Based on the gut–heart axis: Polygonum capitatum improves atherosclerosis by modulating gut microbiota and TMAO, supporting MCPIP1/p53-associated endothelial protection

Fig.1. Metabolomic analysis of drug-containing serum metabolites using a mass spectrometry platform. A OPLS-DA score plot. B Volcano plot showing upregulated and downregulated differential metabolites. C Pie chart illustrating the classification of serum metabolites. D KEGG pathway visualization of differential metabolites analyzed by MetaboAnalyst. E–H Common targets of differential metabolites were identified through database retrieval, followed by GO, KEGG, and PPI analyses, resulting in the construction of compound–target–disease networks

Fig.2. Prediction of AS-related diseases by combining UPLC-MS/MS component analysis with network pharmacology. A Total ion current chromatograms from UPLC–MS analysis in negative and positive ion modes. B Predicted key targets of PC in AS. C Pie chart of main chemical components. D Venn diagram showing the overlap of targets between PC and AS. E Top 20 enriched CC. F Top 20 enriched BP. G Top 20 enriched MF. H Enriched KEGG pathways

Fig.3. Pharmacological effects of PC in high-fat, high-choline diet–induced hamsters. A Serum lipid profiles, including TG, TC, LDL-C, and HDL-C. B Serum TMAO levels. C Pathological changes in the aorta assessed by H&E staining. D Pathological changes in the aorta assessed by Oil Red O staining. E Quantification of apoptotic regions detected by TUNEL fluorescence staining. F–H Serum oxidative stress indicators, including ROS, SOD, and MDA. I, J Serum apoptosis-related factors MCP-1 and IL-1β. K The expression of TUNEL is shown as a bar chart. All the data significance was analyzed by ANOVA by Graph Pad Prism Software, Data are expressed as the mean ± standard deviation of three or more independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig.4. PC restores MCPIP1 expression and attenuates TMAO-induced oxidative stress and apoptosis in hamster aortic tissue. A–I Expression of p47phox, NOX2, eNOS, iNOS, MCPIP1, p53, MDM2, and cleaved Caspase-3 proteins in AS hamsters. Western blot signals were quantified and normalized to β-Actin, with results shown as bar charts. Data are presented as mean ± standard deviation from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. J Localization of MCPIP1 and p53 in aortic tissue determined by in situ fluorescence analysis. K Localization of FMO3 in liver tissue determined by in situ IHC. L, M The expression of MCPIP1 and p53 is shown as a bar chart. N The expression of FMO3 is shown as a bar chart. Data are presented as mean ± standard deviation from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.O Expression of TMAO in the liver. All the data significance was analyzed by ANOVA by Graph Pad Prism Software, Data are presented as mean ± standard deviation from six independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig.5. Increased fecal microbial diversity in golden hamster treated with PC. A The Rarefraction curves of α diversity analysis. B Violin plot of α diversity analysis, C Principal Component Analysis (PCA) of β diversity analysis, D Community similarity analysis,The dots in the figure represent large discrete values with special markings. PCG, Atorvastatin intervention; PC_H, high dose PC treated diet-induced hamster; PC_M, median dose PC treated diet-induced hamster; PC_L, low dose PC treated diet-induced hamster; Model, diet-induced hamster without PC treatment; control, non-diet hamster. Multivariate statistical analysis was performed using principal component analysis (PCA) from the Ropls package in R language

