Arginine depletion-induced autophagy and metabolic dysregulation are involved in the disease severity of hand, foot, and mouth disease

Figure 1.. Precise plasma AA concentrations in the plasma of study participants. (a) The concentrations of AA metabolites with statistical difference between groups. The changes of these metabolites were also summed up and displayed at the bottom. Dot plot depicting mean for the tyrosine/phenylalanine concentration ratio (b) and the valine/glycine concentration ratio (c), and scatterplots between tyrosine (y-axis) and phenylalanine (x-axis) in each healthy control (d), mild case (e) and severe case (f). *p < 0.05, **p < 0.01, ***p < 0.001, ns: no statistical difference.

Figure 2.. Arginine metabolism in severe paediatric patients with HFMD. Under different comparison schemes, (a) HCs vs mild, (b) HCs vs severe, (c) HCs vs all HFMD patients, (d) mild vs severe, scatter plots of the most relevant metabolic pathways from KEGG library arranged by adjusted p values on the y-axis, and pathway impact values on the x-axis. (e) Schematic map of the arginine and taurine metabolism (left), tryptophan metabolism (right).

Figure 3.. The potential of plasma AAs as indicators for severe outcomes related to HFMD. The diagnosis power of aminoadipic acid (a), proline (b), isoleucine (c), kynurenine (d), and arginine (e) were evaluated by ROC analysis. The Logistic Regression model (f), bayesian model (g), artificial neural networks (h), random forest model (i), were applied to enhance the diagnosis power of plasma AAs. In each panel, the left is the ROC of diagnostic efficiency and the right is the ROC of the cross validation.

Figure 4.. Involvement of arginine depletion and metabolic dysregulation in the development of HFMD severity in a mouse model. Non-targeted metabolomics results showed score plot of PCA (a), PLS-DA (b), OPLS-DA (c) analysis. (d) Scree plot of metabolites. The horizontal axis (M/Z) represents the ratio of the mass number to the charge number of electric particles. (e) Heatmap visualization and clustering analysis of differential metabolites. (f) The normalized intensity levels of the down-regulated metabolites. (h) Network analysis of differential metabolites. n = 6 per group. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5.. In vivo experiments to assess the benefits of L-arginine supplementation on HFMD severity. (a) The experimental design of L-arginine intervention. Body weight (b), mean clinical scores (c) and survival rates (d) of mice were monitored and recorded daily (n = 8 per group). (e) The serum L-arginine levels of three groups after supplementing L-arginine at 5 dpi (n = 6 per group). (f) Pathological changes of brain slices and the distribution of VP1 antigens (×400). Histopathological score (g) and the viral loads (h) in the brains of mice (n = 3–4 per group). (i) Colocalization of LC3B and VP1 in brain slices was observed by immunofluorescence assay. The white arrows indicated cells with two proteins colocalization. (j) The expression levels of p-mTOR, p62, LC3B. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6.. The effect of L-arginine supplementation on pathological autophagy. (a) N: nucleus. Lysosomes (red arrows): monolayer organelles with high electron density. Autophagosome (green arrows): vesicles with double-membrane structures engulfing cytoplasmic material. The scale bar has been indicated in the figures. The number of autophagy lysosome (b) and autophagosome (c) (n = 3 per group). *p < 0.05, **p < 0.01, ***p < 0.001.