining of mouse liver under 400× magnification and statistics on percentage of fatty areas between groups (n ≥ 5). (D,H) Oil red staining of whole arterial and statistics of lesion areas between groups (n ≥ 5). (E,I) HE staining of plaques within the aortic arch under 100× magnification and statistics of plaque area between groups (n ≥ 5). * p < 0.05 and ** p < 0.01.

Figure 7

Observation of blood lipid levels and inflammatory-related factor expression levels in AS mouse model following channel blockage with 5-HD. (A–C) Serum levels of total cholesterol/LDL/HDL (n ≥ 6). (D,E) Serum levels of TNF-α and IL-10 in AS model obtained using ELISA (n ≥ 6). (F–I) TNF-α/IL-6/IL-10/ARG1 mRNA relative expressions levels in aortic arch obtained using RT-qPCR (n ≥ 6). * p < 0.05 and ** p < 0.01.