Palmitic Acid, A Critical Metabolite, Aggravates Cellular Senescence Through Reactive Oxygen Species Generation in Kawasaki Disease

Abstract

Coronary artery lesions (CALs) are severe complications of Kawasaki disease (KD), resulting in stenosis and thrombogenesis. Metabolomic profiling of patients' plasma could assist in elucidating the pathogenesis of CALs and identifying diagnostic biomarkers, which are imperative for clinical treatment. The metabolic profiles between KD patients with CALs and without CALs (non-coronary artery lesion, or NCAL, group) indicated the most significantly differentially expressed metabolite, palmitic acid (PA), showed the most massive fold change at 9.879. Furthermore, PA was proven to aggravate endothelial cellular senescence by increasing the generation of reactive oxygen species (ROS) in KD, and those two phenotypes were confirmed to be enriched among the differentially expressed genes between KD and normal samples from GEO datasets. Collectively, our findings indicate that cellular senescence may be one of the mechanisms of vascular endothelial damage in KD. PA may be a biomarker and potential therapeutic target for predicting the occurrence of CALs in KD patients. All things considered, our findings confirm that plasma metabolomics was able to identify promising biomarkers and potential pathogenesis mechanisms in KD. To conclude, Palmitic acid could be a novel target in future studies of CALs in patients with KD.

Keywords: cellular senescence; coronary disease; kawasaki disease; metabolomics; oxidative stress.

FIGURE 1

Comprehensive Metabolomic Characterization of CALs in KD. (A,B) Principal component analysis (PCA) score plots; (C) Orthogonal projections to latent structures discriminant analysis (OPLS-DA) score plots (R²Y = 0.867 and Q²Y = 0.787); (D) Permutation test (n = 200) of OPLS-DA; (E) Disrupted pathways identified in positive and negative mode; (F) Heatmap of the 16 differentially expressed metabolites between the CAL and NCAL groups; red represents a high relative concentration, and blue represents a low relative concentration; (G) Volcano plots of different metabolites; (H) The network of 14 different metabolites with diverse expressions between the CAL and NCAL groups. The width of each edge represents the correlation coefficient and correlation values; the red edges represent positive correlations, and the blue ones represent negative correlations. Larger nodes represent more connections; (I) ROC curve for a panel of biomarkers compared between patients in the CAL and NCAL groups. CAL, coronary artery lesion; NCAL, non-coronary artery lesion; FC, fold change.

FIGURE 2

Functional enrichment analyses of DEGs between KD and Normal group. (A) Statistical table from the three microarray databases searched in the GEO database; (B,C) The Venn diagram of the intersection among DEGs from all three GEO data sets: (B) Upregulated genes; (C) Downregulated genes; (D) Bubble diagram from the GO terms, KEGG pathway, and Reactome pathway of significantly enriched DEGs. DEG, differentially expressed gene; GEO, gene expression omnibus.

FIGURE 3

PA affected cell activity by inducing autophagy and cell senescence (A,B) HUVECs and HASMCs were treated with diffferent concentrations of PA. CCK8 assay established that cellular activities was inhibited; (C–E) HUVECs and HASMCs were treated with PA at the concentrations of 250 and 500 μM. Control groups were treated with 1%BSA. Incubated for 24 h, total proteins were probed for P16, pRB, P62 and LC3II proteins. GAPDH was used as the loading control (C); Relative protein expression levels of P16, pRB, P62, and LC3II compared with GAPDH in HUVECs (D); Relative protein expression levels of P16, pRB, P62, and LC3II compared with GAPDH in HASMCs (E); (F,G) HUVECs were treated with PA under the concentrations of 250 and 500 μM. The presence of SA-β-gal activity in HUVECs indicated cellular senescence was detected (F); Quantification of SA-β-gal staining. The number of SA-β-gal positive cells under 250 and 500 μM PA concentrations were significantly higher than in the control group (G). PA, Palmitic acid. (n = 6; Data shown as Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to control groups).

FIGURE 4

PA aggravated cellular senescence of endothelial cells in Kawasaki disease. (A,B) HUVECs were co-treated with 500 μM PA and 10% KD serum (line 4), or only PA (line 2), or only KD (line 3). Control groups were co-treated with 1%BSA and 10%FBS (line 1). Incubated for 24 h, total proteins were probed for P16 and pRB proteins; Relative protein expression levels of P16 and pRB compared with GAPDH; (C,D) HUVECs were treated with 500 μM PA or 10%KD serum or both compared with control group. The staining of SA-β-gal activity indicated PA aggravated cell senescence in HUVECs (C); Quantification of SA-β-gal staining (D); (E,F) HUVECs were treated with 500 μM PA or 10%KD serum or both, and compared with the control group. Endothelial cell function was detected by cell migration (E); the bar graph represented the quantification of the scratch assay (F). (n = 6; Data shown as Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

FIGURE 5

PA also increased intracellular ROS accumulation in endothelial cells in KD. (A,B) HUVECs were co-treated with 500 μM PA and 10% KD serum (line 4), PA only (line 2), or KD only (line 3). Control groups were co-treated with 1% BSA and 10% FBS (line 1). Incubated for 24 h, total proteins were probed for SOD1 and SOD2 proteins; Relative protein expression levels of SOD1 and SOD2 compared with GAPDH; (C,D) Immunofluorescent images of DHE-stained HUVECs after treatment for 24 h (C). The quantitative data from Immunofluorescent images (D); (E) MDA tests from the supernatant of HUVECs after treatment for 24 h (n = 6; Data shown as Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

FIGURE 6

PA aggravates cell senescence by mediating ROS production. (A,B) HUVECs were co-treated under 10% KD serum with 500 μM PA (line 2) or 500 μM PA and NAC (line 3), compared with control groups co-treated with 1% BSA and 10% KD serum (line 1). Incubated for 24 h, total proteins were probed for P16, pRB, SOD1, and SOD2 proteins (A); Relative protein expression levels of P16, pRB, SOD1, and SOD2, compared with GAPDH (B); (C,D) Immunofluorescent images of DHE-stained HUVECs after treatment for 24 h (C). The quantitative data from immunofluorescent images (D); (E,F) The staining of SA-β-gal activity indicated that decreased ROS production by NAC lessened cellular senescence induced by PA in HUVECs (C); Quantification of SA-β-gal staining. (n = 6; Data shown as Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; scale bar = 100 μm).

FIGURE 7

Palmitic acid, a critical metabolite, aggravates cell senescence through reactive oxygen species generation in Kawasaki disease.