To the Editor: Coronary atherosclerotic heart disease (CAD) arises from coronary atherosclerosis (CAS) plaque buildup, resulting in coronary stenosis (CS) and occlusion, ultimately causing myocardial ischemia, hypoxia, or necrosis. Studies showed that the high-grade CS may be an important predictor of ST segment elevation myocardial infarction in subsequent months.[1]

Recent research has underscored the significant impact of alterations in oral and gut flora on CAS. Dysbiosis, for instance, triggers oxidative stress, inflammatory responses, and metabolic disturbances, thereby initiating the atherosclerotic process.[2] However, a systematic study is still needed to elucidate the distinctions in oral and gut microbial traits and metabonomic features among CAS patients with different degrees of coronary artery stenosis, and to confirm the relationship between them, which may provide novel approach to the early diagnosis and precise treatment of CAS.

To address these concerns, we conducted an analysis of the oral and gut microbial traits in 63 CAS patients and 31 controls using 16S ribosomal RNA (rRNA) amplicon sequencing. Blood samples were collected on the first day of hospitalization, and untargeted metabolomics analysis was performed using Liquid Chromatography combined with Tandem Mass Spectrometry (LC-MS/MS). Genomic DNA was extracted from oral and fecal samples and used for amplifying the gene sequence in the V3–V4 region of bacterial 16S rRNA. Raw reads were obtained and subjected to quality analysis. Furthermore, we employed a multi-omics approach to explore the potential cooperative regulation of metabolism by oral and gut microbiota in CAS patients with varying degrees of stenosis. Wilcoxon rank-sum test was used for analysis, one-way analysis of variance and pairwise comparisons were performed among groups, and a P value <0.05 was considered statistically significant. This study obtained ethical approval from the Second Hospital of Shanxi Medical University Ethics Committee (No. 2022-YX-084). Informed consent forms were signed by the patients or their families.

The participants who underwent coronary angiography were divided into three groups as follows: Minimal and mild coronary stenosis (MMCS) group (n = 33), with stenosis <50% in at least one coronary segment; moderate and severe coronary stenosis (MSCS) group (n = 30), with stenosis >50% in at least one coronary segment;[3] and the control group (negative results in coronary computed tomography or coronary angiography, n = 31). The clinical features of the subjects are summarized in Supplementary Table 1, https://links.lww.com/CM9/B877. Trained dentists evaluated periodontal health indexes [Supplementary Table 2, https://links.lww.com/CM9/B877].

Intra-community alpha diversity, representing microbial richness and evenness, was analyzed, and there was no statistically significant difference in oral microbial community alpha diversity among the three groups. Beta-diversity assessed whether there were significant differences in microbial community between groups. While, the weighted-unifrac-based beta diversity of the oral microbial community differed significantly between the MMCS and control groups (P <0.001), as well as between the MSCS and control groups (P <0.001) [Figure 1A].

Figure 1:

Oral and intestinal microbiology analysis. (A) Weighted-unifrac-based beta diversity of the oral microbial community among three groups. (B) Evaluation of alpha diversity of the intestinal microbial community using sobs indices among three groups. (C) Weighted-unifrac-based beta diversity of the intestinal microbial community among three groups. Cladogram using the LEfSe method indicating the phylogenetic distribution of the oral microbiotas (D,E) and gut microbiotas (F,G). The oral microbiotas (H,I) and gut microbiota (J,K) with significant differences between two groups (LDA score >2.0). *P <0.05, †P <0.01. LEfSe: Linear discriminant analysis effect size; LDA: linear discriminant analysis; MMCS: Minimal and mild coronary stenosis; MSCS: Moderate and severe coronary stenosis; NS: Not significant.

Regarding gut microbial community alpha diversity, the sobs index of the control group was the highest and statistically significantly different from the other two groups (control group vs. MMCS group, P = 0.041; control group vs. MSCS group, P = 0.005; MMCS group vs. MSCS group, P = 0.027) [Figure 1B]. The weighted-unifrac-based beta diversity of the gut microbial community showed statistically significant differences between the MMCS and control group, the MSCS and control group, and the MMCS and MSCS group (control group vs. MMCS group, P <0.001; control group vs. MSCS group, P <0.001; MMCS group vs. MSCS group, P <0.001) [Figure 1C].

Based on the abundance profiles, the features with significantly differential abundance across groups were determined using the Wilcoxon rank-sum test. Linear discriminant analysis effect size (LEfSe) analysis was utilized to detect biomarkers in saliva and feces [Figure 1D–K] for distinguishing MMCS and MSCS from normal coronary arteries.

In saliva samples, compared to the control group, the MMCS group exhibited significantly increased levels of genus Mobiluncus, and significantly decreased levels of the genus Tessaracoccus. (P <0.05). Similarly, the MSCS group, when compared to the control group, showed significantly increased levels of the genus Howardella in saliva. Moreover, in the saliva of MSCS patients, compared to the control group, the order Cardiobacteriales and Burkholderiales, family Cardiobacteriaceae and Burkholderiaceae, genus Cardiobacterium and Lautropia, were significantly lower (P <0.05).

