Characterization of P. gingivalis outer membrane vesicles (OMVs). (A) Transmission electron microscope (TEM) micrographs showing P. gingivalis OMVs. Electron microscopy imaging at 100 kv. Scale bar: 100 nm. (B) Size characteristics of P. gingivalis OMVs with a nanoparticle size analyzer (NSA). The dilution factor is 1000. The average diameter of P. gingivalis OMVs was about 72.28 nm with a concentration of 8.19 × 10¹⁰ particles/mL. (C) Confocal imaging results of P. gingivalis OMVs under 63x oil lens. Red, P. gingivalis OMVs labeled with PKH26. Scale bar: 10 μm

Analysis of the impact of P. gingivalis OMVs on the cardiac system. (A) Schematic diagram of the operation of the P. gingivalis OMVs for zebrafish treatment. Approximately 4 nL of PBS or OMVs were injected into the common cardinal vein of zebrafish larvae at 30 hpf using an Eppendorf microinjector (FemtoJet 4i, Eppendorf). (B) Mortality of zebrafish treated with P. gingivalis OMVs with different concentrations (0, 1.25, 2.5 and 4.5 µg/µL) at 2, 24 and 48 h post injection (hpi). n = 30, each group being replicated thrice. (C) Representative diagram of cardiac phenotype of zebrafish larvae at 48 hpi. Red arrow indicates pericardial position. The zebrafish larvae treated with OMVs showed three degrees of pericardial edema, that is normal, slight pericardial edema and severe pericardial edema. Scale bar: 0.5 mm. (D) Heartbeat statistics for different treatment groups (0, 1.25, 2.5 and 4.5 µg/µL) at 48 hpi. The unit is beats/min. n = 15, each group being replicated thrice. ***p ≤ 0.001. (E) Percentage of pericardial edema at concentrations of 0, 1.25, 2.5 and 4.5 µg/µL in 2 hpi. n = 30, each group being replicated thrice. (F) Percentage of pericardial edema at concentrations of 0, 1.25, 2.5 and 4.5 µg/µL in 24 hpi. n = 30, each group being replicated thrice. (G) Percentage of pericardial edema at concentrations of 0, 1.25, 2.5 and 4.5 µg/µL in 48 hpi. n = 30, each group being replicated thrice. (H) Expression results of gata4 and nkx 2.5 genes in 48 hpi zebrafish larvae. The relative change of mRNA levels was calculated by the 2 − ΔΔCt method and β-actin. Data were shown as the mean ± standard deviation (SD), and P values < 0.05 were considered statistically significant, ***p ≤ 0.001

Analysis of the impact of P. gingivalis OMVs on the vascular system. (A) Representation of confocal image of zebrafish larver at 0 hpi with microinjection of PBS or 2.5 µg/µL P. gingivalis OMVs in that heart region of the body. Bright, Bright channel, Red, P. gingivalis OMVs; Green, vascular endothelial cells. Scale bar: 100 μm. (B) Image of vascular fluorescence at 2 hpi after treated with PBS or 2.5 µg/µL P. gingivalis OMVs. Live imaging was performed under a stereoscopic fluorescence microscope (SMZ800N) in the green channel. Scale bar: 0.5 mm. (C) Analysis results of vascular fluorescence according to picture taken in Fig. 3B. n = 15, each group being replicated thrice. The unit is pixel, Mann Whitney test was used to analyze the significant difference between groups. (D) Image of vascular fluorescence at 24 hpi after treated with PBS or 2.5 µg/µL P. gingivalis OMVs. Scale bar: 0.5 mm. (E) Analysis results of vascular fluorescence according to picture taken in Fig. 3D. n = 15, each group being replicated thrice. Mann Whitney test was used to analyze the significant difference between groups, ***p ≤ 0.001. (F) Image of vascular fluorescence at 48 hpi after treated with PBS or 2.5 µg/µL P. gingivalis OMVs. Scale bar: 0.5 mm. (G) Analysis results of vascular fluorescence according to picture taken in Fig. 3F. n = 15, each group being replicated thrice. Mann Whitney test was used to analyze the significant difference between groups, ***p ≤ 0.001. (H) Expression results of cdh5 genes in 48 hpi zebrafish larvae. The relative change of mRNA levels was calculated by the 2 − ΔΔCt method and β-actin. Data were shown as the mean ± standard deviation (SD), and P values < 0.05 were considered statistically significant, ***p ≤ 0.001

