Fucosylated oligosaccharide Lacto-N-fucopentaose I ameliorates enterovirus 71 infection by inhibiting apoptosis

Figures

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  Fig. 1. Cytotoxicity of LNFPI and its inhibition of EV71 in RD cells. (a, b) Cytotoxicity and anti-EV71 effect of LNFPI were measured using a CCK-8 kit. (c) Dyed crystal violet assay was performed in EV71-induced RD cells pre-treated with LNFPI. (d) The bar graph shows the change in lactic dehydrogenase activity in EV71-induced RD cells with and without LNFPI pre-treatment. (e) The time-of-addition experiment revealed the anti-EV71 effect of LNFPI at different times (*p < 0.05, **p < 0.01 compared with the normal group; #p < 0.05, ##p < 0.01 compared with the model group).

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  Fig. 2. LNFPI inhibited the attachment of EV71 to RD cells. (a) The total RNA of EV71-induced RD cells pre-treated with LNFPI was extracted for the analysis of VP1 expression using reverse-transcription polymerase chain reaction. (b–d) The molecule structure of LNFPI was visualised via 3D draw. AutoDock exhibited the potential binding sites between EV71 and LNFPI (*p < 0.05, **p < 0.01 compared with the normal group; #p < 0.05, ##p < 0.01 compared with the model group).

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  Fig. 3. LNFPI reduced EV71-induced oxidative stress in RD cells. (a, b) The SOD activity and MDA level in EV71-induced RD cells that could affect stress reaction were measured to explore the inhibition of oxidative stress by LNFPI. (c, d) The level of ROS in EV71-induced RD cells was evaluated using DCFH-DA and observed under the fluorescence microscope after LNFPI pre-treatment (*p < 0.05, **p < 0.01 compared with the normal group; #p < 0.05, ##p < 0.01 compared with the model group).

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  Fig. 4. LNFPI mitigated EV71-associated cell apoptosis in mitochondria-dependent and death-receptor manner. (a) The Hoechst 33,258 assay was performed in EV71-induced RD cells with or without LNFPI pre-treatment and observed using fluorescence microscopy with × 100 and × 200 magnifications. (b) LNFPI affected apoptosis rates of RD cells subjected to PI and Annexin V staining after EV71 invasion using flow cytometry. (c) The caspase activity with and without LNFPI pre-treatment was examined. (d, e) The mRNA expressions of apoptosis-associated genes were calculated and displayed in bar graphs (*p < 0.05, **p < 0.01 compared with the normal group; #p < 0.05, ##p < 0.01 compared with the model group).

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  Fig. 5. LNFPI leads to the recovery of EV71-induced S phase arrest in RD cells. (a, b) Flow cytometry was used to analyse EV71-induced RD cells stained with PI to investigate the influence of LNFPI on the cell cycle. (c, d) Western blot assay was employed to explore the effect of LNFPI on cyclin-related protein after EV71 invasion. The bar graph shows quantification of the cyclin E/β-actin and CDK2/β-actin ratios (*p < 0.05, **p < 0.01 compared with the normal group; #p < 0.05, ##p < 0.01 compared with the model group).

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  Fig. 6. Anti-apoptotic effect of LNFPI treatment on C. elegans and its intestinal flora. (a) Acridine orange staining assay showed the anti-apoptotic effect of LNFPI, and bar graphs indicated the relative mean fluorescence. (b) Bar graphs showed the relative mRNA expression of apoptotic genes. (c) The change in Chao1 and Shannon indices of intestinal flora after LNFPI treatment. (d) Difference in bacteria at the phylum level between C. elegans with or without LNFPI pre-treatment. (e) Top abundant bacteria at the genus level. (f, g) Spearman correlation analysis of apoptosis-related genes and their intestinal flora. Red means positive and blue means negative (*p < 0.05, **p < 0.01 compared with the normal group; #p < 0.05, ##p < 0.01 compared with the model group).