Fig.6. Phylogenetic profiles of intestinal flora. A–C Species composition of fecal microbiota at different taxonomic levels: phylum, family, and genus. D Bubble plot of Indicator Species Analysis. PCG, atorvastatin intervention; PC_H, high-dose PC-treated diet-induced hamster; PC_M, medium-dose PC-treated diet-induced hamster; PC_L, low-dose PC-treated diet-induced hamster; Model, diet-induced hamster without PC treatment; Control, non-diet hamster. E Relative abundance levels of specific differential taxa. F Changes in F/B ratio. G Increased microbial communities at the genus level in non-diet, untreated, and high-dose PC-treated hamsters analyzed by LEfSe. H Functional prediction of gut microbial genes using PICRUSt2. I Cluster correlation analysis heat map. All the data significance was analyzed by ANOVA by Graph Pad Prism Software, Data are presented as mean ± standard deviation from six independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig.7. Successful establishment of gnotobiotic and FMT-treated groups. A α-Diversity analysis of gnotobiotic rodents. B β-Diversity analysis of gnotobiotic rodents. C Heatmap analysis of microbial abundance comparing gnotobiotic with pre-and post-FMT groups. D α-Diversity analysis of FMT-treated rodents. E β-Diversity analysis of FMT-treated rodents. Utilize Anosim (Analysis of Similarities) for similarity analysis, based on unweighted unifrac, weighted unifrac, Jaccard, and Bray–Curtis algorithms. All the data significance was analyzed by ANOVA by Graph Pad Prism Software, Data are presented as mean ± standard deviation from six independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig.8. Phylogenetic profiles of intestinal flora. A–D Species composition of fecal microbiota at different taxonomic levels: phylum, class, family, and genus. E Cluster correlation analysis heat map. F Relative abundance levels of specific differential taxa. G, H Changes in the Firmicutes/Bacteroidota (F/B) ratio, serum and liver TMAO levels. I Heatmap showing microbial abundance distribution after transplantation in each group. All the data significance was analyzed by ANOVA by Graph Pad Prism Software, Data are presented as mean ± standard deviation from three or more independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig.9. Levels of various indicators in FMT-treated groups. A Blood lipid levels. B ROS levels in hamster serum. C, D SOD and MDA levels in hamster serum. E, F IL-1β and MCP-1 levels in serum. G–J Expression of apoptosis-related proteins, including p53, MDM2, and Caspase-3. K–M Expression levels of MCPIP1 and p53 in aortic tissue of the GFT group. All the data significance was analyzed by ANOVA by Graph Pad Prism Software, Data are presented as mean ± standard deviation from three or more independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig.10. Proteomic analysis of TMAO-induced endothelial cells. A Venn diagram showing overlapping protein expression among Con, TMAO, and Si-MCPIP1 + TMAO groups. B PCA plot of principal component distribution. C Volcano plot of differentially expressed proteins between TMAO and Con groups. D Top 20 enriched GO terms for biological process BP,CC, and MF. E KEGG pathways identified based on differentially expressed proteins. F KEGG pathway enrichment analysis of quantitative proteins using GSEA

Fig.11. The role of MCPIP1 in oxidative stress and apoptosis was investigated by linking TMAO-MCPIP1-P53 and using Si-RNA. A Docking structure of MCPIP1 and TMAO, predicted by AlphaFold 3. B Results of MCPIP1 protein purification. C Analysis of Physical Binding Level of TMAO and MCPIP1 by SPR Technology. D Co-IP validation of the interaction between MCPIP1 and p53. E CCK8 endothelial cell viability caused by dose-gradient PC.F Transfection efficiency of MCPIP1 siRNAs were determined by reduction of MCPIP1 protein abundance. G, H released SOD and MDA in cell culture supernatant. I ROS in cell culture supernatant. J, K Secreted IL-1β and MCP-1 in cell culture supernatant. All the data significance was analyzed by ANOVA by Graph Pad Prism Software, Data are presented as mean ± standard deviation from three or more independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig.12. Apoptosis rate of endothelial cells and expression of apoptosis-related proteins in MCPIP1-depleted endothelial cells following PC and TMAO treatment. A–I Expression of p47phox, NOX2, eNOS, iNOS, MCPIP1, p53, MDM2, and cleaved Caspase-3 proteins. Western blot signals were quantified and normalized to β-Actin, with results shown as bar charts. Data are presented as mean ± standard deviation from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. J, K Flow cytometry analysis of endothelial cell apoptosis. The APC-A channel represents Annexin V–stained cells undergoing apoptosis, while the 7-AAD-A channel represents 7-AAD–stained dead cells. All the data significance was analyzed by ANOVA by Graph Pad Prism Software, Data are presented as mean ± standard deviation from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001