Analysis of the impact of P. gingivalis OMVs on the neutrophils. (A) Representation of confocal image at 0 hpi with microinjection of PBS or 2.5 µg/µL P. gingivalis OMVs in that heart region of the body. Bright, Bright channel, Red, P. gingivalis OMVs; Green, neutrophils. Scale bar: 100 μm. (B) Image of neutrophils fluorescence at 2 hpi after treated with PBS or 2.5 µg/µL P. gingivalis OMVs. Live imaging was performed under a stereoscopic fluorescence microscope (SMZ800N) in the green channel. Scale bar: 0.5 mm. (C) Analysis results of neutrophils fluorescence according to picture taken in Fig. 4B. n = 15, each group being replicated thrice. The unit is pixel, Mann Whitney test was used to analyze the significant difference between groups. *p < 0.05. (D) Image of neutrophils fluorescence at 24 hpi after treated with PBS or 2.5 µg/µL P. gingivalis OMVs. Scale bar: 0.5 mm. (E) Analysis results of neutrophils fluorescence according to picture taken in Fig. 4D. n = 15, each group being replicated thrice. Mann Whitney test was used to analyze the significant difference between groups, **p < 0.01. (F) Image of neutrophils fluorescence at 48 hpi after treated with PBS or 2.5 µg/µL P. gingivalis OMVs. Scale bar: 0.5 mm. (G) Analysis results of neutrophils fluorescence according to picture taken in Fig. 4F. n = 15, each group being replicated thrice. Mann Whitney test was used to analyze the significant difference between groups, **p < 0.01

Analysis of the impact of P. gingivalis OMVs on the nfκb signaling pathway. (A) Representation of confocal image at 0 hpi with microinjection of PBS or 2.5 µg/µL P. gingivalis OMVs in that heart region of the body. Bright, Bright channel, Red, P. gingivalis OMVs; Green, nfκb signaling. Scale bar: 100 μm. (B) Image of green fluorescence at 2 hpi after treated with PBS or 2.5 µg/µL P. gingivalis OMVs. Live imaging was performed under a stereoscopic fluorescence microscope (SMZ800N) in the green channel. Scale bar: 0.5 mm. (C) Analysis results of green fluorescence according to picture taken in Fig. 5B. n = 15, each group being replicated thrice. The unit is pixel, Mann Whitney test was used to analyze the significant difference between groups. *p < 0.05. (D) Image of green fluorescence at 24 hpi after treated with PBS or 2.5 µg/µL P. gingivalis OMVs. Scale bar: 0.5 mm. (E) Analysis results of green fluorescence according to picture taken in Fig. 5D. n = 15, each group being replicated thrice. Mann Whitney test was used to analyze the significant difference between groups, ***p ≤ 0.001. (F) Image of green fluorescence at 48 hpi after treated with PBS or 2.5 µg/µL P. gingivalis OMVs. Scale bar: 0.5 mm. (G) Analysis results of green fluorescence according to picture taken in Fig. 5F. n = 15, each group being replicated thrice. Mann Whitney test was used to analyze the significant difference between groups, ***p ≤ 0.001. (H) Expression results of tnfa, tnfb and il6 genes in 48 hpi zebrafish larvae. The relative change of mRNA levels was calculated by the 2 − ΔΔCt method and β-actin. Data were shown as the mean ± standard deviation (SD), and P values < 0.05 were considered statistically significant, **p < 0.01, ***p ≤ 0.001

Transcriptomics results and validation. (A) Heat map analysis for RNA-seq data of samples at 48 hpi from the zebrafish larvae injected with PBS and P. gingivalis OMVs. (B) Volcano plot of differential expression analysis of PBS and P. gingivalis OMVs treated zebrafish larvae showing the relationship between P-value and log fold changes. Red shows upregulated genes and blue downregulated genes. Relative to the control, 332 differentially expressed genes (DEGs), including 250 up-regulated and 82 down-regulated, were identified. (C) Scatterplot of enriched top 30 GO pathways for DEGs. The Y-axis represented the GO pathways, and the X-axis represented the Rich factor. (D) Scatterplot of enriched top 20 KEGG pathways for DEGs. The Y-axis represented the KEGG pathways, and the X-axis represented the Rich factor. (E) Expression results of lama3, lamb3, lamc2, sv2a and thbs1a genes in 48 hpi zebrafish larvae. The relative change of mRNA levels was calculated by the 2 − ΔΔCt method and β-actin. Data were shown as the mean ± standard deviation (SD), and P values < 0.05 were considered statistically significant, *p < 0.05, **p < 0.01, ***p ≤ 0.001