In fecal samples, compared to the control group, the MMCS group exhibited significantly increased levels of the genus Fusicatenibacter, and significantly decreased levels of the genus Olsenella, Anaerococcus and Intestinibacter (P <0.05). On the other hand, in the fecal samples of the MSCS group compared to the control group, the genus Desulfovibrio, Moraxella, and Actinomyces were significantly increased, while genus Ilumatobacter, Aeromonas and, Litorilinea, Loktanella were significantly decreased (P <0.05). The data presented in the study are deposited in the NCBI Sequence Read Archive repository (Nos. PRJNA1033397 and PRJNA1042880).

Based on the orthogonal partial least-squares discriminant analysis (OPLS-DA) models of metabolite profiling data, we observed significantly different metabolite profiles in patients with MMCS and MSCS compared to control subjects [Supplementary Figure 1A and 1B, https://links.lww.com/CM9/B877]. These models identified 64 different metabolites in the MMCS group and 170 different metabolites in the MSCS group (screening criteria for differential metabolites: level ≤4, variable importance in projection ≥1, fold change ≥1.2 or ≤0.83, P <0.05) [Supplementary Figure 1C and 1D, https://links.lww.com/CM9/B877]. Next, we analyzed the metabolites identified in the MMCS and MSCS groups using the receiver operating characteristic (ROC) curve to find the metabolic biomarkers of MMCS and MSCS in serum. The results of the area under the curve (AUC) are shown in Supplementary Table 3, https://links.lww.com/CM9/B877. Some of the differential metabolites with predictive value for MMCS included verrucarol, 3-hydroxytetradecanedioic acid (3-HA), and geranylacetone, among others. Similarly, the differential metabolites with predictive value for MSCS included glucuronic acid-3,6-lactone, 3-HA, 1-oleoyl-rac-glycerol, and athamantin, among others.

The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results in MMCS group and MSCS group are showed in Supplementary Figure 2A and 2B, https://links.lww.com/CM9/B877.

Inflammation serves as the primary mechanism in the regulation of CAS. Studies showed that the pathogenic bacteria involved in periodontitis may participate in the pathogenesis of CAS through direct inflammatory response.[2] Our studies also confirmed the results. Compared to the control group, the clinical attachment level (CAL) which reflects periodontal health increased in the MMCS group, and the CAL further increased in the MSCS group. Interleukin-6 (IL-6) is a pro-inflammatory cytokine.[2] Studies showed that elevated levels of 3-HA that are associated with dysregulation of fatty acid metabolism, were observed in patients with severe psoriatic arthritis.[4] Docosahexaenoic acid (DHA) can exert potential anti-obesity effects by reducing the secretion of inflammatory adipokines, oxidative stress, lipolysis, and apoptosis.[5] N-arachidonoyl dopamine (N-ADA) possesses anti-inflammatory properties.[6] Our results showed that, compared to the control group, the MMCS and MSCS groups had higher levels of IL-6 and 3-HA and lower levels of N-ADA. DHA was significantly decreased only in the MSCS group but not in the MMCS group. Moreover, DHA is involved in the biosynthesis of the unsaturated fatty acids pathway, which play a role in the regulation of inflammation, while N-ADA is involved in the regulation of inflammatory mediator on TRP channels.

Therefore, we investigated the correlations between the oral and gut microbiome and serum metabolites 3-HA, N-ADA, DHA and IL-6 in MMCS and MSCS patients using Spearman correlation analysis. In the saliva of MMCS patients, the genus Mobiluncus exhibited a positive correlation with the IL-6 and 3-HA (r = 0.43 and r = 0.50, P <0.05), genus Tessaracoccus exhibited a positive correlation with the N-ADA (r = 0.28, P <0.05).

In the feces of MMCS patients, the genus Fusicatenibacter showed positive correlations with the IL-6 (r = 0.36, P <0.05), the genus Anaerococcus exhibited negative correlations with 3-HA and Intestinibacter exhibited positive correlations with N-ADA (r = –0.37 and r = 0.32, P <0.05).

In the saliva of MSCS patients, the genus Howardella exhibited positive correlations with IL-6 and 3-HA. The genus Cardiobacterium and Lautropia exhibited a positive correlation with DHA [Supplementary Figure 2C, https://links.lww.com/CM9/B877].

In the fecal samples of MSCS patients, the genus Actinomyces and Moraxella were positively correlated with the 3-HA and IL-6, and the genus Litorilinea, Loktanella and Ilumatobacter were negatively correlated with 3-HA and IL-6. Additionally, the genus Desulfovibrio displayed negative correlations with DHA, and positive correlations with N-ADA [Supplementary Figure 2D, https://links.lww.com/CM9/B877].

These findings suggested that the composition of oral and intestinal microorganisms, and metabolites were statistically significantly different between controls and patients with CAS-related CS, and the microorganisms and metabolites may further change with the severity of CS. Oral and gut microorganisms may play a role in the development of MMCS and MSCS by interdependently regulating inflammation-related metabolites such as IL-6, 3-HA, DHA, and N-ADA.

Therefore, maintaining the homeostasis of oral and gut microbiota and reducing the abundance of Mobiluncus, Fusicatenibacter, Actinomyces, Moraxella, and Desulfovibrio in patients with CAS may aid in preventing the occurrence of inflammation and progression of CAS. Limitations of our study include its preliminary nature and small sample size. Expanding the study population would enhance the robustness of the results.

Funding

This research received supports from the key project of the National Natural Science Foundation of China (No. 81430011) and the Research Project of the Shanxi Provincial Health and Family Planning Commission (Nos. 2020068 and 2021XM09).

Conflicts of interest

None.

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